Cancer Research recent issues

Astrocyte Elevated Gene-1: Far More Than Just a Gene Regulated in Astrocytes

Sarkar, D., Emdad, L., Lee, S.-G., Yoo, B. K., Su, Z.-z., Fisher, P. B. — Wed, 11 Nov 2009 21:08:16 PST

Since its original cloning by subtraction hybridization in 2002, it is now evident that Astrocyte elevated gene-1 (AEG-1) is a key contributor to the carcinogenic process in diverse organs. AEG-1 protein expression is elevated in advanced stages of many cancers, which correlates with poor survival. In specific cancers, such as breast and liver cancer, the AEG-1 gene itself is amplified, further supporting a seminal role in tumorigenesis. Overexpression and inhibition studies both in in vitro and in in vivo models reveal the importance of AEG-1 in regulating multiple physiologically and pathologically relevant processes including proliferation, invasion, metastasis, and gene expression. AEG-1 is a single-pass transmembrane protein with multiple nuclear localization signals and no known domains or motifs. Although pertinent roles of AEG-1 in the carcinogenic process are established, its potential function (promotion of metastasis only versus functioning as a bona fide oncogene) as well as localization (cell surface versus nucleus) remain areas requiring further clarification. The present review critically evaluates what is currently known about AEG-1 and provides new perspectives relative to this intriguing molecule that may provide a rational target for intervening in the cancer phenotype. [Cancer Res 2009;69(22):8529–35]

Cell Fusion as a Hidden Force in Tumor Progression

Lu, X., Kang, Y. — Wed, 11 Nov 2009 21:08:17 PST

Cell fusion plays an essential role in fertilization, formation of placenta, bone and muscle tissues, immune response, tissue repair, and regeneration. Increasing recognition of cell fusion in somatic cell dynamics has revitalized the century-old hypothesis that cell fusion may contribute to the initiation and progression of cancer. In this review, we discuss findings from experimental and clinical studies that suggest a potentially multifaceted involvement of cell fusion in different stages of tumor progression, including aneuploidy and tumor initiation, origin of cancer stem cells, multidrug resistance, and the acquisition and diversification of metastatic abilities. [Cancer Res 2009;69(22):8536–9]

Aurora B-Mediated Phosphorylation of RASSF1A Maintains Proper Cytokinesis by Recruiting Syntaxin16 to the Midzone and Midbody

Song, S. J., Kim, S. J., Song, M. S., Lim, D.-S. — Wed, 11 Nov 2009 21:08:17 PST

Aurora B is critically involved in ensuring proper cytokinesis and maintaining genomic stability. The tumor suppressor RASSF1A regulates cell cycle progression by regulating mitotic progression, G₁-S transition, and microtubule stability. We previously reported that both Aurora A and Aurora B phosphorylate RASSF1A, and showed that phosphorylation of RASSF1A by Aurora A blocks the inhibitory function of RASSF1A toward anaphase-promoting complex-Cdc20. However, the role of Aurora B–mediated RASSF1A phosphorylation remains unknown. Here, we show that phosphorylation of RASSF1A on Ser203 by Aurora B during late mitosis has a critical role in regulating cytokinesis. Notably, RASSF1A interacts with Syntaxin16, a member of the t-SNARE family, at the midzone and midbody during late mitosis. Aurora B is required for this interaction and for the subsequent recruitment of Syntaxin16 to the midzone and midbody, a prerequisite for the successful completion of cytokinesis. Furthermore, Aurora B depletion results in a failure of Syntaxin16 to properly localize to the midzone and midbody, a mislocalization that was prevented by overexpression of the phosphomimetic RASSF1A (S203D) mutant. Finally, either depletion of Syntaxin16 or expression of the nonphosphorylatable RASSF1A (S203A) mutant results in cytokinesis defects. Our findings implicate Aurora B–mediated phosphorylation of RASSF1A in the regulation of cytokinesis. [Cancer Res 2009;69(22):8540–4]

Loss of Collapsin Response Mediator Protein1, as Detected by iTRAQ Analysis, Promotes Invasion of Human Gliomas Expressing Mutant EGFRvIII

Wed, 11 Nov 2009 21:08:17 PST

Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor. GBMs are characterized by a variety of genetic alterations, among which oncogenic mutations of epidermal growth factor receptor (EGFRvIII) is most common. GBMs harboring EGFRvIII have increased proliferation and invasive characteristics versus those expressing wild-type (wt) EGFR. To identify the molecular basis of this increased tumorgenic phenotype, we used iTRAQ-labeling differential proteomic analysis. Among several differentially expressed proteins, we selected CRMP1, a protein implicated in cellular invasion that was markedly decreased in GBMs expressing EGFRvIII, for further study. The differential expression of CRMP1 was confirmed in a panel of human GBM cell lines and operative specimens that express wtEGFR or mutant EGFRvIII by quantitative real-time PCR, Western blot, and immunohistochemical analysis. In human GBM samples, decreased expression of CRMP1 correlated with EGFRvIII positivity. Knockdown of CRMP1 by siRNA resulted in increased invasion of wtEGFR expressing human GBM cells (U87 and U373) to those found in isogenic GBM cells. Exogenous expression of EGFRvIII in these wtEGFR-expressing GBM cells promoted their ability to invade and was accompanied by decreased expression of CRMP1. Rescuing CRMP1 expression decreased invasion of the EGFRvIII-expressing GBM cells by tilting the balance between Rac and Rho. Collectively, these results show that the loss of CRMP1 contribute to the increased invasive phenotype of human GBMs expressing mutant EGFRvIII. [Cancer Res 2009;69(22):8545–54]

Lin-Sca-1+CD49fhigh Stem/Progenitors Are Tumor-Initiating Cells in the Pten-Null Prostate Cancer Model

Mulholland, D. J., Xin, L., Morim, A., Lawson, D., Witte, O., Wu, H. — Wed, 11 Nov 2009 21:08:17 PST

We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin^–Sca-1⁺CD49f^high (LSC) subpopulation overlaps with CK5⁺;p63⁺ cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model. [Cancer Res 2009;69(22):8555–62]

p73 and p63 Sustain Cellular Growth by Transcriptional Activation of Cell Cycle Progression Genes

Wed, 11 Nov 2009 21:08:17 PST

Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G₁-S and G₂-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G₀-G₁, p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions. [Cancer Res 2009;69(22):8563–71]

Wnt Signaling Stimulates Transcriptional Outcome of the Hedgehog Pathway by Stabilizing GLI1 mRNA

Wed, 11 Nov 2009 21:08:17 PST

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/β-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells. [Cancer Res 2009;69(22):8572–8]

Inhibition of Lipocalin 2 Impairs Breast Tumorigenesis and Metastasis

Wed, 11 Nov 2009 21:08:17 PST

Lipocalin 2 (LCN2; also known as NGAL) is a secreted glycoprotein and its elevated expression has been observed in breast cancers. However, the importance of LCN2 in breast tumorigenesis is unclear. Here, we employed a spontaneous mammary tumor mouse model showing that MMTV-ErbB2(V664E) mice lacking mouse LCN2 had significantly delayed mammary tumor formation and metastasis with reduced matrix metalloproteinase-9 activity in the blood. LCN2 expression is upregulated by HER2/phosphoinositide 3-kinase/AKT/NF-B pathway. Decreasing LCN2 expression significantly reduced the invasion and migration ability of HER2⁺ breast cancer cells. Furthermore, injecting an anti-mouse LCN2 antibody into mice bearing established murine breast tumors resulted in significant blockage of lung metastasis. Our findings indicate that LCN2 is a critical factor in enhancing breast tumor formation and progression possibly in part by stabilizing matrix metalloproteinase-9. Our results suggest that inhibition of LCN2 function by an inhibitory monoclonal antibody has potential for breast cancer therapy, particularly by interfering with metastasis in aggressive types of breast cancer. [Cancer Res 2009;69(22):8579–84]

Motor Protein-Dependent Membrane Trafficking of KCl Cotransporter-4 Is Important for Cancer Cell Invasion

Wed, 11 Nov 2009 21:08:17 PST

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I–induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I– or EGF-dependent cancer spread. [Cancer Res 2009;69(22):8585–93]

Lipid Rafts and Caveolin-1 Are Required for Invadopodia Formation and Extracellular Matrix Degradation by Human Breast Cancer Cells

Yamaguchi, H., Takeo, Y., Yoshida, S., Kouchi, Z., Nakamura, Y., Fukami, K. — Wed, 11 Nov 2009 21:08:17 PST

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA–mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP–dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion. [Cancer Res 2009;69(22):8594–602]

Functional Significance of Wnt Inhibitory Factor-1 Gene in Kidney Cancer

Wed, 11 Nov 2009 21:08:17 PST

Wnt inhibitory factor-1 (WIF-1) has been identified as one of the secreted antagonists that bind Wnt protein. WIF-1 has been described as a tumor suppressor in various types of cancer. However, the molecular function of WIF-1 gene has never been examined in human renal cell carcinoma (RCC). Therefore, we hypothesized that WIF-1 functions as a tumor suppressor gene and overexpression of this gene may induce apoptosis and inhibit tumor growth in RCC cells. Immunohistochemistry and real-time reverse transcription-PCR revealed that WIF-1 was significantly downregulated in RCC samples and RCC cell lines, respectively. Bisulfite sequencing of the WIF-1 promoter region in RCC cell lines showed it to be densely methylated, whereas there was no methylation of WIF-1 promoter in normal kidney. Significant inhibition of cell growth and colony formation in WIF-1–transfected cells compared with controls were observed. WIF-1 transfection significantly induced apoptosis and suppressed in vivo tumor growth. Also, Wnt signaling activity and β-catenin expression were reduced by WIF-1 transfection. In conclusion, this is the first report documenting that the WIF-1 is downregulated by promoter methylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells. [Cancer Res 2009;69(22):8603–10]

NPM-ALK Oncogenic Tyrosine Kinase Controls T-Cell Identity by Transcriptional Regulation and Epigenetic Silencing in Lymphoma Cells

Wed, 11 Nov 2009 21:08:17 PST

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell–specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell–specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor–related signaling molecules, including CD3, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK^K210R, downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling. [Cancer Res 2009;69(22):8611–9]

{beta}1-Integrin Circumvents the Antiproliferative Effects of Trastuzumab in Human Epidermal Growth Factor Receptor-2-Positive Breast Cancer

Wed, 11 Nov 2009 21:08:17 PST

Resistance to trastuzumab, the monoclonal antibody targeting human epidermal growth factor receptor 2 (HER-2), is a major concern for HER-2–positive metastatic breast cancer (MBC) patients. To date, HER-2 status is the only available biomarker for selecting patients for trastuzumab-based therapy. β₁-Integrin, an adhesion molecule involved in cell survival and drug resistance, shares common downstream signaling elements with HER-2, such as the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways. The significance of β₁-integrin expression in HER-2–positive breast cancer and its involvement in a patient's response to trastuzumab-based therapy are unknown. We show here that overexpression of β₁-integrin is an independent negative prognostic factor for tumor progression of HER-2–positive MBC patients treated with trastuzumab-based chemotherapy. Enforced overexpression of β₁-integrin, its small interfering RNA–induced knockdown or treatment with a β₁-integrin–blocking antibody in HER-2–positive breast cancer cells, identified a strong inverse relationship between expression level of β₁-integrin and in vitro sensitivity to trastuzumab. Notably, β₁-integrin overexpression increased the phosphorylation of Akt-Ser473 and ERK1/2, thereby promoting survival and mitogenic signals to bypass the antiproliferative effects of trastuzumab. Our findings show that β₁-integrin provides a novel independent prognostic biomarker of trastuzumab response in HER-2–positive MBC patients and suggest a new target to augment the antiproliferative effects of trastuzumab. [Cancer Res 2009;69(22):8620–8]

Pattern of Antioxidant and DNA Repair Gene Expression in Normal Airway Epithelium Associated with Lung Cancer Diagnosis

Wed, 11 Nov 2009 21:08:17 PST

In previous studies, we reported that key antioxidant and DNA repair genes are regulated differently in normal bronchial epithelial cells of lung cancer cases compared with non–lung cancer controls. In an effort to develop a biomarker for lung cancer risk, we evaluated the transcript expressions of 14 antioxidant, DNA repair, and transcription factor genes in normal bronchial epithelial cells (HUGO names CAT, CEBPG, E2F1, ERCC4, ERCC5, GPX1, GPX3, GSTM3, GSTP1, GSTT1, GSTZ1, MGST1, SOD1, and XRCC1). A test comprising these 14 genes accurately identified the lung cancer cases in two case-control studies. The receiver operating characteristic–area under the curve was 0.82 (95% confidence intervals, 0.68–0.91) for the first case-control set (25 lung cancer cases and 24 controls), and 0.87 (95% confidence intervals, 0.73–0.96) for the second set (18 cases and 22 controls). For each gene included in the test, the key difference between cases and controls was altered distribution of transcript expression among cancer cases compared with controls, with more lung cancer cases expressing at both extremes among all genes (Kolmorogov-Smirnov test, D = 0.0795; P = 0.041). A novel statistical approach was used to identify the lower and upper boundaries of transcript expression that optimally classifies cases and controls for each gene. Based on the data presented here, there is an increased prevalence of lung cancer diagnosis among individuals that express a threshold number of key antioxidant, DNA repair, and transcription factor genes at either very high or very low levels in the normal airway epithelium. [Cancer Res 2009;69(22):8629–35]

Repression of NHE1 Expression by PPAR{gamma} Activation Is a Potential New Approach for Specific Inhibition of the Growth of Tumor Cells In vitro and In vivo

Wed, 11 Nov 2009 21:08:17 PST

Ligand-induced activation of peroxisome proliferator-activated receptor (PPAR) inhibits proliferation in cancer cells in vitro and in vivo; however, the downstream targets remain undefined. We report the identification of a peroxisome proliferator response element in the promoter region of the Na⁺/H⁺ transporter gene NHE1, the overexpression of which has been associated with carcinogenesis. Exposure of breast cancer cells expressing high levels of PPAR to its natural and synthetic agonists resulted in downregulation of NHE1 transcription as well as protein expression. Furthermore, the inhibitory effect of activated PPAR on tumor colony-forming ability was abrogated on overexpression of NHE1, whereas small interfering RNA–mediated gene silencing of NHE1 significantly increased the sensitivity of cancer cells to growth-inhibitory stimuli. Finally, histopathologic analysis of breast cancer biopsies obtained from patients with type II diabetes treated with the synthetic agonist rosiglitazone showed significant repression of NHE1 in the tumor tissue. These data provide evidence for tumor-selective downregulation of NHE1 by activated PPAR in vitro and in pathologic specimens from breast cancer patients and could have potential implications for the judicious use of low doses of PPAR ligands in combination chemotherapy regimens for an effective therapeutic response. [Cancer Res 2009;69(22):8636–44]

The Fibroblast Growth Factor Receptor Inhibitor PD173074 Blocks Small Cell Lung Cancer Growth In vitro and In vivo

Wed, 11 Nov 2009 21:08:17 PST

Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2–induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham–treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3'-deoxy-3'-[¹⁸F]fluorothymidine–positron emission tomography ([¹⁸F]FLT-PET) showed decreased intratumoral proliferation in live animals treated with the compound at 7 to 14 days. Our results suggest that clinical trials of FGFR inhibitors should be undertaken in patients with SCLC and that [¹⁸F]FLT-PET imaging could provide early in vivo evidence of response. [Cancer Res 2009;69(22):8645–51]

Constitutive Activation of the DNA Damage Signaling Pathway in Acute Myeloid Leukemia with Complex Karyotype: Potential Importance for Checkpoint Targeting Therapy

Wed, 11 Nov 2009 21:08:17 PST

Genomic instability in solid tumors participates in the oncogenetic process and is associated with the activation of the DNA damage response pathway. Here, we report the activation of the constitutive DNA damage and checkpoint pathway associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML). We show that antagonizing CHK1 kinase with a small inhibitory compound or by RNA interference strongly reduces the clonogenic properties of high–DNA damage level AML samples, particularly those with complex karyotypes. Moreover, we observe a beneficial effect of CHK1 inhibition in high–DNA damage level AML samples treated with 1-β-d-arabinofuranosylcytosine. In contrast, CHK1 inhibition has no effect on the clonogenic properties of normal hematopoietic progenitors. All together, our results indicate that CHK1 inhibition may represent an attractive therapeutic opportunity in AML with complex karyotype. [Cancer Res 2009;69(22):8652–61]

De-N-acetyl GM3 Promotes Melanoma Cell Migration and Invasion through Urokinase Plasminogen Activator Receptor Signaling-Dependent MMP-2 Activation

Wed, 11 Nov 2009 21:08:17 PST

We have recently discovered that de-N-acetyl GM3 [NeuNH₂LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82–95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3–positive) and poorly invasive (d-GM3–negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases. [Cancer Res 2009;69(22):8662–9]

Heat Shock Protein 90 Inhibitors: New Mode of Therapy to Overcome Endocrine Resistance

Wong, C., Chen, S. — Wed, 11 Nov 2009 21:08:17 PST

Aromatase inhibitors are important drugs to treat estrogen receptor (ER)–positive postmenopausal breast cancer patients. However, development of resistance to aromatase inhibitors has been observed. We examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of aromatase inhibitor–resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. Aromatase inhibitor–responsive MCF-7aro and aromatase inhibitor–resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G₂ cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ER, the ER transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Collectively, results from this study provide data to support that HSP90 inhibitors may be an effective therapy to treat aromatase inhibitor–resistant breast cancers and that improved efficacy can be achieved by combined use of a HSP90 inhibitor and an AKT inhibitor. [Cancer Res 2009;69(22):8670–7]

A Novel Role of Interleukin-13 Receptor {alpha}2 in Pancreatic Cancer Invasion and Metastasis

Fujisawa, T., Joshi, B., Nakajima, A., Puri, R. K. — Wed, 11 Nov 2009 21:08:17 PST

Whereas interleukin-13 receptor 2 chain (IL-13R2) is overexpressed in a variety of human solid cancers including pancreatic cancer, we investigated its significance in cancer invasion and metastasis. We used two pancreatic cancer cell lines, IL-13R2–negative HPAF-II and IL-13R2–positive HS766T, and generated IL-13R2 stably transfected HPAF-II as well as IL-13R2 RNA interference knocked-down HS766T cells. Ability of invasion and signal transduction was compared between IL-13R2–negative and IL-13R2–positive cells and tumor metastasis was assessed in murine model for human pancreatic cancer with orthotopic implantation of tumors. IL-13 treatment enhanced cell invasion in IL-13R2–positive cancer cell lines but not in IL-13R2–negative cell lines. Furthermore, gene transfer of IL-13R2 in negative cell lines enhanced invasion, whereas its silencing downmodulated invasion of pancreatic cell lines in a Matrigel invasion assay. In vivo study revealed that IL-13R2–positive cancer metastasized to lymph nodes, liver, and peritoneum at a significantly higher rate compared with IL-13R2–negative tumors. The expression of IL-13R2 in metastatic lesions was found to be increased compared with primary tumors, and mice with IL-13R2–positive cancer displayed cachexia and poor prognosis. Invasion and metastasis also correlated with increased matrix metalloproteinase protease activity in these cells. Mechanistically, IL-13 activated extracellular signal-regulated kinase 1/2 and activator protein-1 nuclear factors in IL-13R2–positive pancreatic cancer cell lines but not in IL-13R2–negative cell lines. Taken together, our results show for the first time that IL-13 can signal through IL-13R2 in pancreatic cancer cells and IL-13R2 may serve as a prognostic biomarker of invasion and metastasis in pancreatic cancer. [Cancer Res 2009;69(22):8678–85]

Targeted Disruption of the S1P2 Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation

Wed, 11 Nov 2009 21:08:17 PST

S1P₂ sphingosine 1-phosphate receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S1P₂^–/– mice develop clonal B-cell lymphomas with age, such that approximately half of the animals display this neoplasm by 1.5 to 2 years of age. Histologic, immunophenotypic, and molecular analyses revealed a uniform tumor phenotype with features of germinal center (GC)–derived diffuse large B-cell lymphoma (DLBCL). Tumor formation was preceded by increases in GC B cells and CD69⁺ T cells, as well as an increased formation of spontaneous GCs, suggesting that S1P₂ loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S1P₂ gene disruption is remarkably restricted to DLBCL. In humans, 28 of 106 (26%) DLBCL samples were found to harbor multiple somatic mutations in the 5' sequences of the S1P₂ gene. Mutations displayed features resembling those generated by the IgV-associated somatic hypermutation mechanism, but were not detected at significant levels in normal GC B cells, indicating a tumor-associated aberrant function. Collectively, our data suggest that S1P₂ signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S1P₂^–/– mice, its onset at old age, and the relative lack of other neoplasms identify these mice as a novel, and potentially valuable, model for this highly prevalent and aggressive human malignancy. [Cancer Res 2009;69(22):8686–92]

Enhanced Antitumor Activity Induced by Adoptive T-Cell Transfer and Adjunctive Use of the Histone Deacetylase Inhibitor LAQ824

Wed, 11 Nov 2009 21:08:17 PST

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes. [Cancer Res 2009;69(22):8693–9]

Interleukin 12 Stimulates IFN-{gamma}-Mediated Inhibition of Tumor-Induced Regulatory T-Cell Proliferation and Enhances Tumor Clearance

Wed, 11 Nov 2009 21:08:17 PST

To define the factors that modulate regulatory T (Treg) cells in the tumor setting, we cocultured various tumor cells with either purified Treg cells, or with unfractionated splenocytes. We found that Treg expansion occurred only with unfractionated splenocytes, suggesting that accessory cells and/or factors produced by them play an essential role in tumor-induced Treg expansion. We performed gene expression profiling on tumor-associated Treg cells to identify candidate signaling molecules and studied their effects on tumor-induced Treg expansion. We inadvertently discovered that interleukin (IL)-12 treatment blocked Treg expansion in an IL-12 receptor–dependent fashion. Additional studies showed that IL-12 acts by stimulating IFN- mediated inhibition of Treg cell proliferation, which may partially account for the antitumor effects of IL-12. Furthermore, IL-12 treatment was found to decrease IL-2 production, which may lead to IFN-–independent inhibition of Treg cells, as IL-2 is required for their survival and expansion. Mechanistic studies revealed that IFN- signaling directly causes cell cycle arrest in Treg cells. This study shows that an IL-12–IFN- axis can suppress tumor-induced Treg proliferation. This mechanism may counteract the ability of Treg cells to promote tumor growth in vivo. [Cancer Res 2009;69(22):8700–9]

Toll-like Receptors 3 and 7 Agonists Enhance Tumor Cell Lysis by Human {gamma}{delta} T Cells

Wed, 11 Nov 2009 21:08:17 PST

Toll-like receptor (TLR) agonists are considered adjuvants in clinical trials of cancer immunotherapy. Here, we investigated the modulation of T cell–mediated tumor cell lysis by TLR ligands. T-cell cytotoxicity and granzyme A/B production were enhanced after pretreatment of tumor cells with TLR3 [poly(I:C)] or TLR7 ligand (imiquimod). We examined TLR3- and TLR7-expressing pancreatic adenocarcinomas, squamous cell carcinomas of head and neck and lung carcinomas. Poly(I:C) treatment of pancreatic adenocarcinomas followed by coculture with T cells resulted in an upregulation of CD54 on the tumor cells. The interaction of CD54 and the corresponding ligand CD11a/CD18 expressed on T cells is responsible for triggering effector function in T cells. Moreover, treatment with imiquimod downregulated MHC class I molecules on tumor cells possibly resulting in a reduced binding affinity for inhibitory receptor NKG2A expressed on T cells. These results indicate that TLR3 or TLR7 ligand stimulation of tumor cells enhances the cytotoxic activity of expanded T cells of cancer patients in vitro. [Cancer Res 2009;69(22):8710–7]

Epigenetic Silencing of Interferon-{kappa} in Human Papillomavirus Type 16-Positive Cells

Wed, 11 Nov 2009 21:08:17 PST

We have investigated interferon- (IFN-) regulation in the context of human papillomavirus (HPV)–induced carcinogenesis using primary human foreskin keratinocytes (HFK), immortalized HFKs encoding individual oncoproteins of HPV16 (E6, E7, and E6/E7), and cervical carcinoma cells. Here, IFN- was suppressed in the presence of E6, whereas its expression was not affected in HFKs or E7-immortalized HFKs. Transcription could be reactivated after DNA demethylation but was decreased again upon drug removal. Partial reactivation could also be accomplished when E6 was knocked down, suggesting a contribution of E6 in IFN- de novo methylation. We identified a single CpG island near the transcriptional start site as being involved in selective IFN- expression. To prove the functional relevance of IFN- in building up an antiviral response, IFN- was ectopically expressed in cervical carcinoma cells where protection against vesicular stomatitis virus–mediated cytolysis could be achieved. Reconstitution of IFN- was accompanied by an increase of p53, MxA, and IFN-regulatory factors, which was reversed by knocking down either IFN- or p53 by small interfering RNA. This suggests the existence of a positive feedback loop between IFN-, p53, and components of IFN signaling pathway to maintain an antiviral state. Our in vitro findings were further corroborated in biopsy samples of cervical cancer patients, in which IFN- was also downregulated when compared with normal donor tissue. This is the first report showing an epigenetic silencing of type I IFN after HPV16 oncogene expression and revealing a novel strategy on how high-risk HPVs can abolish the innate immune response in their genuine host cells. [Cancer Res 2009;69(22):8718–25]

p27Kip1 Stabilization Is Essential for the Maintenance of Cell Cycle Arrest in Response to DNA Damage

Wed, 11 Nov 2009 21:08:18 PST

One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27^Kip1 and retinoblastoma tumor suppressors and is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance, but not the initiation, of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ataxia telangiectasia mutated–dependent and ataxia telangiectasia mutated and Rad3-related–dependent DDR but downstream of p38 mitogen-activated protein kinase. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important tumor suppressors in human cancer. [Cancer Res 2009;69(22):8726–32]

Retinoblastoma Family Proteins Have Distinct Functions in Pulmonary Epithelial Cells In vivo Critical for Suppressing Cell Growth and Tumorigenesis

Simpson, D. S., Mason-Richie, N. A., Gettler, C. A., Wikenheiser-Brokamp, K. A. — Wed, 11 Nov 2009 21:08:18 PST

Lung cancer is the leading cause of cancer deaths, accounting for more deaths than breast, colon, and prostate cancer combined. The retinoblastoma (Rb)/p16 tumor suppressive pathway is deregulated in most cancers. Loss of p16 occurs more frequently than Rb loss, suggesting that p16 suppresses cancer by regulating Rb as well as the related proteins p107 and p130. However, direct evidence demonstrating that p130 or p107 cooperate with Rb to suppress epithelial cancers associated with p16 loss is currently lacking. Moreover, the roles of p130 and p107 in lung cancer are not clear. In the present studies, Rb ablation was targeted to the lung epithelium in wild-type, p107, or p130 null mice to determine unique and overlapping Rb family functions critical in tumor suppression. Rb ablation during development resulted in marked epithelial abnormalities despite p107 upregulation. In contrast, p130 and p107 were not required during development but had distinct functions in the Rb-deficient epithelium: p107 was required to suppress proliferation, whereas a novel proapoptotic function was identified for p130. Adult Rb-ablated lungs lacked the epithelial phenotype seen at birth and showed compensatory p107 upregulation and p16 induction in epithelial cell lineages that share phenotypic characteristics with human non–small cell lung cancers (NSCLC) that frequently show p16 loss. Importantly, Rb/p107-deficient, but not Rb/p130-deficient, lungs developed tumors resembling NSCLC. Taken together, these studies identify distinct Rb family functions critical in controlling epithelial cell growth, and provide direct evidence that p107 cooperates with Rb to protect against a common adult cancer. [Cancer Res 2009;69(22):8733–41]

Inhibition of Rho-Associated Kinase Signaling Prevents Breast Cancer Metastasis to Human Bone

Liu, S., Goldstein, R. H., Scepansky, E. M., Rosenblatt, M. — Wed, 11 Nov 2009 21:08:18 PST

Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility. Aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Here, we show that ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines compared with nonmetastatic tumors and cell lines. Overexpression of ROCK confers a metastatic phenotype on the nonmetastatic MCF-7 cell line. Inhibition of ROCK activity, by either a specific ROCK inhibitor (Y27632) or ROCK-targeted small interfering RNAs, reduces cell migration and proliferation in vitro and metastasis to bone in vivo using a novel "human breast cancer metastasis to human bone" mouse model. Expression of the c-Myc–regulated miR-17-92 cluster is shown to be elevated in metastatic breast cancer cells compared with nonmetastatic cells and diminished by Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. Together, these findings suggest that augmented ROCK signaling contributes to breast cancer metastasis. The effects of ROCK on tumor cell invasion/motility and growth may derive from regulating cytoskeletal actin-myosin contraction and modulating the c-Myc pathway, including c-Myc–dependent microRNAs. Inhibition of ROCK or the pathway it stimulates, therefore, may represent a novel approach for treatment of breast cancer metastases. [Cancer Res 2009;69(22):8742–51]

Ubiquitination and Downregulation of BRCA1 by Ubiquitin-Conjugating Enzyme E2T Overexpression in Human Breast Cancer Cells

Wed, 11 Nov 2009 21:08:18 PST

Breast cancer is generated through a multistep genetic and epigenetic process including activations of oncogenes and inactivations of tumor suppressor genes. Here, we report a critical role of ubiquitin-conjugating enzyme E2T (UBE2T), an E2 ubiquitin-conjugating enzyme, in mammary carcinogenesis. Immunocytochemical staining and in vitro binding assay revealed that UBE2T interacted and colocalized with the BRCA1/BRCA1-associated RING domain protein (BARD1) complex. Knocking down of UBE2T expression with small interfering RNA drastically suppressed the growth of breast cancer cells. Interestingly, in vivo ubiquitination assay indicated BRCA1 to be polyubiquitinated by incubation with wild-type UBE2T protein, but not with C86A-UBE2T protein, an E2 activity–dead mutant, in which the 86th residue of cysteine was replaced with alanine. Furthermore, knocking down of UBE2T protein induced upregulation of BRCA1 protein in breast cancer cells, whereas its overexpression caused the decrease of the BRCA1 protein. Our data imply a critical role of UBE2T in development and/or progression of breast cancer through the interaction with and the regulation of the BRCA1/BARD1 complex. [Cancer Res 2009;69(22):8752–60]

Regulation of Protein Citrullination through p53/PADI4 Network in DNA Damage Response

Tanikawa, C., Ueda, K., Nakagawa, H., Yoshida, N., Nakamura, Y., Matsuda, K. — Wed, 11 Nov 2009 21:08:18 PST

Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. Here, we report the novel role of p53 in the regulation of protein citrullination. p53 transactivated peptidylarginine deiminase type 4 (PADI4) through an intronic p53-binding site. The PADI4 gene encodes an enzyme catalyzing the citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4-dependent manner. We found that PADI4 citrullinated the histone chaperone protein, nucleophosmin (NPM1), at the arginine 197 residue in vivo under physiologic conditions. Citrullination of NPM1 by PADI4 resulted in its translocation from the nucleoli to the nucleoplasm, whereas PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly, the knockdown of PADI4 attenuated p53-mediated growth-inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway.[Cancer Res 2009;69(22):8761–9]

Mammary Tumor Development in Dogs Is Associated with BRCA1 and BRCA2

Wed, 11 Nov 2009 21:08:18 PST

Breast cancer is a major contributor to overall morbidity and mortality in women. Several genes predisposing to breast cancer have been identified, but the majority of risk factors remain unknown. Even less is known about the inherited risk factors underlying canine mammary tumors (CMT). Clear breed predispositions exist, with 36% of English springer spaniels (ESS) in Sweden being affected. Here, we evaluate 10 human breast cancer genes (BRCA1, BRCA2, CHEK2, ERBB2, FGFR2, LSP1, MAP3K1, RCAS1, TOX3, and TP53) for association with CMTs. Sixty-three single-nucleotide polymorphisms (SNPs; four to nine SNPs per gene) were genotyped by iPLEX in female ESS dogs, 212 CMT cases and 143 controls. Two genes, BRCA1 and BRCA2, were significantly associated with CMT (Bonferroni corrected P = 0.005 and P = 0.0001, respectively). Borderline association was seen for FGFR2. Benign and malignant cases were also analyzed separately. Those findings supported the association to BRCA1 and BRCA2 but with a stronger association to BRCA1 in malignant cases. Both BRCA1 and BRCA2 showed odds ratios of ~4. In conclusion, this study indicates that BRCA1 and BRCA2 contribute to the risk of CMT in ESS, suggesting that dogs may serve as a good model for human breast cancer. [Cancer Res 2009;69(22):8770–4]

Fanconi Anemia Complementation Group FANCD2 Protein Serine 331 Phosphorylation Is Important for Fanconi Anemia Pathway Function and BRCA2 Interaction

Wed, 11 Nov 2009 21:08:18 PST

Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]

Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic Instability In vivo in Mice

Trouiller, B., Reliene, R., Westbrook, A., Solaimani, P., Schiestl, R. H. — Wed, 11 Nov 2009 21:08:18 PST

Titanium dioxide (TiO₂) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO₂ is chemically inert, TiO₂ nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO₂-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO₂ nanoparticles–induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO₂ nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the -H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO₂ nanoparticles induced 8-hydroxy-2'-deoxyguanosine, -H2AX foci, micronuclei, and DNA deletions. The formation of -H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO₂ nanoparticles–induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO₂ nanoparticles, these findings raise concern about potential health hazards associated with TiO₂ nanoparticles exposure. [Cancer Res 2009;69(22):8784–9]

Intraoperative Evaluation of Breast Tumor Margins with Optical Coherence Tomography

Wed, 11 Nov 2009 21:08:18 PST

As breast cancer screening rates increase, smaller and more numerous lesions are being identified earlier, leading to more breast-conserving surgical procedures. Achieving a clean surgical margin represents a technical challenge with important clinical implications. Optical coherence tomography (OCT) is introduced as an intraoperative high-resolution imaging technique that assesses surgical breast tumor margins by providing real-time microscopic images up to 2 mm beneath the tissue surface. In a study of 37 patients split between training and study groups, OCT images covering 1 cm² regions were acquired from surgical margins of lumpectomy specimens, registered with ink, and correlated with corresponding histologic sections. A 17-patient training set used to establish standard imaging protocols and OCT evaluation criteria showed that areas of higher scattering tissue with a heterogeneous pattern were indicative of tumor cells and tumor tissue in contrast to lower scattering adipocytes found in normal breast tissue. The remaining 20 patients were enrolled into the feasibility study. Of these lumpectomy specimens, 11 were identified with a positive or close surgical margin and 9 were identified with a negative margin under OCT. Based on histologic findings, 9 true positives, 9 true negatives, 2 false positives, and 0 false negatives were found, yielding a sensitivity of 100% and specificity of 82%. These results show the potential of OCT as a real-time method for intraoperative margin assessment in breast-conserving surgeries. [Cancer Res 2009;69(22):8790–6]

Microenvironmental Independence Associated with Tumor Progression

Wed, 11 Nov 2009 21:08:18 PST

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments. [Cancer Res 2009;69(22):8797–806]

TP53 Cancerous Mutations Exhibit Selection for Translation Efficiency

Waldman, Y. Y., Tuller, T., Sharan, R., Ruppin, E. — Wed, 11 Nov 2009 21:08:18 PST

The tumor suppressor gene TP53 is known to be a key regulator in cancer, and more than half of human cancers exhibit mutations in this gene. Recent evidence shows that point mutations in TP53 not only disrupt its function but also possess gain-of-function and dominant-negative effects on wild-type copies, thus making the mutated gene an oncogene. Hence, this brings about the possibility that TP53 mutations may be under selection for increasing the overall translation efficiency (TE) of defected TP53 in cancerous cells. Here, we perform the first large-scale analysis of TE in human cancer mutated TP53 variants, identifying a significant increase in TE that is correlated with the frequency of TP53 mutations. Furthermore, mutations with a known oncogenic effect significantly increase their TE compared with the other TP53 mutations. Further analysis shows that TE may have influence both on selecting the location of the mutation and on its outcome: codons with lower TE show stronger selection toward nonsynonymous mutations and, for each codon, frequent mutations show stronger increase in TE compared with less frequent mutations. Additionally, we find that TP53 mutations have significantly higher TE increase in progressive versus primary tumors. Finally, an analysis of TP53 NCI-60 cell lines points to a coadaptation between the mutations and the tRNA pool, increasing the overall TP53 TE. Taken together, these results show that TE plays an important role in the selection of TP53 cancerous mutations. [Cancer Res 2009;69(22):8807–13]

Paradoxical Dependencies of Tumor Dormancy and Progression on Basic Cell Kinetics

Wed, 11 Nov 2009 21:08:18 PST

Even after a tumor is established, it can early on enter a state of dormancy marked by balanced cell proliferation and cell death. Disturbances to this equilibrium may affect cancer risk, as they may cause the eventual lifetime clinical presentation of a tumor that might otherwise have remained asymptomatic. Previously, we showed that cell death, proliferation, and migration can play a role in shifting this dynamic, making the understanding of their combined influence on tumor development essential. We developed an individual cell-based computer model of the interaction of cancer stem cells and their nonstem progeny to study early tumor dynamics. Simulations of tumor growth show that three basic components of tumor growth—cell proliferation, migration, and death—combine in unexpected ways to control tumor progression and, thus, clinical cancer risk. We show that increased proliferation capacity in nonstem tumor cells and limited cell migration overall lead to space constraints that inhibit proliferation and tumor growth. By contrast, increasing the rate of cell death produces the expected tumor size reduction in the short term, but results ultimately in paradoxical accelerated long-term growth owing to the liberation of cancer stem cells and formation of self-metastases.[Cancer Res 2009;69(22):8814–21]

Noninvasive Multiparametric Imaging of Metastasis-Permissive Microenvironments in a Human Prostate Cancer Xenograft

Wed, 11 Nov 2009 21:08:18 PST

Metastasis continues to be one of the major causes of mortality from prostate cancer. Because human malignant cell lines metastasize more readily from orthotopic sites than from heterotopic sites, to identify metastasis-permissive tumor microenvironments, we used noninvasive imaging to compare the in vivo vascular, metabolic, and physiologic characteristics of a human prostate cancer xenograft implanted orthotopically in the prostate or s.c. in the flank. Hypoxia was detected in these xenografts by placing an enhanced green fluorescence protein optical reporter under the control of a hypoxia response element. A multiparametric analysis of hypoxia, extracellular pH, vascularization, and metabolism provided a characterization of environments that are permissive for metastasis to occur. We found that orthotopic tumors, which metastasized more easily, were characterized by higher vascular volume, permeability, and total choline and a more acidic extracellular pH. Interestingly, metastatic deposits in the lymph nodes as well as cancer cells in ascites fluid were found to be hypoxic, explaining, in part, the refractory nature of metastatic disease. These results also provide the basis for clinically translatable noninvasive imaging markers for predicting metastatic risk in prostate cancer. [Cancer Res 2009;69(22):8822–9]

Comment re: Vitamin C Antagonizes the Cytotoxic Effects of Chemotherapy

Espey, M. G., Chen, Q., Levine, M. — Wed, 11 Nov 2009 21:08:18 PST

Heaney, M. L., Gardner, J. R., O'Connor, O. A. — Wed, 11 Nov 2009 21:08:18 PST

Correction: RLIP76: A Target for Kidney Cancer Therapy

Wed, 11 Nov 2009 21:08:18 PST

Correction: Parathyroid Hormone-Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

Wed, 11 Nov 2009 21:08:18 PST

Correction: Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission

Wed, 11 Nov 2009 21:08:18 PST

Nuclear Receptor Coregulators in Cancer Biology

O'Malley, B. W., Kumar, R. — Wed, 28 Oct 2009 21:08:14 PDT

Coregulators (coactivators and corepressors) occupy the driving seat for actions of all nuclear receptors, and consequently, selective receptor modulator drugs. The potency and selectivity for subreactions of transcription reside in the coactivators, and thus, they are critically important for tissue-selective gene function. Each tissue has a "quantitative finger print" of coactivators based on its relative inherited concentrations of these molecules. When the cellular concentration of a coactivator is altered, genetic dysfunction usually leads to a pathologic outcome. For example, many cancers overexpress "growth coactivators." In this way, the cancer cell can hijack these coactivator molecules to drive proliferation and metastasis. The present review contains summaries of selective coactivators and corepressors that have been demonstrated to play important roles in the malignant process and emphasizes their importance for future therapeutic interventions. [Cancer Res 2009;69(21):8217–22]

Hijacking the Chromatin Remodeling Machinery: Impact of SWI/SNF Perturbations in Cancer

Weissman, B., Knudsen, K. E. — Wed, 28 Oct 2009 21:08:14 PDT

There is increasing evidence that alterations in chromatin remodeling play a significant role in human disease. The SWI/SNF chromatin remodeling complex family mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics whose functional specificity is driven by combinatorial assembly of a central ATPase and association with 10 to 12 unique subunits. Although the biochemical consequence of SWI/SNF in model systems has been extensively reviewed, the present article focuses on the evidence linking SWI/SNF perturbations to cancer initiation and tumor progression in human disease. [Cancer Res 2009;69(21):8223–30]

A Role for BRCA1 in Uterine Leiomyosarcoma

Wed, 28 Oct 2009 21:08:14 PDT

Uterine leiomyosarcoma (ULMS) is a rare gynecologic malignancy with a low survival rate. Currently, there is no effective treatment for ULMS. Infrequent occurrences of human ULMS hamper the understanding of the initiation and progression of the disease, thereby limiting the ability to develop efficient therapies. To elucidate the roles of the p53 and BRCA1 tumor suppressor genes in gynecologic malignancies, we generated mice in which p53 and/or BRCA1 can be conditionally deleted using anti-Müllerian hormone type II receptor (Amhr2)–driven Cre recombinase. We showed that conditional deletion of p53 in mice results in the development of uterine tumors that resemble human ULMS and that concurrent deletion of p53 and BRCA1 significantly accelerates the progression of these tumors. This finding led to our hypothesis that BRCA1 may play a role in human ULMS development. Consistent with this hypothesis, we showed that the BRCA1 protein is absent in 29% of human ULMS and that BRCA1 promoter methylation is the likely mechanism of BRCA1 downregulation. These data indicate that the loss of BRCA1 function may be an important step in the progression of ULMS. Our findings provide a rationale for investigating therapies that target BRCA1 deficiency in ULMS. [Cancer Res 2009;69(21):8231–5]

Presence of the Carcinogen N'-Nitrosonornicotine in the Urine of Some Users of Oral Nicotine Replacement Therapy Products

Wed, 28 Oct 2009 21:08:14 PDT

N'-nitrosonornicotine (NNN) is a strong carcinogen present in unburned tobacco and cigarette smoke. We here analyze data obtained in two studies, in which a biomarker of exposure to NNN—the sum of NNN and its pyridine-N-glucuronide, called total NNN—was quantified in the urine of people who had stopped smoking and used various nicotine replacement therapy (NRT) products. In 13 of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after biochemically confirmed smoking cessation was comparable with, or considerably higher than, the baseline levels. For most of the subjects who used the nicotine patch as a smoking cessation aid, urinary total NNN at all post–quit time points was <37% of their mean baseline levels. These results indicate that endogenous formation of significant amounts of NNN may occur sporadically in some users of oral NRT. Given the carcinogenicity of NNN and the frequent use of nicotine gum as a smoking cessation aid, further studies are needed so that preventive measures can be developed. [Cancer Res 2009;69(21):8236–40]

Evidence for Cancer Stem Cells in Human Endometrial Carcinoma

Hubbard, S. A., Friel, A. M., Kumar, B., Zhang, L., Rueda, B. R., Gargett, C. E. — Wed, 28 Oct 2009 21:08:14 PDT

Emerging evidence indicates that the highly regenerative human endometrium harbors rare populations of epithelial progenitor cells. In tumors of other regenerative epithelial tissues, rare cancer stem cells (CSC) have been identified that may have originated from normal epithelial stem/progenitor cells. We hypothesized that CSC are responsible for epithelial neoplasia associated with endometrial carcinoma, the most common gynecologic malignancy in women. Stem cell characteristics of single cells isolated from endometrial carcinoma tissues from women ages 62 ± 11.8 years (n = 34) were assessed. Twenty-five of 28 endometrial carcinoma samples contained a small population of clonogenic cells [0.24% (0-1.84%)], with no significant difference in cloning efficiency between the three grades of endometrial carcinoma or between endometrial carcinoma and normal endometrial epithelial samples. Isolated endometrial carcinoma cells transplanted under the kidney capsule of immunocompromised mice in serial dilution (2 x 10⁶-1 x 10⁴ cells) generated tumors in 8 of 9 samples with morphologies similar to the parent tumors. These tumors recapitulated cytokeratin, vimentin, estrogen receptor-, and progesterone receptor expression of the parent tumor, indicating that tumor-initiating cells likely differentiated into cells comprising the endometrial carcinoma tissue. Individual clones underwent serial clonal subculture 2.5 to 4 times, with a trend of increasing number of subclonings with increasing tumor grade, indicating increasing self-renewal with greater malignancy. Clonally derived endometrial carcinoma cells also expressed the self-renewal genes BMI-1, NANOG, and SOX-2. Isolated cells from primary tumors were serially transplanted 3 to 5 times in nonobese diabetic/severe combined immunodeficient mice, showing self-renewal in vivo. This evidence of cells with clonogenic, self-renewing, differentiating, and tumorigenic properties suggests that a CSC population may be responsible for production of endometrial carcinoma tumor cells. [Cancer Res 2009;69(21):8241–8]

Altered Runx1 Subnuclear Targeting Enhances Myeloid Cell Proliferation and Blocks Differentiation by Activating a miR-24/MKP-7/MAPK Network

Wed, 28 Oct 2009 21:08:14 PDT

Disruption of Runx1/AML1 subnuclear localization, either by a single amino acid substitution or by a chromosomal translocation [e.g., t(8;21)], is linked to the etiology of acute myeloid leukemia (AML). Here, we show that this defect induces a select set of micro-RNAs (miR) in myeloid progenitor cells and AML patients with t(8;21). Both Runx1 and the t(8;21)-encoded AML1-ETO occupy the miR-24-23-27 locus and reciprocally control miR-24 transcription. miR-24 directly downregulates mitogen-activated protein kinase (MAPK) phosphatase-7 and enhances phosphorylation of both c-jun-NH₂-kinase and p38 kinases. Expression of miR-24 stimulates myeloid cell growth, renders proliferation independent of interleukin-3, and blocks granulocytic differentiation. Thus, compromised Runx1 function induces a miR-dependent mechanism that, through MAPK signaling, enhances myeloid proliferation but blocks differentiation—key steps that contribute to leukemia. [Cancer Res 2009;69(21):8249–55]

Activation of the PI3K/AKT Pathway Induces Urothelial Carcinoma of the Renal Pelvis: Identification in Human Tumors and Confirmation in Animal Models

Wed, 28 Oct 2009 21:08:14 PDT

Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type and identified a phosphatidylinositol 3-kinase (PI3K)/AKT activation expression signature in 76.9% (n = 13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear-cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phosphorylated mammalian target of rapamycin (mTOR; 63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than 1 year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses showed increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g., mTOR inhibitor) may serve as effective therapeutic agents. [Cancer Res 2009;69(21):8256–64]

CXC Receptor-1 Silencing Inhibits Androgen-Independent Prostate Cancer

Shamaladevi, N., Lyn, D. A., Escudero, D. O., Lokeshwar, B. L. — Wed, 28 Oct 2009 21:08:14 PDT

The CXC receptor-1 (CXCR1) is a coreceptor for interleukin-8 (IL-8) and is expressed on both normal and tumor cells. The function of CXCR1 in prostate cancer was investigated by silencing its expression, using RNA interference. We established stable cell colonies of PC-3 cells, depleted of CXCR1, using lentiviral plasmids (pLK0.1puro) generating small hairpin RNA (shRNA) against CXCR1 mRNA. Stable shRNA transfectants (PLK1–PLK5) that express significantly reduced CXCR1 mRNA (≥90% down) and protein (≥43% down) or vector-only transfectants (PC-3V) were characterized. PLK cells showed reduced cell proliferation (down, ≥66%), due to cell cycle arrest at G₁-S phase, decreases in Cyclin D1, CDK4, phosphorylated Rb, and extracellular signal-regulated kinase 1/2 levels compared with those in PC-3V cells. CXCR1 depletion lead to increases in spontaneous apoptosis by mitochondria-mediated intrinsic mechanism and increases in proapoptotic proteins (BAD, 40%; BAX, 12%), but decreases in antiapoptotic proteins (BCL2, down 38%; BCL_xL, 20%). PLK2 cells grew as slow-growing tumors (decrease of 54%), compared with that of PC3V tumors in athymic mice. Ex vivo analyses of PLK2 tumor tissues showed reduced expression of Cyclin D1 and vascular endothelial growth factor, and increased apoptosis activity. Other IL-8–expressing prostate cancer cell lines also exhibited similar phenotypes when CXCR1 was depleted by CXCR1 shRNA transfection. In contrast to these cells, CXCR1 depletion had little effect on IL-8 ligand–deficient LNCaP cells. RNA interference rescue using mutated CXCR1 plasmids reversed the silencing effect of PLK2, thus demonstrating the specificity of phenotypic alteration by CXCR1 shRNA. These studies establish that CXCR1 promotes IL-8–mediated tumor growth. [Cancer Res 2009;69(21):8265–74]

Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Wed, 28 Oct 2009 21:08:14 PDT

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor–independent proliferation, which was confirmed by the resistance of the PTEN^–/– MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel. [Cancer Res 2009;69(21):8275–83]

Role for Kruppel-Like Factor 4 in Determining the Outcome of p53 Response to DNA Damage

Zhou, Q., Hong, Y., Zhan, Q., Shen, Y., Liu, Z. — Wed, 28 Oct 2009 21:08:14 PDT

Cells are incessantly exposed to many sources of genotoxic stress. A critical unresolved issue is how the resulting activation of the p53 tumor suppressor can lead to either cell cycle arrest or apoptosis depending on the extent of DNA damage. The present study shows that the level of Krüppel-like factor 4 (KLF4) expression is inversely correlated with the extent of DNA damage. KLF4 is activated by p53 following cytostatic, mild DNA damage, whereas it is strongly repressed via enhanced turnover of mRNA on severe DNA damage that irreversibly drives cells to apoptosis. Blocking the repression of KLF4 on severe DNA damage suppresses p53-mediated apoptosis, whereas ablation of the KLF4 induction on mild DNA damage shifts the p53 response from cell cycle arrest to cell death. Our results suggest that coordinate regulation of KLF4 expression depending on the extent of DNA damage may be an important mechanism that dictates the life and death decisions of p53. [Cancer Res 2009;69(21):8284–92]

Identification of Compounds Selectively Killing Multidrug-Resistant Cancer Cells

Wed, 28 Oct 2009 21:08:14 PDT

There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance (MDR) in cancer. We catalogued the National Cancer Institute's DTP drug repository in search of compounds showing increased toxicity in MDR cells. By comparing the sensitivity of parental cell lines with MDR derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR. [Cancer Res 2009;69(21):8293–301]

Concordant Gene Expression Signatures Predict Clinical Outcomes of Cancer Patients Undergoing Systemic Therapy

Wed, 28 Oct 2009 21:08:14 PDT

Conventional development of multivariate gene expression models (GEM) predicting therapeutic response of cancer patients is based on analysis of patients treated with specific regimens, which limits generalization to different or novel drug combinations. We overcome this limitation by developing GEMs based on in vitro drug sensitivities and microarray analyses of the NCI-60 cancer cell line panel. These GEMs were evaluated in blind fashion as predictors of tumor response and/or patient survival in seven independent cohorts of patients with breast (n = 275), bladder (n = 59), and ovarian (n= 143) cancer treated with multiagent chemotherapy, of which 233 patients were from prospectively enrolled clinical trials. In all studies, GEMs effectively stratified tumor response and patient survival independent of established clinical and pathologic tumor variables. In bladder cancer patients treated with neoadjuvant methotrexate, vinblastine, Adriamycin (doxorubicin), and cisplatin, the 3-year overall survival for those with favorable GEM scores was 81% versus 33% for those with less favorable scores (P = 0.002). GEMs for breast cancer patients treated with 5-fluorouracil, Adriamycin (doxorubicin), and cyclophosphamide and ovarian cancer patients treated with platinum-containing regimens also stratified patient survival [5-year overall survival 100% versus 74% (P = 0.05) and 3-year overall survival 68% versus 43% (P = 0.008), respectively]. Importantly, clinical prediction using our in vitro GEM was superior to that of conventionally derived GEMs. We show a facile yet effective approach to GEM derivation that identifies patients most likely to benefit from selected multiagent therapy. Use of such in vitro–based GEMs may provide a robust and generalizable approach to personalized cancer therapy. [Cancer Res 2009;69(21):8302–9]

Enhanced Leukemia Cell Detection Using a Novel Magnetic Needle and Nanoparticles

Wed, 28 Oct 2009 21:08:14 PDT

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a "magnetic needle" biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34–conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care. [Cancer Res 2009;69(21):8310–6]

The BRAFV600E Oncogene Induces Transforming Growth Factor {beta} Secretion Leading to Sodium Iodide Symporter Repression and Increased Malignancy in Thyroid Cancer

Wed, 28 Oct 2009 21:08:14 PDT

The activating mutation BRAF^V600E is a frequent genetic event in papillary thyroid carcinomas (PTC) that predicts a poor prognosis, leading to loss of sodium/iodide symporter (NIS) expression and subsequent radioiodide-refractory metastatic disease. The molecular basis of such an aggressive behavior induced by BRAF remains unclear. Here, we show a mechanism through which BRAF induces NIS repression and promotes epithelial to mesenchimal transition and invasion based on the operation of an autocrine transforming growth factor (TGF)β loop. BRAF induces secretion of functional TGFβ and blocking TGFβ/Smad signaling at multiple levels rescues BRAF-induced NIS repression. Although this mechanism is MAP/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK independent, secreted TGFβ cooperates with MEK-ERK signaling in BRAF-induced cell migration, Matrigel invasion, and EMT. Consistent with this process, TGFβ and other key components of TGFβ signaling, such as TβRII and pSmad2, are overexpressed in human PTC, suggesting a widespread activation of this pathway by locally released TGFβ. Moreover, this high TGFβ/Smad activity is associated with PTC invasion, nodal metastasis, and BRAF status. Interestingly, TGFβ is overexpressed in the invasive front, whereas NIS is preferentially expressed in the central regions of the tumors, suggesting that this negative correlation between TGFβ and NIS occurs locally inside the tumor. Our study describes a novel mechanism of NIS repression in thyroid cancer and provides evidence that TGFβ may play a key role in promoting radioiodide resistance and tumor invasion during PTC progression. [Cancer Res 2009;69(21):8317–25]

Three-Dimensional Imaging and Quantification of Both Solitary Cells and Metastases in Whole Mouse Liver by Magnetic Resonance Imaging

Wed, 28 Oct 2009 21:08:14 PDT

The metastatic cell population, ranging from solitary cells to actively growing metastases, is heterogeneous and unlikely to respond uniformly to treatment. However, quantification of the entire experimental metastatic cell population in whole organs is complicated by requirements of an imaging modality with the large field of view and high spatial resolution necessary to detect both single cells and metastases in the same organ. Thus, it is difficult to assess differential responses of these distinct metastatic populations to therapy. Here, we develop a magnetic resonance imaging (MRI) technique capable of quantifying the full population of metastatic cells in a secondary organ. B16F1 mouse melanoma cells were labeled with micron-sized iron oxide particles (MPIO) and injected into mouse liver via the mesenteric vein. Livers were removed immediately or at day 9 or 11, following doxorubicin or vehicle control treatment, and imaged using a 3T clinical magnetic resonance scanner and custom-built gradient coil. Both metastases (>200 µm) and MPIO-labeled single cells were detected and quantified from MR images as areas of hyperintensity or hypointensity (signal voids), respectively. We found that 1mg/kg doxorubicin treatment inhibited metastasis growth (n = 11 per group; P = 0.02, t test) but did not decrease the solitary metastatic cell population in the same livers (P > 0.05). Thus, the technique presented here is capable of quickly quantifying the majority of the metastatic cell population, including both growing metastases and solitary cells, in whole liver by MRI and can identify differential responses of growing metastases and solitary cells to therapy. [Cancer Res 2009;69(21):8326–31]

Widespread Estrogen-Dependent Repression of microRNAs Involved in Breast Tumor Cell Growth

Wed, 28 Oct 2009 21:08:15 PDT

Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17β-estradiol (E₂). We show that the expression of a broad set of miRNAs decreases following E₂ treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E₂-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E₂-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer. [Cancer Res 2009;69(21):8332–40]

Lung Adenocarcinoma with EGFR Amplification Has Distinct Clinicopathologic and Molecular Features in Never-Smokers

Wed, 28 Oct 2009 21:08:15 PDT

In a subset of lung adenocarcinomas, the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. For this study, we selected 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion–mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, and low and high polysomy (100% versus 54%, P = 0.009). EGFR amplification occurred invariably on the mutated and not the wild-type allele (median mutated/wild-type ratios 14.0 versus 0.33, P = 0.003), was associated with solid histology (P = 0.008), and advanced clinical stage (P = 0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival, 16 versus 31 months, P = 0.01) and when adjusted for stage (P = 0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and show distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology. [Cancer Res 2009;69(21):8341–8]

Height, Body Mass Index, and Physical Activity in Relation to Glioma Risk

Wed, 28 Oct 2009 21:08:15 PDT

Whether energy balance during early life and/or adulthood is related to glioma risk is unknown. We therefore investigated height, body mass index (BMI), and physical activity in relation to glioma risk in the prospective NIH-AARP Diet and Health Study. Participants completed a baseline questionnaire (sent in 1995-1996) inquiring about height, weight, and potential confounders. A second questionnaire (sent in 1996) inquired about physical activity during ages 15 to 18, 19 to 29, and 35 to 39 years and the past 10 years and body weight at ages 18, 35, and 50 years. During follow-up from 1995/1996 to 2003, we documented 480 cases of glioma among 499,437 respondents to the baseline questionnaire and 257 cases among 305,681 respondents to the second questionnaire. Glioma risk among tall persons (≥1.90 m) was twice that of short persons [<1.60 m; multivariate relative risk (RR), 2.12; 95% confidence interval (95% CI), 1.18-3.81; P_trend = 0.006]. Risk among participants who were obese (BMI 30.0-34.9 kg/m²) at age 18 years was nearly four times that of persons of normal weight (BMI 18.5-24.9 kg/m²) at age 18 years (RR, 3.74; 95% CI, 2.03-6.90; P_trend = 0.003); 11 cases were obese at age 18 years. Risk among participants who were active during ages 15 to 18 years was 36% lower than that of persons who were inactive during ages 15 to 18 years (RR, 0.64; 95% CI, 0.44-0.93; P_trend = 0.02). BMI and physical activity after age 18 years were unrelated to glioma risk. Adult height, BMI during adolescence, and physical activity during adolescence were each associated with glioma risk, supporting a role for early-life energy balance in glioma carcinogenesis. [Cancer Res 2009;69(21):8349–55]

The Cell Death-Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Wed, 28 Oct 2009 21:08:15 PDT

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide–induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356–65]

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia

Wed, 28 Oct 2009 21:08:15 PDT

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC₅₀ of 0.55 µmol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV_H genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15–induced apoptosis. Pharmacologic inhibition of c-jun NH₂-terminal kinase inhibited PBOX-15–induced apoptosis in mutated IgV_H and ZAP-70^– CLL cells but not in unmutated IgV_H and ZAP-70⁺ cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential. [Cancer Res 2009;69(21):8366–75]

Pivotal Roles of Snail Inhibition and RKIP Induction by the Proteasome Inhibitor NPI-0052 in Tumor Cell Chemoimmunosensitization

Baritaki, S., Yeung, K., Palladino, M., Berenson, J., Bonavida, B. — Wed, 28 Oct 2009 21:08:15 PDT

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052–mediated sensitization may result from NF-B inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-B and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-B inhibition in sensitization was corroborated with the NF-B inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052–mediated sensitization. These findings show that NPI-0052 modifies the NF-B-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance. [Cancer Res 2009;69(21):8376–85]

Inhibition of the Androgen Receptor as a Novel Mechanism of Taxol Chemotherapy in Prostate Cancer

Wed, 28 Oct 2009 21:08:15 PDT

Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for Taxol are not fully understood. Here, we showed that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)–activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that Taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the phosphoinositide 3-kinase inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further showed that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor, and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interfering RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1, and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of Taxol in CRPC cells that may play an important role in Taxol-mediated inhibition of CRPC growth. [Cancer Res 2009;69(21):8386–94]

Pegylated Kunitz Domain Inhibitor Suppresses Hepsin-Mediated Invasive Tumor Growth and Metastasis

Wed, 28 Oct 2009 21:08:15 PDT

The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCaP-34 tumors were treated with a PEGylated form of Kunitz domain-1, a potent hepsin active site inhibitor derived from hepatocyte growth factor activator inhibitor-1 (K_i^app 0.30 ± 0.02 nmol/L). Treatment of established tumors with PEGylated Kunitz domain-1 decreased contralateral prostate invasion (46% weight reduction) and lymph node metastasis (50% inhibition). Moreover, serum prostate-specific antigen level remained reduced during the entire treatment period, reaching a maximal reduction of 76% after 5 weeks of dosing. The findings show that hepsin promotes invasive prostate tumor growth and metastasis and suggest that active site-directed hepsin inhibition could be effective in prostate cancer therapy. [Cancer Res 2009;69(21):8395–402]

HDAC Inhibitor SNDX-275 Induces Apoptosis in erbB2-Overexpressing Breast Cancer Cells via Down-regulation of erbB3 Expression

Huang, X., Gao, L., Wang, S., Lee, C.-K., Ordentlich, P., Liu, B. — Wed, 28 Oct 2009 21:08:15 PDT

Breast cancer is a highly heterogeneous disease with distinct histologic subtypes. Targeted therapies such as endocrine therapy and growth factor receptor inhibitors have had a significant impact on the treatment of metastatic breast cancer patients. Unfortunately, resistance to these agents eventually occurs, and currently represents a significant clinical problem in the management of breast cancers. Inhibitors of histone deacetylases (HDACi) exhibit anticancer activity in a variety of tumor cell models and have been shown to target mechanisms of resistance to a number of targeted agents. It is unclear, however, if there are specific breast cancer subtypes for which an HDACi may be more or less effective. Here, we report that the class I isoform–selective HDACi entinostat (SNDX-275) preferentially inhibits cell proliferation/survival and inactivates downstream signaling in erbB2-overexpressing compared with basal breast cancer cells. SNDX-275 reduces the levels of both erbB2 and erbB3, as well as significantly decreases P-erbB2, P-erbB3, P-Akt, and P-MAPK in erbB2-overexpressing cells. Additionally, SNDX-275 promotes apoptosis and induces cell cycle arrest predominantly at G₁ phase in erbB2-overexpressing cells, whereas SNDX-275 mainly induces G₂-M arrest in basal breast cancer cells. The cellular bias of SNDX-275 is shown to be related partly to the levels of erbB3 expression that directly impact the ability of SNDX-275 to inhibit proliferation/survival of the erbB2-overexpressing breast cancer cells. These findings show that SNDX-275 may be developed as a novel therapeutic agent to treat breast cancers with coexpression of both erbB2 and erbB3. [Cancer Res 2009;69(21):8403–11]

Simultaneous Infiltration of Polyfunctional Effector and Suppressor T Cells into Renal Cell Carcinomas

Wed, 28 Oct 2009 21:08:15 PDT

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4⁺ Th1, Th2, and Th17 and CD8⁺ Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patient's peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4⁺ cells of the Th1 lineage and in effector memory CD8⁺ cells. Additionally, several populations of CD4⁺ and CD8⁺ regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses. [Cancer Res 2009;69(21):8412–9]

Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer-Specific Immune Responses and Their Correlation to Prognosis

Wed, 28 Oct 2009 21:08:15 PDT

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN- ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu–specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN- and reduced transforming growth factor (TGF)β1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFβ1, reduced IFN-, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis. [Cancer Res 2009;69(21):8420–8]

Whole-Body Sleeping Beauty Mutagenesis Can Cause Penetrant Leukemia/Lymphoma and Rare High-Grade Glioma without Associated Embryonic Lethality

Wed, 28 Oct 2009 21:08:15 PDT

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis. [Cancer Res 2009;69(21):8429–37]

Identification of Key Regions and Genes Important in the Pathogenesis of Sezary Syndrome by Combining Genomic and Expression Microarrays

Wed, 28 Oct 2009 21:08:15 PDT

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-B pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS. [Cancer Res 2009;69(21):8438–46]

Epigenetic Inactivation of the Circadian Clock Gene BMAL1 in Hematologic Malignancies

Wed, 28 Oct 2009 21:08:15 PDT

Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation–associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock. [Cancer Res 2009;69(21):8447–54]

Eukaryotic Initiation Factor 4E Binding Protein Family of Proteins: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

Wed, 28 Oct 2009 21:08:15 PDT

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here, we show that the primary negative regulators of these oncogenic initiation factors—the 4E-BP protein family—operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK), 4ebp1^–/–/4ebp2^–/– mice showed increased sensitivity to tumorigenesis compared with their wild-type counterparts. The 4E-BP–deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape, with translational activation of genes governing angiogenesis, growth, and proliferation, and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. [Cancer Res 2009;69(21):8455–62]

p53 Pre- and Post-Binding Event Theories Revisited: Stresses Reveal Specific and Dynamic p53-Binding Patterns on the p21 Gene Promoter

Millau, J.-F., Bastien, N., Bouchard, E. F., Drouin, R. — Wed, 28 Oct 2009 21:08:15 PDT

p53 is a master transcription factor that prevents neoplasia and genomic instability. It is an important target for anticancer drug design. Understanding the molecular mechanisms behind its transcriptional activities in normal cells is a prerequisite to further understand the deregulation effected by mutant p53 in cancerous cells. Currently, how p53 coordinates transcription programs in response to stress remains unclear. One theory proposes that stresses induce pre-binding events that direct p53 to bind to specific response elements, whereas a second posits that, in response to stress, p53 binds most response elements and post-binding events then regulate transcription initiation. It is critical to establish the relevance of both theories and investigate whether stresses induce specific p53-binding patterns correlated with effector gene induction. Using unique in cellulo genomic footprinting experiments, we studied p53 binding to the five response elements of p21 in response to stresses and monitored p21 mRNA variant transcription. We show clear footprints of p53 bound to response elements in living cells and reveal that the binding of p53 to response elements is transient, subject to dynamic changes during stress responses, and influenced by response element pentamer orientations. We show further that stresses lead to specific p53-binding patterns correlated with particular p21 mRNA variant transcription profiles and that p53 binding is necessary but not sufficient to induce p21 transcription. Our results indicate that pre- and post-binding events act together to regulate adapted stress responses; this paves the way to the unification of pre- and post-binding event theories. [Cancer Res 2009;69(21):8463–71]

Distinct MicroRNA Alterations Characterize High- and Low-Grade Bladder Cancer

Wed, 28 Oct 2009 21:08:15 PDT

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype–specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy. [Cancer Res 2009;69(21):8472–81]

Progressive Chromatin Repression and Promoter Methylation of CTNNA1 Associated with Advanced Myeloid Malignancies

Wed, 28 Oct 2009 21:08:15 PDT

Complete loss or deletion of the long arm of chromosome 5 is frequent in myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). The putative gene(s) deleted and responsible for the pathogenesis of these poor prognosis hematologic disorders remain controversial. This study is a comprehensive analysis of previously implicated and novel genes for epigenetic inactivation in AML and MDS. In 146 AML cases, methylation of CTNNA1 was frequent, and more common in AML patients with 5q deletion (31%) than those without 5q deletion (14%), whereas no methylation of other 5q genes was observed. In 31 MDS cases, CTNNA1 methylation was only found in high-risk MDS (≥RAEB2), but not in low-risk MDS (CTNNA1 methylation might be important in the transformation of MDS to AML. CTNNA1 expression was lowest in AML/MDS patients with CTNNA1 methylation, although reduced expression was found in some patients without promoter methylation. Repressive chromatin marks (H3K27me3) at the promoter were identified in CTNNA1-repressed AML cell lines and primary leukemias, with the most repressive state correlating with DNA methylation. These results suggest progressive, acquired epigenetic inactivation at CTNNA1, including histone modifications and promoter CpG methylation, as a component of leukemia progression in patients with both 5q– and non-5q– myeloid malignancies. [Cancer Res 2009;69(21):8482–90]

ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors

Wed, 28 Oct 2009 21:08:15 PDT

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription. [Cancer Res 2009;69(21):8491–8]

Oncogenic KRAS and BRAF Differentially Regulate Hypoxia-Inducible Factor-1{alpha} and -2{alpha} in Colon Cancer

Kikuchi, H., Pino, M. S., Zeng, M., Shirasawa, S., Chung, D. C. — Wed, 28 Oct 2009 21:08:15 PDT

KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. We showed previously that K-ras can interact with hypoxia to activate multiple signaling pathways. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1 and HIF-2, and we sought to define the roles of mutant KRAS and BRAF in the induction of HIF-1 and HIF-2 in colon cancer cells. Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1, whereas mutant BRAF enhanced both HIF-1 and HIF-2. Knockout or knockdown of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of only HIF-1. HIF-1 mRNA levels were comparable in cells with and without a KRAS mutation. However, the rate of HIF-1 protein synthesis was higher in cells with a KRAS mutation, and this was suppressed by the phosphoinositide 3-kinase inhibitor LY294002. In contrast, knockdown of mutant BRAF in HT29 cells suppressed both HIF-1 and HIF-2. Although BRAF regulated mRNA levels of both HIF-1 and HIF-2, knockdown of BRAF or treatment with the MEK inhibitor PD98059 impaired the translation of only HIF-2. Our data reveal that oncogenic KRAS and BRAF mutations differentially regulate the hypoxic induction of HIF-1 and HIF-2 in colon cancer, and this may potentially contribute to the phenotypic differences of KRAS and BRAF mutations in colon tumors. [Cancer Res 2009;69(21):8499–506]

Tumor Growth Instability and Its Implications for Chemotherapy

Castorina, P., Carco, D., Guiot, C., Deisboeck, T. S. — Wed, 28 Oct 2009 21:08:15 PDT

Optimal delivery of chemotherapy intensity is dependent on host- and tumor-specific characteristics. In this article, the chemotherapy late intensity schedule is revised to account for tumor growth instability, where a small tumor cell fraction emerges that exhibits a higher proliferation rate than the parent strain. Modeling this instability as simplified two-population dynamics, we find that: (a) if this instability precedes the onset of treatment, the slope of the linear increase of the drug concentration for the standard "Norton-Simon late intensity schedule" changes and the initial value of the dose strongly depends on the ratio of the two tumor cell populations and on their distinct growth rates; and (b) if the instability trails the initial treatment, the effective chemotherapeutic drug concentration changes as well. Both cases point toward testable potential refinements of the Norton-Simon late intensity schedule. [Cancer Res 2009;69(21):8507–15]

Host Lymphodepletion Enhances the Therapeutic Activity of an Oncolytic Vaccinia Virus Expressing 4-1BB Ligand

Kim, H. S., Kim-Schulze, S., Kim, D. W., Kaufman, H. L. — Wed, 28 Oct 2009 21:08:15 PDT

Oncolytic viral vectors have shown promise as antitumor therapeutic agents but their effectiveness is complicated by induction of antiviral antibody responses and rapid host clearance of recombinant vectors. We developed a recombinant oncolytic vaccinia virus expressing the 4-1BBL T-cell costimulatory molecule (rV-4-1BBL) and showed modest tumor regression in the poorly immunogenic B16 murine melanoma model. To improve the therapeutic potential of this vector, we tested the antitumor activity of local intratumoral injection in the setting of host lymphodepletion, which has been shown to augment vaccination and adoptive T-cell therapy. In this model, rV-4-1BBL injection in the setting of lymphodepletion promoted MHC class I expression, reduced antiviral antibody titers, promoted viral persistence, and rescued effector-memory CD8⁺ T cells, significantly improving the therapeutic effectiveness of the oncolytic vector. These data suggest that vaccination with rV-4-1BBL in the setting of host nonmyeloablative lymphodepletion represents a logical strategy for improving oncolytic vaccination in melanoma, and perhaps other cancers as well. [Cancer Res 2009;69(21):8516–25]

Correction: Development and Cancer: At the Crossroads of Nodal and Notch Signaling

Wed, 28 Oct 2009 21:08:15 PDT

Correction: Cucurbitacin B Induces Apoptosis by Inhibition of the JAK/STAT Pathway and Potentiates Antiproliferative Effects of Gemcitabine on Pancreatic Cancer Cells

Wed, 28 Oct 2009 21:08:15 PDT

Differences in Tumor Regulatory T-Cell Localization and Activation Status Impact Patient Outcome

Menetrier-Caux, C., Gobert, M., Caux, C. — Wed, 14 Oct 2009 21:07:37 PDT

The presence of regulatory T cells (Treg) has been described in a large panel of solid tumors. However, their impact on tumor progression differs according to the tumor type analyzed. We recently obtained evidence in breast carcinoma that Treg localized within lymphoid aggregates, but not in the tumor bed, have a negative impact on patients' survival. Moreover, we showed selective Treg recruitment through CCR4/CCL22 in the lymphoid aggregates upon contact with dendritic cells (DC), where they became strongly and selectively activated (ICOS^high) and block conventional T-cell response. Here, we discuss the meaning and potential implication of these novel findings. [Cancer Res 2009;69(20):7895–8]

Revving the Throttle on an Oncogene: CDK8 Takes the Driver Seat

Firestein, R., Hahn, W. C. — Wed, 14 Oct 2009 21:07:37 PDT

The Wnt/β-catenin pathway plays an important role in initiation in most, if not all, colon cancers. Prior work has provided important insights into the regulation of β-catenin stability in the cytoplasm; however, relatively little is known about the mechanism by which β-catenin activates gene transcription in the nucleus. Using genetic approaches, studies in human colon cancers and Drosophila have identified CDK8 as a colon cancer oncogene that regulates β-catenin transcriptional activity. These convergent observations provide new insights into the regulation of nuclear β-catenin activity and identify a novel therapeutic target for β-catenin-driven malignancies. [Cancer Res 2009;69(20):OF7899–901]

Frontiers of Biomedical Imaging Science 2009: Workshop Report and Research Opportunities

Wed, 14 Oct 2009 21:07:37 PDT

Direct Evidence that Bevacizumab, an Anti-VEGF Antibody, Up-regulates SDF1{alpha}, CXCR4, CXCL6, and Neuropilin 1 in Tumors from Patients with Rectal Cancer

Wed, 14 Oct 2009 21:07:37 PDT

Clinical studies converge on the observation that circulating cytokines are elevated in most cancer patients by anti–vascular endothelial growth factor (VEGF) therapy. However, the source of these molecules and their relevance in tumor escape remain unknown. We examined the gene expression profiles of cancer cells and tumor-associated macrophages in tumor biopsies before and 12 days after monotherapy with the anti-VEGF antibody bevacizumab in patients with rectal carcinoma. Bevacizumab up-regulated stromal cell–derived factor 1 (SDF1), its receptor CXCR4, and CXCL6, and down-regulated PlGF, Ang1, and Ang2 in cancer cells. In addition, bevacizumab decreased Ang1 and induced neuropilin 1 (NRP1) expression in tumor-associated macrophages. Higher SDF1 plasma levels during bevacizumab treatment significantly associated with distant metastasis at three years. These data show that VEGF blockade up-regulates inflammatory pathways and NRP1, which should be evaluated as potential targets for improving anti-VEGF therapy. [Cancer Res 2009;69(20):7905–10]

A Novel PTEN-Dependent Link to Ubiquitination Controls FLIPS Stability and TRAIL Sensitivity in Glioblastoma Multiforme

Panner, A., Crane, C. A., Weng, C., Feletti, A., Parsa, A. T., Pieper, R. O. — Wed, 14 Oct 2009 21:07:37 PDT

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP_S, and confers resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP_S protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP_S polyubiquitination. FLIP_S half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP_S E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP_S or with the FLIP_S-containing death inducing signal complex. Small interfering RNA–mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP_S ubiquitination, prolonged FLIP_S half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP_S steady-state levels, and suppressed FLIP_S ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP_S ubiquitination, FLIP_S stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability. [Cancer Res 2009;69(20):7911–6]

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Wed, 14 Oct 2009 21:07:38 PDT

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age. [Cancer Res 2009;69(20):7917–24]

In vivo, Noninvasive, Label-Free Detection and Eradication of Circulating Metastatic Melanoma Cells Using Two-Color Photoacoustic Flow Cytometry with a Diode Laser

Galanzha, E. I., Shashkov, E. V., Spring, P. M., Suen, J. Y., Zharov, V. P. — Wed, 14 Oct 2009 21:07:38 PDT

The circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. However, its clinical utility for metastasis prevention remains unclear, because metastases may already be present at the time of initial diagnosis with existing assays. Their sensitivity ex vivo is limited by a small blood sample volume, whereas in vivo examination of larger blood volumes may be clinically restricted by the toxicity of labels used for targeting of CTCs. We introduce a method for in vivo photoacoustic blood cancer testing with a high-pulse-repetition-rate diode laser that, when applied to melanoma, is free of this limitation. It uses the overexpression of melanin clusters as intrinsic, spectrally-specific cancer markers and signal amplifiers, thus providing higher photoacoustic contrast of melanoma cells compared with a blood background. In tumor-bearing mouse models and melanoma-spiked human blood samples, we showed a sensitivity level of 1 CTC/mL with the potential to improve this sensitivity 10³-fold in humans in vivo, which is impossible with existing assays. Additional advances of this platform include decreased background signals from blood through changes in its oxygenation, osmolarity, and hematocrit within physiologic norms, assessment of CTCs in deep vessels, in vivo CTC enrichment, and photoacoustic-guided photothermal ablation of CTCs in the bloodstream. These advances make feasible the early diagnosis of melanoma during the initial parallel progression of primary tumor and CTCs, and laser blood purging using noninvasive or hemodialysis-like schematics for the prevention of metastasis. [Cancer Res 2009;69(20):7926–34]

EBV-Induced Human CD8+ NKT Cells Suppress Tumorigenesis by EBV-Associated Malignancies

Wed, 14 Oct 2009 21:07:38 PDT

The underlying mechanism of the protective and suppressive role of NKT cells in human tumor immunosurveillance remains to be fully elucidated. We show that the frequencies of CD8⁺ NKT cells in patients with EBV-associated Hodgkin's lymphoma or nasopharyngeal carcinoma are significantly lower than those in healthy EBV carriers. These CD8⁺ NKT cells in tumor patients are also functionally impaired. In human-thymus-severe combined immunodeficient (hu-thym-SCID) chimeras, EBV challenge efficiently promotes the generation of IFN-–biased CD8⁺ NKT cells. These cells are strongly cytotoxic, drive syngeneic T cells into a Th1 bias, and enhance T-cell cytotoxicity to EBV-associated tumor cells. Interleukin-4–biased CD4⁺ NKT cells are predominately generated in unchallenged chimeras. These cells are noncytotoxic, drive syngeneic T cells into a Th2 bias, and do not affect T-cell cytotoxicity. In humanized xenogeneic tumor-transplanted hu-thym-SCID chimeras, adoptive transfer with EBV-induced CD8⁺ NKT cells significantly suppresses tumorigenesis by EBV-associated malignancies. EBV-induced CD8⁺ NKT cells are necessary and sufficient to enhance the T-cell immunity to EBV-associated malignancies in the hu-thym-SCID chimeras. CD4⁺ NKT cells are synergetic with CD8⁺ NKT cells, leading to a more pronounced T-cell antitumor response in the chimeras cotransferred with CD4⁺ and CD8⁺ NKT cells. Thus, immune reconstitution with EBV-induced CD8⁺ NKT cells could be a useful strategy in management of EBV-associated malignancies. [Cancer Res 2009;69(20):7935–44]

Magnetic Resonance Imaging Defines Cervicovaginal Anatomy, Cancer, and VEGF Trap Antiangiogenic Efficacy in Estrogen-Treated K14-HPV16 Transgenic Mice

Wed, 14 Oct 2009 21:07:38 PDT

Noninvasive detection of dysplasia provides a potential platform for monitoring the efficacy of chemopreventive therapy of premalignancy, imaging the tissue compartments comprising dysplasia: epithelium, microvasculature, and stromal inflammatory cells. Here, using respiratory-gated magnetic resonance imaging (MRI), the anatomy of premalignant and malignant stages of cervical carcinogenesis in estrogen-treated K14-HPV16 transgenic mice was noninvasively defined. Dynamic contrast enhanced (DCE)-MRI was used to quantify leakage across premalignant dysplastic microvasculature. Vascular permeability as measured by DCE-MRI, K^trans, was similar in transgenic (0.053 ± 0.020 min^–1; n = 32 mice) and nontransgenic (0.056 ± 0.029 min^–1; n = 17 mice) animals despite a 2-fold increase in microvascular area in the former compared with the latter. DCE-MRI did detect a significant decrease in vascular permeability accompanying diminution of dysplastic microvasculature by the antiangiogenic agent, vascular endothelial growth factor Trap (K^trans = 0.052 ± 0.013 min^–1 pretreatment; n = 6 mice versus K^trans = 0.019 ± 0.008 min^–1 post-treatment; n = 5 mice). Thus, we determined that the threshold of microvessel leakage associated with cervical dysplasia was <17 kDa and highlighted the potential of DCE-MRI to noninvasively monitor the efficacy of antiangiogenic drugs or chemoprevention regimens targeting the vasculature in premalignant cervical dysplasia. [Cancer Res 2009;69(20):7945–52]

Sox11 Prevents Tumorigenesis of Glioma-Initiating Cells by Inducing Neuronal Differentiation

Hide, T., Takezaki, T., Nakatani, Y., Nakamura, H., Kuratsu, J.-i., Kondo, T. — Wed, 14 Oct 2009 21:07:38 PDT

Recent findings have shown that malignant tumors contain cancer-initiating cells (CIC), which self-renew and are tumorigenic. However, CICs have not been characterized properly due to lack of specific markers. We recently established a mouse glioma cell line, NSCL61, by overexpressing an oncogenic HRas^L61 in p53-deficient neural stem cells. Using limiting dilution assays, we show that only 2 of 24 NSCL61 clones retained their tumorigenicity in vivo, although the others also expressed oncogenic HRas^L61 and could proliferate in culture. A comparison of the gene expression profiles of tumorigenic and nontumorigenic clones showed that the tumorigenic clones had lost Sox11 expression. We show that overexpression of sox11 prevented tumorigenesis of NSCL61s by inducing their neuronal differentiation accompanied with decreased levels of plagl1. We also show that overexpression of plagl1 abolished neuronal commitment of nontumorigenic cells and induced them to become tumorigenic. Moreover, we show that human glioma-initiating cells lost sox11 expression, and overexpression of sox11 prevented their tumorigenesis in vivo. Together with the clinical evidence showing that downregulation of sox11 mRNA correlates with a significant decrease in survival, these findings suggest that Sox11 prevents gliomagenesis by blocking the expression of oncogenic plagl1. [Cancer Res 2009;69(20):7953–9]

Phosphorylation and Degradation of MdmX Is Inhibited by Wip1 Phosphatase in the DNA Damage Response

Zhang, X., Lin, L., Guo, H., Yang, J., Jones, S. N., Jochemsen, A., Lu, X. — Wed, 14 Oct 2009 21:07:38 PDT

MdmX and Mdm2 regulate p53 tumor suppressor functions by controlling p53 transcriptional activity and/or stability in cells exposed to DNA damage. Accumulating evidence indicates that ATM-mediated phosphorylation and degradation of Mdm2 and MdmX may be the initial driving force that induces p53 activity during the early phase of the DNA damage response. We have recently determined that a novel protein phosphatase, Wip1 (or PPM1D), contributes to p53 regulation by dephosphorylating Mdm2 to close the p53 activation loop initiated by the ATM/ATR kinases. In the present study, we determine that Wip1 directly dephosphorylates MdmX at the ATM-targeted Ser403 and indirectly suppresses phosphorylation of MdmX at Ser342 and Ser367. Wip1 inhibits the DNA damage–induced ubiquitination and degradation of MdmX, leading to the stabilization of MdmX and reduction of p53 activities. Our data suggest that Wip1 is an important component in the ATM-p53-MdmX regulatory loop. [Cancer Res 2009;69(20):7960–8]

Intravital Imaging Reveals Transient Changes in Pigment Production and Brn2 Expression during Metastatic Melanoma Dissemination

Wed, 14 Oct 2009 21:07:38 PDT

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)β2 was up-regulated in the poorly pigmented cells. Furthermore, TGFβ signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis. [Cancer Res 2009;69(20):7969–77]

The Cytoskeleton Protein Filamin-A Is Required for an Efficient Recombinational DNA Double Strand Break Repair

Yue, J., Wang, Q., Lu, H., Brenneman, M., Fan, F., Shen, Z. — Wed, 14 Oct 2009 21:07:38 PDT

The human actin-binding protein filamin-A (also known as ABP-280) cross-links actin into a dynamic three-dimensional structure. It interacts with >45 proteins of diverse functions, serving as the scaffold in various signaling networks. BRCA2 is a protein that regulates RAD51-dependent recombinational repair of DNA double strand breaks (DSB). Proximate to the COOH terminus of the BRCA2 protein, a conserved and DNA binding domain (BRCA2-DBD) interacts with filamin-A and BCCIP. In this study, we sought to test the hypothesis that filamin-A influences homologous recombinational repair of DSB and the maintenance of genomic stability. We used three pairs of cell lines with normal and reduced filamin-A expression, including breast cancer and melanoma cells. We found that lack or reduction of filamin-A sensitizes cells to ionizing radiation, slows the removal of DNA damage–induced H2AX nuclear foci, reduces RAD51 nuclear focus formation and recruitment to chromatin in response to irradiation, and results in a 2-fold reduction of homologous recombinational repair of DSB. Furthermore, filamin-A–deficient cells have increased frequencies of micronucleus formation after irradiation. Our data illustrate the importance of the cytoskeleton structure in supporting the homologous recombinational DNA repair machinery and genome integrity, and further implicate a potential of filamin-A as a marker for prognosis in DNA damage–based cancer therapy. [Cancer Res 2009;69(20):7978–85]

Glioblastoma Cells Require Glutamate Dehydrogenase to Survive Impairments of Glucose Metabolism or Akt Signaling

Wed, 14 Oct 2009 21:07:38 PDT

Oncogenes influence nutrient metabolism and nutrient dependence. The oncogene c-Myc stimulates glutamine metabolism and renders cells dependent on glutamine to sustain viability ("glutamine addiction"), suggesting that treatments targeting glutamine metabolism might selectively kill c-Myc–transformed tumor cells. However, many current or proposed cancer therapies interfere with the metabolism of glucose, not glutamine. Here, we studied how c-Myc–transformed cells maintained viability when glucose metabolism was impaired. In SF188 glioblastoma cells, glucose deprivation did not affect net glutamine utilization but elicited a switch in the pathways used to deliver glutamine carbon to the tricarboxylic acid cycle, with a large increase in the activity of glutamate dehydrogenase (GDH). The effect on GDH resulted from the loss of glycolysis because it could be mimicked with the glycolytic inhibitor 2-deoxyglucose and reversed with a pyruvate analogue. Furthermore, inhibition of Akt signaling, which facilitates glycolysis, increased GDH activity whereas overexpression of Akt suppressed it, suggesting that Akt indirectly regulates GDH through its effects on glucose metabolism. Suppression of GDH activity with RNA interference or an inhibitor showed that the enzyme is dispensable in cells able to metabolize glucose but is required for cells to survive impairments of glycolysis brought about by glucose deprivation, 2-deoxyglucose, or Akt inhibition. Thus, inhibition of GDH converted these glutamine-addicted cells to glucose-addicted cells. The findings emphasize the integration of glucose metabolism, glutamine metabolism, and oncogenic signaling in glioblastoma cells and suggest that exploiting compensatory pathways of glutamine metabolism can improve the efficacy of cancer treatments that impair glucose utilization. [Cancer Res 2009;69(20):7986–93]

Candidate Gene Association Study of Esophageal Squamous Cell Carcinoma in a High-Risk Region in Iran

Wed, 14 Oct 2009 21:07:38 PDT

There is a region with a high risk for esophageal squamous cell carcinoma (ESCC) in the northeast of Iran. Previous studies suggest that hereditary factors play a role in the high incidence of cancer in the region. We selected 22 functional variants (and 130 related tagSNPs) from 15 genes that have been associated previously with the risk of ESCC. We genotyped a primary set of samples from 451 Turkmens (197 cases and 254 controls). Seven of 152 variants were associated with ESCC at the P = 0.05 level; these single nucleotide polymorphisms were then studied in a validation set of 549 cases and 1,119 controls, which included both Turkmens and non-Turkmens. The association observed for a functional variant in ADH1B was confirmed in the validation set, and that of a tagSNP in MGMT, the association was borderline significant in the validation set, after correcting for multiple testing. The other 5 variants that were associated in the primary set were not significantly associated in the validation set. The histidine allele at codon 48 of ADH1B gene was associated with a significantly decreased risk of ESCC in the joint data set (primary and validation set) under a recessive model (odds ratio, 0.41; 95% confidence interval, 0.29-0.76; P = 4 x 10^–4). The A allele of the rs7087131 variant of MGMT gene was associated with a decreased risk of ESCC under a dominant model (odds ratio, 0.79; 95% confidence interval, 0.64-0.96; P = 0.02). These results support the hypothesis that genetic predisposition plays a role in the high incidence of ESSC in Iran. [Cancer Res 2009;69(20):7994–8000]

Apolipoprotein E/C1 Locus Variants Modify Renal Cell Carcinoma Risk

Wed, 14 Oct 2009 21:07:38 PDT

Lipid peroxidation is considered a unifying mechanistic pathway through which known risk factors induce renal cell carcinoma (RCC). We hypothesized that genes selected a priori for their role in lipid peroxidation would modify cancer risk. We genotyped 635 single nucleotide polymorphisms (SNP) in 38candidate genes in 777 Caucasian RCC cases and 1,035 controls enrolled in a large European case-control study. Top candidate SNPs were confirmed among 718 Caucasian cases and 615 controls in a second study in the United States. Two of the three SNPs (rs8106822 and rs405509) that replicated in the U.S. study were within a regulatory region of the APOE promoter. The OR for rs8106822 A>G variant was 1.22_AG and 1.41_GG (P_trend = 0.01) in the European study, 1.05_AG and 1.51_GG (P_trend = 0.03) in the U.S. study, and 1.15_AG and 1.44_GG (P_trend = 0.001) among 1,485 cases and 1,639 controls combined. The rs405509 G>T variant was associated with risk in the European (OR, 0.87_TG; OR, 0.71_TT; P_trend = 0.02), the U.S. (OR, 0.68_TG; OR, 0.71_TT; P_trend = 0.02), and both studies combined (OR_TG, 0.79; OR_TT, 0.71; P_trend = 0.001), as was the G-G haplotype (r² = 0.64; P= 4.7 x 10^–4). This association is biologically plausible as SNP rs405509 was shown to modify protein binding and transcriptional activity of the APOE protein in vitro and is in linkage disequilibrium with key known variants defining the e2, e3, and e4 alleles that modify risk of atherosclerosis, Alzheimer's disease risk, and progression to AIDS. In two large case-control studies, our findings further define a functional region of interest at the APOE locus that increases RCC susceptibility. [Cancer Res 2009;69(20):8001–8]

Inhibition of Tumor Cell Growth, Invasion, and Metastasis by EXEL-2880 (XL880, GSK1363089), a Novel Inhibitor of HGF and VEGF Receptor Tyrosine Kinases

Wed, 14 Oct 2009 21:07:38 PDT

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in a wide variety of human malignancies. Vascular endothelial growth factor (VEGF) receptors are expressed on the surface of vascular endothelial cells and cooperate with Met to induce tumor invasion and vascularization. EXEL-2880 (XL880, GSK1363089) is a small-molecule kinase inhibitor that targets members of the HGF and VEGF receptor tyrosine kinase families, with additional inhibitory activity toward KIT, Flt-3, platelet-derived growth factor receptor β, and Tie-2. Binding of EXEL-2880 to Met and VEGF receptor 2 (KDR) is characterized by a very slow off-rate, consistent with X-ray crystallographic data showing that the inhibitor is deeply bound in the Met kinase active site cleft. EXEL-2880 inhibits cellular HGF-induced Met phosphorylation and VEGF-induced extracellular signal-regulated kinase phosphorylation and prevents both HGF-induced responses of tumor cells and HGF/VEGF-induced responses of endothelial cells. In addition, EXEL-2880 prevents anchorage-independent proliferation of tumor cells under both normoxic and hypoxic conditions. In vivo, these effects produce significant dose-dependent inhibition of tumor burden in an experimental model of lung metastasis. Collectively, these data indicate that EXEL-2880 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of invasion and angiogenesis mediated by HGF and VEGF receptors. [Cancer Res 2009;69(20):8009–16]

A Novel Experimental Heme Oxygenase-1-Targeted Therapy for Hormone-Refractory Prostate Cancer

Wed, 14 Oct 2009 21:07:38 PDT

Heme oxygenase-1 (HO-1), a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress. Herein, we identify HO-1 as a biomarker and potential therapeutic target for advanced prostate cancer (PCA). Immunohistochemical analysis of prostate tissue using a progression tissue microarray from patients with localized PCA and across several stages of disease progression revealed a significant elevation of HO-1 expression in cancer epithelial cells, but not in surrounding stromal cells, from hormone-refractory PCA (HRPCA) compared with hormone-responsive PCA and benign tissue. Silencing the ho-1 gene in HRPCA cells decreased the HO-1 activity, oxidative stress, and activation of the mitogen-activated protein kinase–extracellular signal-regulated kinase/p38 kinase. This coincided with reduced cell proliferation, cell survival, and cell invasion in vitro, as well as inhibition of prostate tumor growth and lymph node and lung metastases in vivo. The effect of ho-1 silencing on these oncogenic features was mimicked by exposure of cells to a novel selective small-molecule HO-1 inhibitor referred to as OB-24. OB-24 selectively inhibited HO-1 activity in PCA cells, which correlated with a reduction of protein carbonylation and reactive oxygen species formation. Moreover, OB-24 significantly inhibited cell proliferation in vitro and tumor growth and lymph node/lung metastases in vivo. A potent synergistic activity was observed when OB-24 was combined with Taxol. Together, these results establish HO-1 as a potential therapeutic target for advanced PCA. [Cancer Res 2009;69(20):8017–24]

Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases

Wed, 14 Oct 2009 21:07:38 PDT

Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addition, GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH₂-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we determined the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compound is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a -complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isozymes determine the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile¹⁰⁴ in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile¹⁰⁴ into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile¹⁰⁴Val and Ile¹⁰⁴Ala variants display a 4-fold higher affinity for the compound. Remarkably, the GSTP1-1/Ile¹⁰⁴Ala structure in complex with NBDHEX shows a considerable shift of the compound inside the H-site. These data might be useful for the development of new anticancer compounds. [Cancer Res 2009;69(20):8025–34]

Overexpression of Kinesins Mediates Docetaxel Resistance in Breast Cancer Cells

De, S., Cipriano, R., Jackson, M. W., Stark, G. R. — Wed, 14 Oct 2009 21:07:38 PDT

Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumor-derived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes. [Cancer Res 2009;69(20):8035–42]

Tpl2 Is a Key Mediator of Arsenite-Induced Signal Transduction

Lee, K. M., Lee, K. W., Bode, A. M., Lee, H. J., Dong, Z. — Wed, 14 Oct 2009 21:07:38 PDT

Arsenite is a well-known human carcinogen that especially targets skin. The tumor progression locus 2 (Tpl2) gene encodes a serine/threonine protein kinase that is overexpressed in various cancer cells. However, the relevance of Tpl2 in arsenite-induced carcinogenesis and the underlying mechanisms remain to be explored. We show that arsenite increased Tpl2 kinase activity and its phosphorylation in mouse epidermal JB6 P+ cells in a dose- and time-dependent manner. Exposure to arsenite resulted in a marked induction of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂), important mediators of inflammation and tumor promotion. Treatment with a Tpl2 kinase inhibitor or Tpl2 short hairpin RNA suppressed COX-2 expression and PGE₂ production induced by arsenite treatment, suggesting that Tpl2 is critical in arsenite-induced carcinogenesis. We also found that arsenite-induced phosphorylation of extracellular signal-regulated kinases (ERK) or c-Jun NH₂-terminal kinases (JNK) was markedly suppressed by Tpl2 kinase inhibitor or Tpl2 short hairpin RNA. Inhibition of arsenite-induced ERK or JNK signaling using a pharmacologic inhibitor of ERK or JNK substantially blocked COX-2 expression. Furthermore, inhibition of Tpl2 reduced the arsenite-induced promoter activity of NF-B and activator protein-1 (AP-1), indicating that NF-B and AP-1 are downstream transducers of arsenite-triggered Tpl2. Our results show that Tpl2 plays a key role in arsenite-induced COX-2 expression and PGE₂ production and further elucidate the role of Tpl2 in arsenite signals that activate ERK/JNK and NF-B/AP-1 in JB6 P+ cells. [Cancer Res 2009;69(20):8043–9]

Anticancer Chemotherapy Inhibits MHC Class I-Related Chain A Ectodomain Shedding by Downregulating ADAM10 Expression in Hepatocellular Carcinoma

Wed, 14 Oct 2009 21:07:38 PDT

MHC class I–related chain A (MICA) is a ligand for the NKG2D-activating immunoreceptor that mediates activation of natural killer (NK) cells. The ectodomain of MICA is shed from tumor cells, which may be an important means of evading antitumor immunity. We previously reported that patients with hepatocellular carcinoma (HCC) display high levels of soluble MICA in circulation, which could be downregulated by chemotherapy. The present study shows that anti-HCC drugs suppress MICA ectodomain shedding by inhibiting expression of a disintegrin and metalloproteinase 10 (ADAM10). Both ADAM10 and CD44, a typical substrate of the ADAM10 protease, were expressed in human HCC tissues and HCC cells but not in normal liver tissues or cultured hepatocytes. Small interfering RNA–mediated knockdown experiments revealed that ADAM10 is a critical sheddase for both MICA and CD44 in HCC cells. Of interest is the finding that epirubicin clearly downregulated ADAM10 expression and MICA shedding in HCC cells; its suppressive effect on MICA shedding was abolished in ADAM10-depleted cells. Epirubicin treatment also enhanced the NKG2D-mediated NK sensitivity of HCC cells. Patients with HCC had significantly higher levels of serum-soluble CD44, which correlated well with serum-soluble MICA levels, thus suggesting a close link between ADAM10 activity and MICA shedding in these patients. Soluble MICA and CD44 levels were downregulated with a significant correlation in patients treated by transarterial chemoembolization using epirubicin. In conclusion, anticancer drugs can modulate expression of ADAM10, which is critically involved in MICA ectodomain shedding. Epirubicin therapy may have a previously unrecognized effect on antitumor immunity in HCC patients. [Cancer Res 2009;69(20):8050–7]

Immunoselection of Breast and Ovarian Cancer Cells with Trastuzumab and Natural Killer Cells: Selective Escape of CD44high/CD24low/HER2low Breast Cancer Stem Cells

Wed, 14 Oct 2009 21:07:38 PDT

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell–mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44^highCD24^low "cancer stem cell–like" phenotype and expressed significantly less HER2 compared with non–stem cells. Likewise, the CD44^highCD24^low population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44^highCD24^low cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants. [Cancer Res 2009;69(20):8058–66]

Kupffer Cell Suppression of CD8+ T Cells in Human Hepatocellular Carcinoma Is Mediated by B7-H1/Programmed Death-1 Interactions

Wu, K., Kryczek, I., Chen, L., Zou, W., Welling, T. H. — Wed, 14 Oct 2009 21:07:38 PDT

B7-H1 is a recently identified B7 family member that, along with one of its receptors, programmed death-1 (PD-1), has been involved in multiple immunopathologic scenarios. However, the nature of B7-H1 and PD-1 in human hepatocellular carcinoma (HCC) remains poorly defined. We investigated the expression and functional relevance of this pathway in patients with HCC. We showed that B7-H1 expression on Kupffer cells (KC) was increased in tumor tissues compared with surrounding nontumor liver tissues in patients with HCC and this correlated with poorer survival. Coculture of HCC cells with monocytes showed that tumor-associated interleukin-10 contributed to the induction of B7-H1 in the HCC environment. We further observed that the levels of PD-1⁺CD8⁺ T cells were higher in tumor tissues than in nontumor tissues. B7-H1⁺ KCs and PD-1⁺ T cells were colocalized in the HCC stroma. PD-1⁺CD8⁺ T cells had decreased proliferative ability and effector function as shown by reduced granule and cytokine expression compared with PD-1^– T cells. Importantly, blocking KC B7-H1 interaction with PD-1⁺CD8⁺ cells using neutralizing antibodies recovered effector T-cell function. Our data indicate that the B7-H1/PD-1 axis contributes to immune suppression in human HCC, with blockade of this pathway carrying important therapeutic implications. [Cancer Res 2009;69(20):8067–75]

Human Suppressor of Cytokine Signaling 1 Controls Immunostimulatory Activity of Monocyte-Derived Dendritic Cells

Hong, B., Ren, W., Song, X.-T., Evel-Kabler, K., Chen, S.-Y., Huang, X. F. — Wed, 14 Oct 2009 21:07:38 PDT

Dendritic cell (DC)–based tumor vaccines have only achieved limited clinical efficacy, underscoring the limitation of stimulatory strategies to elicit effective cytotoxic T lymphocyte (CTL) responses against self-tumor–associated antigens. Here, we investigate the role of human suppressor of cytokine signaling 1 (SOCS1), a feedback inhibitor of the Janus-activated kinase/signal transducer and activator of transcription signaling pathway, in regulating antigen presentation by human DCs (hDC). We find that human SOCS1 (hSOCS1)–silenced DCs have an enhanced stimulatory ability to prime self-antigen–specific CTLs in vitro and in a severe combined immunodeficient-hu mouse model. Human CTLs activated by SOCS1-silenced DCs, but not wild-type DCs, have an active lytic activity to natural antigen-expressing tumor cells. We further find that the capacity of hDCs to prime CTLs is likely controlled by SOCS1-restricted production and signaling of proinflammatory cytokines, such as interleukin-12. These results indicate a critical role of hSOCS1 in negatively regulating the immunostimulatory capacity of DCs and imply a translational potential of this alternative SOCS1 silencing strategy to develop effective DC vaccines. [Cancer Res 2009;69(20):8076–84]

Tumor Antigen-Specific FOXP3+ CD4 T Cells Identified in Human Metastatic Melanoma: Peptide Vaccination Results in Selective Expansion of Th1-like Counterparts

Wed, 14 Oct 2009 21:07:38 PDT

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A_26-35(A27L) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6–restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6–restricted Melan-A–specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells. [Cancer Res 2009;69(20):8085–93]

Oncogenesis Caused by Loss of the SNF5 Tumor Suppressor Is Dependent on Activity of BRG1, the ATPase of the SWI/SNF Chromatin Remodeling Complex

Wed, 14 Oct 2009 21:07:38 PDT

Alterations in chromatin play an important role in oncogenic transformation, although the underlying mechanisms are often poorly understood. The SWI/SNF complex contributes to epigenetic regulation by using the energy of ATP hydrolysis to remodel chromatin and thus regulate transcription of target genes. SNF5, a core subunit of the SWI/SNF complex, is a potent tumor suppressor that is specifically inactivated in several types of human cancer. However, the mechanism by which SNF5 mutation leads to cancer and the role of SNF5 within the SWI/SNF complex remain largely unknown. It has been hypothesized that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. Here, we show, however, distinct effects for inactivation of Snf5 and the ATPase subunit Brg1 in primary cells. Further, using both human cell lines and mouse models, we show that cancer formation in the absence of SNF5 does not result from SWI/SNF inactivation but rather that oncogenesis is dependent on continued presence of BRG1. Collectively, our results show that cancer formation in the absence of SNF5 is dependent on the activity of the residual BRG1-containing SWI/SNF complex. These findings suggest that, much like the concept of oncogene addiction, targeted inhibition of SWI/SNF ATPase activity may be an effective therapeutic approach for aggressive SNF5-deficient human tumors. [Cancer Res 2009;69(20):8094–101]

Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81

Wed, 14 Oct 2009 21:07:38 PDT

ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site–driven reporter androgen inducible, and, on the other hand, ETV1 superinduced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy. [Cancer Res 2009;69(20):8102–10]

Runt-Related Transcription Factor RUNX3 Is a Target of MDM2-Mediated Ubiquitination

Wed, 14 Oct 2009 21:07:38 PDT

The p14^ARF-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14^ARF and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14^ARF-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14^ARF-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation. [Cancer Res 2009;69(20):8111–9]

ARTEMIS Nuclease Facilitates Apoptotic Chromatin Cleavage

Britton, S., Frit, P., Biard, D., Salles, B., Calsou, P. — Wed, 14 Oct 2009 21:07:38 PDT

One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis. [Cancer Res 2009;69(20):8120–6]

Azacytidine Inhibits RNA Methylation at DNMT2 Target Sites in Human Cancer Cell Lines

Schaefer, M., Hagemann, S., Hanna, K., Lyko, F. — Wed, 14 Oct 2009 21:07:38 PDT

The cytosine analogues azacytidine and decitabine are currently being developed as drugs for epigenetic cancer therapy. Although various studies have shown that both drugs are effective in inhibiting DNA methylation, it has also become clear that their mode of action is not limited to DNA demethylation. Because azacytidine is a ribonucleoside, the primary target of this drug may be cellular RNA rather than DNA. We have now analyzed the possibility that azacytidine inhibits the RNA methyltransferase DNMT2. We found that DNMT2 is variably expressed in human cancer cell lines. RNA bisulfite sequencing showed that azacytidine, but not decitabine, inhibits cytosine 38 methylation of tRNA^Asp, a major substrate of DNMT2. Azacytidine caused a substantially stronger effect than decitabine on the metabolic rate of all the cancer cell lines tested, consistent with an effect of this drug on RNA metabolism. Of note, drug-induced loss of RNA methylation seemed specific for DNMT2 target sites because we did not observe any significant demethylation at sites known to be methylated by other RNA methyltransferases. Our results uncover a novel and quantifiable drug activity of azacytidine and raise the possibility that tRNA hypomethylation might contribute to patient responses. [Cancer Res 2009;69(20):8127–32]

Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism

Wed, 14 Oct 2009 21:07:38 PDT

A number of epidemiologic studies have indicated a strong association between dietary fat intake and prostate cancer development, suggesting that lipid metabolism plays some important roles in prostate carcinogenesis and its progression. In this study, through our genome-wide gene expression analysis of clinical prostate cancer cells, we identified a novel lipogenic gene, ELOVL7, coding a possible long-chain fatty acid elongase, as overexpressed in prostate cancer cells. ELOVL7 expression is regulated by the androgen pathway through SREBP1, as well as other lipogenic enzymes. Knockdown of ELOVL7 resulted in drastic attenuation of prostate cancer cell growth, and it is notable that high-fat diet promoted the growth of in vivo tumors of ELOVL7-expressed prostate cancer. In vitro fatty acid elongation assay and fatty acid composition analysis indicated that ELOVL7 was preferentially involved in fatty acid elongation of saturated very-long-chain fatty acids (SVLFA, C20:0~). Lipid profiles showed that knockdown of ELOVL7 in prostate cancer cells affected SVLFAs in the phospholipids and the neutral lipids, such as cholesterol ester. Focusing on cholesterol ester as a source of de novo steroid synthesis, we show that ELOVL7 affected de novo androgen synthesis in prostate cancer cells. These findings suggest that EVOLV7 could be involved in prostate cancer growth and survival through the metabolism of SVLFAs and their derivatives, could be a key molecule to elucidate the association between fat dietary intake and prostate carcinogenesis, and could also be a promising molecular target for development of new therapeutic or preventive strategies for prostate cancers. [Cancer Res 2009;69(20):8133–40]

Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Wed, 14 Oct 2009 21:07:38 PDT

Androgen receptor (AR) is known to be overexpressed in castration-resistant prostate cancer. To interrogate the functional significance of the AR level, we established two LNCaP cell sublines expressing in a stable fashion two to four times (LNCaP-ARmo) and four to six times (LNCaP-ARhi) higher level of AR than the parental cell line expressing the empty vector (LNCaP-pcDNA3.1). LNCaP-ARhi cell line grew faster than the control line in low concentrations, especially in 1 nmol/L 5-dihydrotestosterone (DHT). Microarray-based transcript profiling and subsequent unsupervised hierarchical clustering showed that LNCaP-ARhi cells clustered together with VCaP cells, containing endogenous AR gene amplification and overexpression, indicating the central role of AR in the overall regulation of gene expression in prostate cancer cells. Two hundred forty genes showed >2-fold changes on DHT treatment in LNCaP-ARhi at 4 h time point, whereas only 164 and 52 showed changes in LNCaP-ARmo and LNCaP-pcDNA3.1, respectively. Many androgen-regulated genes were upregulated in LNCaP-ARhi at 10-fold lower concentration of DHT than in control cells. DHT (1 nmol/L) increased expression of several cell cycle–associated genes in LNCaP-ARhi cells. ChIP-on-chip assay revealed the presence of chromatin binding sites for AR within ±200 kb of most of these genes. The growth of LNCaP-ARhi cells was also highly sensitive to cyclin-dependent kinase inhibitor, roscovitine, at 1nmol/L DHT. In conclusion, our results show that overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens. The activity of AR signaling pathway is regulated by the levels of both ligand and the receptor. [Cancer Res 2009;69(20):8141–9]

A Modified Sleeping Beauty Transposon System That Can Be Used to Model a Wide Variety of Human Cancers in Mice

Wed, 14 Oct 2009 21:07:38 PDT

Recent advances in cancer therapeutics stress the need for a better understanding of the molecular mechanisms driving tumor formation. This can be accomplished by obtaining a more complete description of the genes that contribute to cancer. We previously described an approach using the Sleeping Beauty (SB) transposon system to model hematopoietic malignancies in mice. Here, we describe modifications of the SB system that provide additional flexibility in generating mouse models of cancer. First, we describe a Cre-inducible SBase allele, RosaSBase^LsL, that allows the restriction of transposon mutagenesis to a specific tissue of interest. This allele was used to generate a model of germinal center B-cell lymphoma by activating SBase expression with an Aid-Cre allele. In a second approach, a novel transposon was generated, T2/Onc3, in which the CMV enhancer/chicken β-actin promoter drives oncogene expression. When combined with ubiquitous SBase expression, the T2/Onc3 transposon produced nearly 200 independent tumors of more than 20 different types in a cohort of 62 mice. Analysis of transposon insertion sites identified novel candidate genes, including Zmiz1 and Rian, involved in squamous cell carcinoma and hepatocellular carcinoma, respectively. These novel alleles provide additional tools for the SB system and provide some insight into how this mutagenesis system can be manipulated to model cancer in mice. [Cancer Res 2009;69(20):8150–6]

microRNA-21 Negatively Regulates Cdc25A and Cell Cycle Progression in Colon Cancer Cells

Wed, 14 Oct 2009 21:07:38 PDT

microRNAs (miRNA) are small noncoding RNAs that participate in diverse biological processes by suppressing target gene expression. Altered expression of miR-21 has been reported in cancer. To gain insights into its potential role in tumorigenesis, we generated miR-21 knockout colon cancer cells through gene targeting. Unbiased microarray analysis combined with bioinformatics identified cell cycle regulator Cdc25A as a miR-21 target. miR-21 suppressed Cdc25A expression through a defined sequence in its 3'-untranslated region. We found that miR-21 is induced by serum starvation and DNA damage, negatively regulates G₁-S transition, and participates in DNA damage–induced G₂-M checkpoint through down-regulation of Cdc25A. In contrast, miR-21 deficiency did not affect apoptosis induced by a variety of commonly used anticancer agents or cell proliferation under normal cell culture conditions. Furthermore, miR-21 was found to be underexpressed in a subset of Cdc25A-overexpressing colon cancers. Our data show a role of miR-21 in modulating cell cycle progression following stress, providing a novel mechanism of Cdc25A regulation and a potential explanation of miR-21 in tumorigenesis. [Cancer Res 2009;69(20):8157–65]

Combination of Sulindac and Antimicrobial Eradication of Helicobacter pylori Prevents Progression of Gastric Cancer in Hypergastrinemic INS-GAS Mice

Wed, 14 Oct 2009 21:07:39 PDT

Helicobacter pylori infection causes severe dysplasia manifested as gastrointestinal intraepithelial neoplasia (GIN) after 28 weeks post–H. pylori infection (WPI) in cancer-prone, hypergastrinemic male INS-GAS mice. We examined the efficacy of the nonsteroidal anti-inflammatory drug sulindac (400 ppm in drinking water) alone, the CCK2/gastrin receptor antagonist YM022 (45 mg/kg/wk) alone, and sulindac or YM022 combined with H. pylori eradication therapy to prevent H. pylori–associated gastric cancer in male INS-GAS mice. Treatments started at 22 WPI, and mice were euthanized at 28 WPI. In uninfected mice, all treatments significantly delayed development of spontaneous GIN (P < 0.05). In H. pylori–infected mice, sulindac alone or YM022 alone had no protective effect on H. pylori–associated GIN. Importantly, sulindac exacerbated the severity of H. pylori–associated gastritis despite decreased gastric prostaglandin E₂ levels. However, sulindac combined with H. pylori antimicrobial eradication reduced the incidence of GIN (P < 0.05), whereas YM022 combined with antimicrobial eradication did not reduce GIN. In infected mice, sulindac or YM022 treatment did not alter gastric expression of the proinflammatory cytokines Ifn- and Tnf- and mucosal cell proliferation. Sulindac or YM022 combined with antimicrobial eradication down-regulated mRNA levels of Ifn- and Tnf- and mucosal cell proliferation (P < 0.05). We conclude that sulindac enhances H. pylori gastritis and may promote inflammation-mediated gastric carcinogenesis. The combination of sulindac and antimicrobial H. pylori eradication was beneficial for reducing proinflammatory cytokine mRNA in the stomach and preventing progression from severe dysplasia to gastric cancer in H. pylori–infected INS-GAS mice. [Cancer Res 2009;69(20):8166–74]

Inhibition of Azoxymethane-Induced Colorectal Cancer by CP-31398, a TP53 Modulator, Alone or in Combination with Low Doses of Celecoxib in Male F344 Rats

Wed, 14 Oct 2009 21:07:39 PDT

Tumor suppressor p53 plays a major role in colorectal cancer development. The present study explores the effects of p53-modulating agent CP-31398 alone and combined with celecoxib on azoxymethane-induced aberrant crypt foci (ACF) and colon adenocarcinomas in F344 rats. Maximum tolerated doses were 400 and 3,000 ppm for CP-31398 and celecoxib, respectively. ACF and tumor efficacy endpoints were carried out on azoxymethane-treated 7-week-old rats (48 per group) fed the control AIN-76A diet. Two weeks after carcinogen treatment, rats were fed the diets containing 0, 150, or 300 ppm CP-31398, 300 ppm celecoxib, or 150 ppm CP-31398 plus 300 ppm celecoxib. ACF and colon adenocarcinomas were determined at 8 and 48 weeks after azoxymethane treatment, respectively. Dietary CP-31398 was shown to suppress mean colonic total ACF by 43% and multicrypt ACF by 63%; dietary CP-31398 at 150 and 300 ppm suppressed adenocarcinoma incidence by 30.4% (P < 0.02) and 44% (P < 0.005), respectively, and adenocarcinoma multiplicity by 51% (P < 0.005) and 65% (P < 0.0001), respectively. Dietary celecoxib suppressed colon adenocarcinoma incidence (60%; P < 0.0003) and multiplicity (70%; P < 0.0001). Importantly, combination of low-dose CP-31398 and celecoxib suppressed colon adenocarcinoma incidence by 78% and multiplicity by 90%. Rats that were fed the high-dose CP-31398 or a combination of low-dose CP-31398 and celecoxib showed considerable enhancement of p53 and p21^WAF1/CIP expression, apoptosis, and reduced tumor cell proliferation in colonic tumors. These observations show, for the first time, that CP-31398 possesses significant dose-dependent chemopreventive activity in a well-established colon cancer model and that a combination of low-dose CP-31398 and celecoxib significantly enhanced colon cancer chemopreventive efficacy. [Cancer Res 2009;69(20):8175–82]

Molecular Consequences of Genetic Variations in the Glutathione Peroxidase 1 Selenoenzyme

Wed, 14 Oct 2009 21:07:39 PDT

Accumulating data have implicated the selenium-containing cytosolic glutathione peroxidase, GPx-1, as a determinant of cancer risk and a mediator of the chemopreventive properties of selenium. Genetic variants of GPx-1 have been shown to be associated with cancer risk for several types of malignancies. To investigate the relationship between GPx-1 enzyme activity and genotype, we measured GPx-1 enzyme activity and protein levels in human lymphocytes as a function of the presence of two common variations: a leucine/proline polymorphism at codon 198 and a variable number of alanine-repeat codons. Differences in GPx activity among these cell lines, as well as in the response to the low-level supplementation of the media with selenium, indicated that factors other than just genotype are significant in determining activity. To restrict the study to genotypic effects, human MCF-7 cells were engineered to exclusively express allelic variants representing a combination of either a codon 198 leucine or proline and either 5 or 7 alanine-repeat codons following transfection of GPx-1 expression constructs. Transfectants were selected and analyzed for GPx-1 enzyme activity and protein levels. GPx-1 with 5 alanines and a leucine at codon 198 showed a significantly higher induction when cells were incubated with selenium and showed a distinct pattern of thermal denaturation as compared with GPx-1 encoded by the other examined alleles. The collective data obtained using both lymphocytes and MCF-7 indicate that both intrinsic and extrinsic factors cooperate to ultimately determine the levels of this enzyme available to protect cells against DNA damage and mutagenesis. [Cancer Res 2009;69(20):8183–90]

RAS Mutations Affect Tumor Necrosis Factor-Induced Apoptosis in Colon Carcinoma Cells via ERK-Modulatory Negative and Positive Feedback Circuits Along with Non-ERK Pathway Effects

Kreeger, P. K., Mandhana, R., Alford, S. K., Haigis, K. M., Lauffenburger, D. A. — Wed, 14 Oct 2009 21:07:39 PDT

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor- (TNF)–induced apoptosis. When the dynamics of phosphorylated ERK response to TNF were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor- autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNF treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence. [Cancer Res 2009;69(20):8191–9]

Protein Kinase CK2 Regulates Cytoskeletal Reorganization during Ionizing Radiation-Induced Senescence of Human Mesenchymal Stem Cells

Wang, D., Jang, D.-J. — Wed, 14 Oct 2009 21:07:39 PDT

Human mesenchymal stem cells (hMSC) are critical for tissue regeneration. How hMSC respond to genotoxic stresses and potentially contribute to aging and cancer remain underexplored. We showed that ionizing radiation induced cellular senescence of hMSC over a period of 10 days, showing a critical transition between days 3 and 6. This was confirmed by senescence-associated β-galactosidase staining, protein expression profiles of key cell cycle regulators (retinoblastoma protein, p53, p21^waf1/Cip1, and p16^INK4A), and senescence-associated secretory phenotypes (interleukin-8, interleukin-12, GRO, and MDC). We observed dramatic cytoskeletal reorganization of hMSC through reduction of myosin-10, redistribution of myosin-9, and secretion of profilin-1. Using a SILAC-based phosphoproteomics method, we detected significant reduction of myosin-9 phosphorylation at Ser¹⁹⁴³, coinciding with its redistribution. Importantly, through treatment with cell-permeable inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) and gene knockdown using RNA interference, we identified CK2, a kinase responsible for myosin-9 phosphorylation at Ser¹⁹⁴³, as a key factor contributing to the radiation-induced senescence of hMSC. We showed that individual knockdown of CK2 catalytic subunits CK2 and CK2' induced hMSC senescence. However, only knockdown of CK2 resulted in morphologic phenotypes resembling those of radiation-induced senescence. These results suggest that CK2 and CK2' play differential roles in hMSC senescence progression, and their relative expression might represent a novel regulatory mechanism for CK2 activity. [Cancer Res 2009;69(20):8200–7]

Aldehyde Dehydrogenase-Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Wed, 14 Oct 2009 21:07:39 PDT

Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells, the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore, the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer, these colitis-derived tumors were capable of propagation as sphere cultures. However, unlike the origins of sporadic colon cancer, the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings, we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting, normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients, suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer. [Cancer Res 2009;69(20):8208–15]

Correction: Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Wed, 14 Oct 2009 21:07:39 PDT

Correction: Glioma Tumor Stem-Like Cells Promote Tumor Angiogenesis and Vasculogenesis via Vascular Endothelial Growth Factor and Stromal-Derived Factor 1

Wed, 14 Oct 2009 21:07:39 PDT

Correction: Genetic Alterations in the Phosphoinositide 3-Kinase/Akt Signaling Pathway Confer Sensitivity of Thyroid Cancer Cells to Therapeutic Targeting of Akt and Mammalian Target of Rapamycin

Wed, 14 Oct 2009 21:07:39 PDT

Correction: Elevated Epithelial Insulin-Like Growth Factor Expression Is a RiskFactor for Lung Cancer Development

Wed, 14 Oct 2009 21:07:39 PDT

Acting Locally and Globally: Myc's Ever-Expanding Roles on Chromatin

Varlakhanova, N. V., Knoepfler, P. S. — Thu, 01 Oct 2009 11:26:04 PDT

Myc regulates key cellular processes including cell cycle, differentiation, and apoptosis. It has long been thought to direct these functions by acting solely as a classic transcription factor regulating expression of a small number of key target genes through discrete chromatin events in their promoters. A recent wave of genomics studies together directly challenge the narrowness of this model. For example, Myc binds to tens of thousands of sites in the human genome. It also regulates histone acetylation at and transcription of a remarkable number of genes, far beyond that expected of a classical transcription factor. The influence of Myc on chromatin also surprisingly extends to both genic and expansive intergenic regions. These studies support an evolving model in which Myc activity on chromatin is far more complex than previously imagined. The ability of Myc to act both locally and globally on chromatin may be responsible for its wide-ranging effects on the biology of stem and tumor cells. [Cancer Res 2009;69(19):7487–90]

c-Myc and eIF4F Constitute a Feedforward Loop That Regulates Cell Growth: Implications for Anticancer Therapy

Lin, C.-J., Malina, A., Pelletier, J. — Thu, 01 Oct 2009 11:26:04 PDT

The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. Recent findings indicate that the role of Myc during cell growth and proliferation is linked to an increase in eIF4F activity in a feedforward relationship, providing a possible molecular mechanism of cell transformation by Myc. Developing therapeutics to inhibit eIF4F and/or Myc could be a potential treatment for a wide range of human cancers. [Cancer Res 2009;69(19):7491–4]

Metastamir: The Field of Metastasis-Regulatory microRNA Is Spreading

Hurst, D. R., Edmonds, M. D., Welch, D. R. — Thu, 01 Oct 2009 11:26:04 PDT

Despite advancements in knowledge from more than a century of metastasis research, the genetic programs and molecular mechanisms required for cancer metastasis are still incompletely understood. Genes that specifically regulate the process of metastasis are useful tools to elucidate molecular mechanisms and may become markers and/or targets for antimetastatic therapy. Recently, several noncoding regulatory RNA genes, microRNA (miRNA), were identified, which play roles in various steps of metastasis, some without obvious roles in tumorigenesis. Understanding how these metastasis-associated miRNA, which we term metastamir, are involved in metastasis will help identify possible biomarkers or targets for the most lethal attribute of cancer: metastasis. [Cancer Res 2009;69(19):7495–8]

Lessons from Applied Ecology: Cancer Control Using an Evolutionary Double Bind

Gatenby, R. A., Brown, J., Vincent, T. — Thu, 01 Oct 2009 11:26:04 PDT

Because the metastatic cascade is largely governed by the ability of malignant cells to adapt and proliferate at the distant tissue site, we propose that disseminated cancers are analogous in many important ways to the evolutionary and ecological dynamics of exotic species. Although pests can be decimated through the application of chemical toxins, this strategy virtually never achieves robust control as evolution of resistant phenotypes typically permits population recovery to pretreatment levels. In general, biological strategies that introduce predators, parasitoids, or pathogens have achieved more durable control of pest populations even after emergence of resistant phenotypes. From this we propose that long term outcome from any treatment strategy for invasive pests, including cancer, is not limited by evolution of resistance, but rather by the phenotypic cost of that resistance. If a cancerous cell's adaptation to therapy is achieved by upregulating xenobiotic metabolism or a redundant signaling pathway, the required investment in resources is small, and the original malignant phenotype remains essentially intact. As a result, the cancer cells' initial high level of fitness is little changed and unconstrained proliferation will resume once resistance evolves. Robust population control is possible if resistance to therapy requires a substantial and costly phenotypic adaptation that also significantly reduces the organism's fitness in its original niche: an evolutionary double bind. [Cancer Res 2009;69(19):7499–502]

Regulatory Myeloid Suppressor Cells in Health and Disease

Thu, 01 Oct 2009 11:26:04 PDT

Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission

Hirsch, H. A., Iliopoulos, D., Tsichlis, P. N., Struhl, K. — Thu, 01 Oct 2009 11:26:04 PDT

The cancer stem cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist chemotherapeutic drugs and can regenerate the various cell types in the tumor, thereby causing relapse of the disease. Thus, drugs that selectively target cancer stem cells offer great promise for cancer treatment, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug for diabetes, inhibits cellular transformation and selectively kills cancer stem cells in four genetically different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent, doxorubicin, kills both cancer stem cells and non–stem cancer cells in culture. Furthermore, this combinatorial therapy reduces tumor mass and prevents relapse much more effectively than either drug alone in a xenograft mouse model. Mice seem to remain tumor-free for at least 2 months after combinatorial therapy with metformin and doxorubicin is ended. These results provide further evidence supporting the cancer stem cell hypothesis, and they provide a rationale and experimental basis for using the combination of metformin and chemotherapeutic drugs to improve treatment of patients with breast (and possibly other) cancers. [Cancer Res 2009;69(19):7507–11]

Phosphorylation-Dependent Lys63-Linked Polyubiquitination of Daxx Is Essential for Sustained TNF-{alpha}-Induced ASK1 Activation

Thu, 01 Oct 2009 11:26:04 PDT

Apoptosis signal–regulating kinase 1 (ASK1) is a key regulatory kinase in the proapoptotic response to various stresses. ASK1 phosphorylation of Daxx, an ASK1 activator protein, increases Daxx accumulation in cells and further enhances ASK1 activity through a positive feedback mechanism. Here, we show that ASK1-dependent phosphorylation of Daxx induces Lys⁶³ (K63)-linked polyubiquitination on Lys¹²² of Daxx. Polyubiquitination is dispensable for Daxx accumulation or Daxx interaction with ASK1 because mutant Daxx deficient in polyubiquitin still exhibits ASK1-dependent accumulation and interaction with cellular ASK1. However, K63-linked Daxx polyubiquitination is required for tumor necrosis factor- (TNF-)–induced activation of ASK1. Therefore, K63-linked polyubiquitination of Daxx functions as a molecular switch to initiate and amplify the stress kinase response in the TNF- signaling pathway. [Cancer Res 2009;69(19):7512–7]

Rearrangements and Amplification of IER3 (IEX-1) Represent a Novel and Recurrent Molecular Abnormality in Myelodysplastic Syndromes

Thu, 01 Oct 2009 11:26:04 PDT

IER3 (formerly IEX-1) encodes a 27-kDa glycoprotein that regulates death receptor–induced apoptosis, interacts with NF-B pathways, and increases expression rapidly in response to cellular stresses such as irradiation. Animal models, gene expression microarray experiments, and functional studies in cell lines have suggested a potential role for IER3 in oncogenesis, but, to date, no abnormalities of IER3 at the DNA level have been reported in patients with neoplasia. Here, we describe breakpoint cloning of a t(6;9)(p21;q34) translocation from a patient with a myelodysplastic syndrome (MDS), facilitated by conversion technology and array-based comparative genomic hybridization, which revealed a rearrangement translocating the IER3 coding region away from critical flanking/regulatory elements and to a transcript-poor chromosomal region, markedly decreasing expression. Using split-signal and locus-specific fluorescence in situ hybridization (FISH) probes, we analyzed 204 patients with diverse hematological malignancies accompanied by clonal chromosome 6p21 abnormalities, and found 8 additional patients with MDS with IER3 rearrangements (translocations or amplification). Although FISH studies on 157 additional samples from patients with MDS and a normal-karyotype were unrevealing, and sequencing the IER3 coding and proximal promoter regions of 74 MDS patients disclosed no point mutations, reverse transcription-PCR results suggested that dysregulated expression of IER3 is common in MDS (61% >4-fold increase or decrease in expression with decreased expression primarily in early MDS and increased expression primarily in later MDS progressing toward leukemia), consistent with findings in previous microarray experiments. These data support involvement of IER3 in the pathobiology of MDS. [Cancer Res 2009;69(19):7518–23]

Contribution of Granulocyte Colony-Stimulating Factor to the Acute Mobilization of Endothelial Precursor Cells by Vascular Disrupting Agents

Thu, 01 Oct 2009 11:26:04 PDT

Vascular disrupting agents (VDA) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow–derived circulating endothelial precursor (CEP) cells could promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here, we show that following treatment of mice with OXi-4503, a second-generation potent prodrug derivative of combretastatin-A4 phosphate, rapid increases in circulating plasma vascular endothelial growth factor, stromal derived factor-1 (SDF-1), and granulocyte colony-stimulating factor (G-CSF) levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R^–/– mice were treated with OXi-4503. We found that as opposed to wild-type controls, G-CSF-R^–/– mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared with tumors treated in wild-type mice. Evidence for rapid elevations in circulating plasma G-CSF, vascular endothelial growth factor, and SDF-1 were also observed in patients with VDA (combretastatin-A4 phosphate)-treated cancer. These results highlight the possible effect of drug-induced G-CSF on tumor regrowth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target. [Cancer Res 2009;69(19):7524–8]

Pulmonary Vascular Destabilization in the Premetastatic Phase Facilitates Lung Metastasis

Huang, Y., Song, N., Ding, Y., Yuan, S., Li, X., Cai, H., Shi, H., Luo, Y. — Thu, 01 Oct 2009 11:26:04 PDT

Before metastasis, certain organs have already been influenced by primary tumors. However, the exact alterations and regulatory mechanisms of the premetastatic organs remain poorly understood. Here, we report that, in the premetastatic stage, angiopoietin 2 (Angpt2), matrix metalloproteinase (MMP) 3, and MMP10 are up-regulated in the lung by primary B16/F10 tumor, which leads to the increased permeability of pulmonary vasculatures and extravasation of circulating tumor cells. Subsequent studies show that Angpt2, MMP3, and MMP10 have a synergistic effect on disrupting vascular integrity in both in vitro and in vivo models. Lentivirus-based in vivo RNA interference of Angpt2, MMP3, and MMP10 attenuates the pulmonary vascular permeability and suppresses the infiltration of myeloid cells in the premetastatic lung. Moreover, knocking down these factors significantly inhibits the spontaneous lung metastasis in the model by orthotopic implantation of MDA-MB-231-Luc-D3H1 cells in nude mice. Further investigations reveal that the malignancy of tumor cells is positively correlated with their capabilities to induce the expression of Angpt2, MMP3, and MMP10. Luciferase reporter assay and chromatin immunoprecipitation assay also suggest that transforming growth factor-β1 and tumor necrosis factor- signaling are involved in the regulation of these premetastatic factors. Our study shows that pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of tumor cells and facilitates lung metastasis, which may provide potential targets for clinical prevention of metastasis. [Cancer Res 2009;69(19):7529–37]

Melanoma Proteoglycan Modifies Gene Expression to Stimulate Tumor Cell Motility, Growth, and Epithelial-to-Mesenchymal Transition

Thu, 01 Oct 2009 11:26:04 PDT

Melanoma chondroitin sulfate proteoglycan (MCSP) is a plasma membrane–associated proteoglycan that facilitates the growth, motility, and invasion of tumor cells. MCSP expression in melanoma cells enhances integrin function and constitutive activation of Erk1,2. The current studies were performed to determine the mechanism by which MCSP expression promotes tumor growth and motility. The results show that MCSP expression in radial growth phase, vertical growth phase, or metastatic cell lines causes sustained activation of Erk1,2, enhanced growth, and motility which all require the cytoplasmic domain of the MCSP core protein. MCSP expression in a radial growth phase cell line also promotes an epithelial-to-mesenchymal transition based on changes in cell morphology and the expression of several epithelial-to-mesenchymal transition markers. Finally, MCSP enhances the expression of c-Met and hepatocyte growth factor, and inhibiting c-Met expression or activation limits the increased growth and motility of multiple melanoma cell lines. The studies collectively show the importance of MCSP in promoting progression by an epigenetic mechanism and they indicate that MCSP could be targeted to delay or inhibit tumor progression in patients. [Cancer Res 2009;69(19):7538–47]

Epidermal Growth Factor Receptor Variant III-Induced Glioma Invasion Is Mediated through Myristoylated Alanine-Rich Protein Kinase C Substrate Overexpression

Thu, 01 Oct 2009 11:26:04 PDT

Glioblastoma multiforme (GBM) is the most common and most malignant adult brain tumor. A characteristic of GBM is their highly invasive nature, making complete surgical resection impossible. The most common gain-of-function alteration in GBM is amplification, overexpression, and mutations of the epidermal growth factor receptor (EGFR). The constitutively activated mutant EGFR variant III (EGFRvIII), found in ~20% of GBM, confers proliferative and invasive advantage. The signaling cascades downstream of aberrant EGFR activation contributing to the invasive phenotype are not completely understood. Here, we show myristoylated alanine-rich protein kinase C substrate (MARCKS), previously implicated in cell adhesion and motility, contributes to EGFR-mediated invasion of human GBM cells. EGFRvIII-expressing or EGF-stimulated human GBM cells increased expression, phosphorylation, and cytosolic translocation of MARCKS in a protein kinase C-–dependent manner. Down-regulation of MARCKS expression with small interfering RNA in GBM cells expressing EGFRvIII led to decreased cell adhesion, spreading, and invasion. Elucidation of mechanisms that promote EGFRvIII-mediated tumorigenesis in GBM, such as MARCKS, provides additional understanding and potential biological targets against this currently terminal human cancer. [Cancer Res 2009;69(19):7548–56]

Molecular Distinctions between Stasis and Telomere Attrition Senescence Barriers Shown by Long-term Culture of Normal Human Mammary Epithelial Cells

Thu, 01 Oct 2009 11:26:04 PDT

Normal human epithelial cells in culture have generally shown a limited proliferative potential of ~10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated β-galactosidase–positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage–associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC. [Cancer Res 2009;69(19):7557–68]

MicroRNA-34a Inhibits Glioblastoma Growth by Targeting Multiple Oncogenes

Thu, 01 Oct 2009 11:26:04 PDT

MicroRNA-34a (miR-34a) is a transcriptional target of p53 that is down-regulated in some cancer cell lines. We studied the expression, targets, and functional effects of miR-34a in brain tumor cells and human gliomas. Transfection of miR-34a down-regulated c-Met in human glioma and medulloblastoma cells and Notch-1, Notch-2, and CDK6 protein expressions in glioma cells. miR-34a expression inhibited c-Met reporter activities in glioma and medulloblastoma cells and Notch-1 and Notch-2 3'-untranslated region reporter activities in glioma cells and stem cells. Analysis of human specimens showed that miR-34a expression is down-regulated in glioblastoma tissues as compared with normal brain and in mutant p53 gliomas as compared with wild-type p53 gliomas. miR-34a levels in human gliomas inversely correlated to c-Met levels measured in the same tumors. Transient transfection of miR-34a into glioma and medulloblastoma cell lines strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion, but transfection of miR-34a into human astrocytes did not affect cell survival and cell cycle status. Forced expression of c-Met or Notch-1/Notch-2 transcripts lacking the 3'-untranslated region sequences partially reversed the effects of miR-34a on cell cycle arrest and cell death in glioma cells and stem cells, respectively. Also, transient expression of miR-34a in glioblastoma cells strongly inhibited in vivo glioma xenograft growth. Together, these findings represent the first comprehensive analysis of the role of miR-34a in gliomas. They show that miR-34a suppresses brain tumor growth by targeting c-Met and Notch. The results also suggest that miR-34a could serve as a potential therapeutic agent for brain tumors. [Cancer Res 2009;69(19):7569–76]

BCL9 Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic, and Angiogenic Properties to Cancer Cells

Thu, 01 Oct 2009 11:26:04 PDT

Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances β-catenin–mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling. [Cancer Res 2009;69(19):7577–86]

Targeted Disruption of Stat3 Reveals a Major Role for Follicular Stem Cells in Skin Tumor Initiation

Kim, D. J., Kataoka, K., Rao, D., Kiguchi, K., Cotsarelis, G., DiGiovanni, J. — Thu, 01 Oct 2009 11:26:04 PDT

The initiation stage of mouse skin carcinogenesis involves the induction of mutations in keratinocyte stem cells (KSC), which confers a selective growth advantage allowing clonal expansion during tumor promotion. Targeted disruption of signal transducer and activator of transcription 3 (Stat3) in bulge region KSCs was achieved by treating K15.CrePR1 x Stat3^fl/fl mice with RU486. Deletion of Stat3 prior to skin tumor initiation with 7,12-dimethylbenz(a)anthracene significantly increased the number of apoptotic KSCs and decreased the frequency of Ha-ras codon 61 A¹⁸²->T transversion mutations in this cell population compared with wild-type littermates. Targeted disruption of Stat3 in bulge region KSCs at the time of initiation also dramatically reduced the number of skin tumors (by ~80%) produced following promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. These results show that Stat3 is required for the survival of bulge region KSCs during tumor initiation. Furthermore, these data provide direct evidence that bulge region KSCs are the primary targets for the initiation of skin tumors in this model system. [Cancer Res 2009;69(19):7587–94]

Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-{beta}-Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4

Caja, L., Sancho, P., Bertran, E., Iglesias-Serret, D., Gil, J., Fabregat, I. — Thu, 01 Oct 2009 11:26:04 PDT

Transforming growth factor-β (TGF-β) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-β in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-β–induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-β–induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-β, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-β and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-β–induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis. [Cancer Res 2009;69(19):7595–602]

Atypical Protein Kinase C{iota} Is Required for Bronchioalveolar Stem Cell Expansion and Lung Tumorigenesis

Regala, R. P., Davis, R. K., Kunz, A., Khoor, A., Leitges, M., Fields, A. P. — Thu, 01 Oct 2009 11:26:04 PDT

Protein kinase C (PKC) is an oncogene required for maintenance of the transformed phenotype of non–small cell lung cancer cells. However, the role of PKC in lung tumor development has not been investigated. To address this question, we established a mouse model in which oncogenic Kras^G12D is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKC gene, Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore, the small molecule PKC inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus, Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore, aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKC as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. [Cancer Res 2009;69(19):7603–11]

Correction of the Abnormal Trafficking of Primary Myelofibrosis CD34+ Cells by Treatment with Chromatin-Modifying Agents

Wang, X., Zhang, W., Ishii, T., Sozer, S., Wang, J., Xu, M., Hoffman, R. — Thu, 01 Oct 2009 11:26:04 PDT

The abnormal trafficking of CD34⁺ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34⁺ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor–mobilized peripheral blood (mPB) CD34⁺ cells into NOD/SCID mice, reduced numbers of PMF CD34⁺ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice, whereas similar numbers of PMF and mPB CD34⁺ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34⁺ cells was associated with reduced expression of CXCR4, but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34⁺ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), but not treatment with small molecule inhibitors of JAK2, resulted in the generation of increased numbers of CD34⁺CXCR4⁺ cells, which was accompanied by enhanced homing of PMF CD34⁺ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment, JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34⁺ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents. [Cancer Res 2009;69(19):7612–8]

A Role for the Clock Gene Per1 in Prostate Cancer

Cao, Q., Gery, S., Dashti, A., Yin, D., Zhou, Y., Gu, J., Koeffler, H. P. — Thu, 01 Oct 2009 11:26:04 PDT

Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbations of these rhythms are associated with pathogenic conditions, such as depression, diabetes, and cancer. Androgens play an important role in both normal development and carcinogenesis of the prostate. In the present study, we investigated a potential role for the core clock factor Per1 in the pathogenesis of prostate cancer. Serum-shocked synchronized prostate cancer cells displayed disrupted circadian rhythms compared with the normal prostate tissue. Using Oncomine to perform a meta-analysis of microarray expression studies, we found that Per1 is down-regulated in human prostate cancer samples compared with normal prostates. Reporter assays showed that Per1 inhibited transactivation of the androgen receptor (AR) both in 293T cells overexpressing the AR and in the prostate cancer cell line LNCaP. Forced expression of Per1 in LNCaP cells diminished the expression of known androgen-sensitive genes following stimulation with dihydrotestosterone. We showed that Per1 physically interacted with AR; in addition, we found that Per1 itself is regulated by androgens in prostate cancer cells. Overexpression of Per1 in prostate cancer cells resulted in significant growth inhibition and apoptosis. Our results support the emerging role of circadian genes as key players in malignant transformation. Further elucidating the connections between clock genes and the AR pathway could benefit the development of new therapeutic strategies for prostate cancer as well as provide insights into chronotherapy as a way to optimize current therapies. [Cancer Res 2009;69(19):7619–25]

Nitrosative Stress-Induced S-Glutathionylation of Protein Disulfide Isomerase Leads to Activation of the Unfolded Protein Response

Thu, 01 Oct 2009 11:26:04 PDT

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively called the unfolded protein response (UPR). Protein disulfide isomerase (PDI) is one of the most abundant ER proteins and maintains a sentinel function in organizing accurate protein folding. Treatment of cells with O²-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) resulted in a dose-dependent increase in intracellular nitric oxide that caused S-glutathionylation of various proteins. Within 4 h, PABA/NO activated the UPR and led to translational attenuation as measured by the phosphorylation and activation of the ER transmembrane kinase, pancreatic ER kinase, and its downstream effector eukaryotic initiation factor 2 in human leukemia (HL60) and ovarian cancer cells (SKOV3). Cleavage of the transcription factor X-box protein 1 and transcriptional activation of the ER resident proteins BiP, PDI, GRP94, and ERO1 (5- to 10-fold induction) also occurred. Immunoprecipitation of PDI showed that whereas nitrosylation was undetectable, PABA/NO treatment caused S-glutathionylation of PDI. Mass spectroscopy analysis showed that single cysteine residues within each of the catalytic sites of PDI had a mass increase [+305.3 Da] consistent with S-glutathionylation. Circular dichroism confirmed that S-glutathionylation of PDI results in alterations in the -helix content of PDI and is concurrent with inhibition of its isomerase activity. Thus, it appears that S-glutathionylation of PDI is an upstream signaling event in the UPR and may be linked with the cytotoxic potential of PABA/NO. [Cancer Res 2009;69(19):7626–34]

Expression of Insulin Receptor Isoform A and Insulin-like Growth Factor-1 Receptor in Human Acute Myelogenous Leukemia: Effect of the Dual-Receptor Inhibitor BMS-536924 In vitro

Thu, 01 Oct 2009 11:26:04 PDT

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are receptor tyrosine kinases that participate in mitogenic and antiapoptotic signaling in normal and neoplastic epithelia. In the present study, immunoblotting and reverse transcription-PCR demonstrated expression of IGF1R and IR isoform A in acute myelogenous leukemia (AML) cell lines as well as in >80% of clinical AML isolates. Treatment with insulin enhanced signaling through the Akt and MEK1/2 pathways as well as survival of serum-starved AML cell lines. Conversely, treatment with BMS-536924, a dual IGF1R/IR kinase inhibitor that is undergoing preclinical testing, inhibited constitutive receptor phosphorylation as well as downstream signaling through MEK1/2 and Akt. These changes inhibited proliferation and, in some AML cell lines, induced apoptosis at submicromolar concentrations. Likewise, BMS-536924 inhibited leukemic colony formation in CD34⁺ clinical AML samples in vitro. Collectively, these results not only indicate that expression of IGF1R and IR isoform A is common in AML but also show that interruption of signaling from these receptors inhibits proliferation in clinical AML isolates. Accordingly, further investigation of IGF1R/IR axis as a potential therapeutic target in AML appears warranted. [Cancer Res 2009;69(19):7635–43]

Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Blockade Is an Effective Radiosensitizing Strategy for the Treatment of Non-Small Cell Lung Cancer Harboring K-RAS Mutations

Thu, 01 Oct 2009 11:26:04 PDT

Non–small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC often harbors oncogenic K-RAS mutations that lead to the aberrant activation of several intracellular networks including the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Oncogenic K-RAS predicts poor prognosis and resistance to treatment with ionizing radiation (IR). Oncogenic K-Ras expression in the respiratory epithelium is sufficient to initiate NSCLC tumorigenesis, which requires the catalytic subunit of PI3K. Thus, effective inhibition of the PI3K signaling should lead to significant antitumor effects. However, therapy with rapamycin analogues has yielded disappointing results due in part to compensatory up-regulation of AKT. We hypothesized that dual PI3K/mTOR blockade would overcome these limitations. We tested this hypothesis with BEZ235, a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. We found that BEZ235 induces a striking antiproliferative effect both in transgenic mice with oncogenic K-RAS–induced NSCLC and in NSCLC cell lines expressing oncogenic K-RAS. We determined that treatment with BEZ235 was not sufficient to induce apoptosis. However, we found that dual PI3K/mTOR blockade effectively sensitizes NSCLC expressing oncogenic K-RAS to the proapoptotic effects of IR both in vitro and in vivo. We conclude that dual PI3K/mTOR blockade in combination with IR may benefit patients with NSCLC expressing oncogenic K-RAS. These findings may have general applicability in cancer therapy, because aberrant activation of PI3K occurs frequently in human cancer. [Cancer Res 2009;69(19):7644–52]

Anticancer Activity of CX-3543: A Direct Inhibitor of rRNA Biogenesis

Thu, 01 Oct 2009 11:26:05 PDT

Hallmark deregulated signaling in cancer cells drives excessive ribosome biogenesis within the nucleolus, which elicits unbridled cell growth and proliferation. The rate-limiting step of ribosome biogenesis is synthesis of rRNA (building blocks of ribosomes) by RNA Polymerase I (Pol I). Numerous kinase pathways and products of proto-oncogenes can up-regulate Pol I, whereas tumor suppressor proteins can inhibit rRNA synthesis. In tumorigenesis, activating mutations in certain cancer-associated kinases and loss-of-function mutations in tumor suppressors lead to deregulated signaling that stimulates Pol I transcription with resultant increases in ribosome biogenesis, protein synthesis, cell growth, and proliferation. Certain anticancer therapeutics, such as cisplatin and 5-fluorouracil, reportedly exert, at least partially, their activity through disruption of ribosome biogenesis, yet many prime targets for anticancer drugs within the ribosome synthetic machinery of the nucleolus remain largely unexploited. Herein, we describe CX-3543, a small molecule nucleolus-targeting agent that selectively disrupts nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting Pol I transcription and inducing apoptosis in cancer cells. CX-3543 is the first G-quadruplex interactive agent to enter human clinical trials, and it is currently under evaluation against carcinoid/neuroendocrine tumors in a phase II clinical trial. [Cancer Res 2009;69(19):7653–61]

The Insulin-like Growth Factor-1 Receptor-Targeting Antibody, CP-751,871, Suppresses Tumor-Derived VEGF and Synergizes with Rapamycin in Models of Childhood Sarcoma

Thu, 01 Oct 2009 11:26:05 PDT

Signaling through the type 1 insulin-like growth factor receptor (IGF-1R) occurs in many human cancers, including childhood sarcomas. As a consequence, targeting the IGF-1R has become a focus for cancer drug development. We examined the antitumor activity of CP-751,871, a human antibody that blocks IGF-1R ligand binding, alone and in combination with rapamycin against sarcoma cell lines in vitro and xenograft models in vivo. In Ewing sarcoma (EWS) cell lines, CP751,871 inhibited growth poorly (<50%), but prevented rapamycin-induced hyperphosphorylation of AKT(Ser473) and induced greater than additive apoptosis. Rapamycin treatment also increased secretion of IGF-1 resulting in phosphorylation of IGF-1R (Tyr1131) that was blocked by CP751,871. In vivo CP-751,871, rapamycin, or the combination were evaluated against EWS, osteosarcoma, and rhabdomyosarcoma xenografts. CP751871 induced significant growth inhibition [EFS(T/C) >2] in four models. Rapamycin induced significant growth inhibition [EFS(T/C) >2] in nine models. Although neither agent given alone caused tumor regressions, in combination, these agents had greater than additive activity against 5 of 13 xenografts and induced complete remissions in one model each of rhabdomyosarcoma and EWS, and in three of four osteosarcoma models. CP751,871 caused complete IGF-1R down-regulation, suppression of AKT phosphorylation, and dramatically suppressed tumor-derived vascular endothelial growth factor (VEGF) in some sarcoma xenografts. Rapamycin treatment did not markedly suppress VEGF in tumors and synergized only in tumor lines where VEGF was dramatically inhibited by CP751,871. These data suggest a model in which blockade of IGF-1R suppresses tumor-derived VEGF to a level where rapamycin can effectively suppress the response in vascular endothelial cells. [Cancer Res 2009;69(19):7662–71]

Preclinical Profile of a Potent {gamma}-Secretase Inhibitor Targeting Notch Signaling with In vivo Efficacy and Pharmacodynamic Properties

Thu, 01 Oct 2009 11:26:05 PDT

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of -secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC₅₀ in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way. [Cancer Res 2009;69(19):7672–80]

Mutant p53 Disrupts the Stress MAPK Activation Circuit Induced by ASK1-Dependent Stabilization of Daxx

Thu, 01 Oct 2009 11:26:05 PDT

Daxx is a regulatory protein for apoptosis signal–regulating kinase 1 (ASK1) which activates c-Jun NH₂-terminal kinase (JNK) and p38 pathways in response to stressors such as tumor necrosis factor- (TNF). Here, we show that TNF treatment induces the accumulation of Daxx protein through ASK1 activation by preventing its proteasome-dependent degradation. ASK1 directly phosphorylates Daxx at Ser¹⁷⁶ and Ser¹⁸⁴ and Daxx is required for the sustained activation of JNK. Tumorigenic mutant p53, which binds to Daxx and inhibits Daxx-dependent activation of ASK1, prevents Daxx phosphorylation and stabilization. When mutant p53 was depleted in cancer cells, Daxx was accumulated and the cell-killing effect of TNF was restored. Our results indicate that Daxx not only activates ASK1 but also is a downstream target of ASK1 and that accumulated Daxx further activates ASK1. Thus, the Daxx-ASK1 positive feedback loop amplifying JNK/p38 signaling plays an important role in the cell-killing effects of stressors, such as TNF. Tumorigenic mutant p53 disrupts this circuit and makes cells more tolerable to stresses, as its gain-of-function mechanism. [Cancer Res 2009;69(19):7681–8]

A Small Molecule Polyamine Oxidase Inhibitor Blocks Androgen-Induced Oxidative Stress and Delays Prostate Cancer Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

Thu, 01 Oct 2009 11:26:05 PDT

High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiologic factor in prostate cancer (CaP) occurrence, recurrence, and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgen-induced polyamine oxidation that produces H₂O₂ could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N'-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells, as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data show that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays CaP progression. [Cancer Res 2009;69(19):7689–95]

Identification of Candidate Biomarkers of Therapeutic Response to Docetaxel by Proteomic Profiling

Thu, 01 Oct 2009 11:26:05 PDT

Docetaxel chemotherapy improves symptoms and survival in men with metastatic hormone-refractory prostate cancer (HRPC). However, ~50% of patients do not respond to Docetaxel and are exposed to significant toxicity without direct benefit. This study aimed to identify novel therapeutic targets and predictive biomarkers of Docetaxel resistance in HRPC. We used iTRAQ-mass spectrometry analysis to identify proteins associated with the development of Docetaxel resistance using Docetaxel-sensitive PC3 cells and Docetaxel-resistant PC3-Rx cells developed by Docetaxel dose escalation. Functional validation experiments were performed using recombinant protein treatment and siRNA knockdown experiments. Serum/plasma levels of the targets in patient samples were measured by ELISA. The IC₅₀ for Docetaxel in the PC3-Rx cells was 13-fold greater than the parent PC-3 cell line (P = 0.004). Protein profiling identified MIC-1 and AGR2 as respectively up-regulated and down-regulated in Docetaxel-resistant cells. PC-3 cells treated with recombinant MIC-1 also became resistant to Docetaxel (P = 0.03). Conversely, treating PC3-Rx cells with MIC-1 siRNA restored sensitivity to Docetaxel (P = 0.02). Knockdown of AGR2 expression in PC3 cells resulted in Docetaxel resistance (P = 0.007). Furthermore, increased serum/plasma levels of MIC-1 after cycle one of chemotherapy were associated with progression of the cancer (P = 0.006) and shorter survival after treatment (P = 0.002). These results suggest that both AGR2 and MIC-1 play a role in Docetaxel resistance in HRPC. In addition, an increase in serum/plasma MIC-1 level after cycle one of Docetaxel may be an indication to abandon further treatment. Further investigation of MIC-1 as a biomarker and therapeutic target for Docetaxel resistance in HRPC is warranted. [Cancer Res 2009;69(19):7696–7703]

c-Jun Protects Hypoxia-Inducible Factor-1{alpha} from Degradation via Its Oxygen-Dependent Degradation Domain in a Nontranscriptional Manner

Yu, B., Miao, Z.-H., Jiang, Y., Li, M.-H., Yang, N., Li, T., Ding, J. — Thu, 01 Oct 2009 11:26:05 PDT

Although hypoxia-inducible factor-1 (HIF-1) has long been intensively investigated as a drug target by interfering with its expression or transcriptional function, the regulatory mechanisms of HIF-1 remain to be further clarified. We report here that c-Jun associates with HIF-1 via its oxygen-dependent degradation domain, masks the sites for ubiquitination, and thus protects HIF-1 from proteasome-executing degradation. All of these together resulted in the stabilization and accumulation of HIF-1, consequently promoting the transcription of its target gene and driving angiogenesis-related events. The stabilization of HIF-1 was dependent on the domains of c-Jun for DNA binding and heterodimerization but independent of the Ser^63/73 phosphorylation that is critical for transcriptional function. These findings highlight a previously unrecognized nontranscriptional function of c-Jun on the one hand and a distinct regulatory mechanism of HIF-1 activity on the other, consequently offering profound mechanistic insights into multiple events simultaneously involving both c-Jun and HIF-1 in tumor progression. [Cancer Res 2009;69(19):7704–12]

Expression of IFN-{beta} Enhances Both Efficacy and Safety of Oncolytic Vesicular Stomatitis Virus for Therapy of Mesothelioma

Thu, 01 Oct 2009 11:26:05 PDT

Our preclinical and clinical trials using a replication-defective adenoviral vector expressing IFN-β have shown promising results for the treatment of malignant mesothelioma. Based on the hypotheses that a replication-competent vesicular stomatitis virus (VSV) oncolytic vector would transduce more tumor cells in vivo, that coexpression of the immunostimulatory IFN-β gene would enhance the immune-based effector mechanisms associated both with regression of mesotheliomas and with VSV-mediated virotherapy, and that virus-derived IFN-β would add further safety to the VSV platform, we tested the use of IFN-β as a therapeutic transgene expressed from VSV as a novel treatment for mesothelioma. VSV-IFN-β showed significant therapy against AB12 murine mesotheliomas in the context of both local and locoregional viral delivery. Biologically active IFN-β expressed from VSV added significantly to therapy compared with VSV alone, dependent in part on host CD8⁺ T-cell responses. Immune monitoring suggested that these antitumor T-cell responses may be due to a generalized T-cell activation rather than the priming of tumor antigen–specific T-cell responses. Finally, IFN-β also added considerable extra safety to the virus by providing protection from off-target viral replication in nontumor tissues and protected severe combined immunodeficient mice from developing lethal neurotoxicity. The enhanced therapeutic index provided by the addition of IFN-β to VSV therefore provides a powerful justification for the development of this virus for future clinical trials. [Cancer Res 2009;69(19):7713–20]

[18F]Fluoromethyl-[1,2-2H4]-Choline: A Novel Radiotracer for Imaging Choline Metabolism in Tumors by Positron Emission Tomography

Thu, 01 Oct 2009 11:26:05 PDT

Current radiotracers for positron emission tomography imaging of choline metabolism have poor systemic metabolic stability in vivo. We describe a novel radiotracer, [¹⁸F]fluoromethyl-[1,2-²H₄]-choline (D4-FCH), that employs deuterium isotope effect to improve metabolic stability. D4-FCH proved more resistant to oxidation than its nondeuterated analogue, [¹⁸F]fluoromethylcholine, in plasma, kidneys, liver, and tumor, while retaining phosphorylation potential. Tumor radiotracer levels, a determinant of sensitivity in imaging studies, were improved by deuterium substitution; tumor uptake values expressed as percent injected dose per voxel at 60 min were 7.43 ± 0.47 and 5.50 ± 0.49 for D4-FCH and [¹⁸F]fluoromethylcholine, respectively (P = 0.04). D4-FCH was also found to be a useful response biomarker. Treatment with the mitogenic extracellular kinase inhibitor PD0325901 resulted in a reduction in tumor radiotracer uptake that occurred in parallel with reductions in choline kinase A expression. In conclusion, D4-FCH is a very promising metabolically stable radiotracer for imaging choline metabolism in tumors. [Cancer Res 2009;69(19):7721–8]

Down-regulation of c-FLIP Enhances Death of Cancer Cells by Smac Mimetic Compound

Cheung, H. H., Mahoney, D. J., LaCasse, E. C., Korneluk, R. G. — Thu, 01 Oct 2009 11:26:05 PDT

Smac mimetic compounds (SMC) are novel small molecules being developed for cancer therapy. The mechanism of SMC-induced sensitivity in cancer cells depends on autocrine release of tumor necrosis factor (TNF); however, potential mechanisms of resistance remain unknown. Here, we investigated the molecular profile and cytotoxic responsiveness of a diverse panel of 51 cancer cell lines to combinations of a dimeric SMC (AEG40730), death ligand TNF, and tumor necrosis factor-related apoptosis-inducing ligand. Synergy was seen in combination with death receptor agonists in some cells, although single-agent activity was limited to a fewsensitive lines. Unexpectedly, the majority of cell lines resistant to combinations of SMC-AEG40730 and death ligands expressed caspase-8, FADD, RIP1, and ligand receptors necessary for apoptosis execution. Furthermore, TNF-mediated ubiquitination of RIP1 was repressed by SMC-AEG40730 treatment, leading to the formation of the proapoptosis complex II. However, in resistant cancer cells, SMC-AEG40730 repressed TNF-mediated c-jun-NH₂-kinase activation and the levels of caspase-8 inhibitor c-FLIP were persistently elevated, in contrast to SMC-responsive cancer cells. Importantly, the silencing of c-FLIP restored SMC sensitivity in previously resistant cancer cells by allowing ligand-mediated activation of caspase-8 and caspase-3 to proceed. Together, these results provide mechanistic insight into the action of SMCs, demonstrating that the deciphering of the relevant molecular signature in cancer cells leads to the prediction of cancer cell responsiveness to SMC treatment. Furthermore, a majority of resistant cancer cells were sensitized to SMC-AEG40730 and TNF by down-regulating c-FLIP, suggesting novel approaches in the use of SMCs and c-FLIP antagonists in treating cancer. [Cancer Res 2009;69(19):7729–38]

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Thu, 01 Oct 2009 11:26:05 PDT

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5'-termini and dephosphorylates DNA 3'-termini. hPNKP is involved in both single- and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to -radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC₅₀ of 0.06 µmol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3'-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 µmol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs. [Cancer Res 2009;69(19):7739–46]

Tumor Vaccines Expressing Flt3 Ligand Synergize with CTLA-4 Blockade to Reject Preimplanted Tumors

Curran, M. A., Allison, J. P. — Thu, 01 Oct 2009 11:26:05 PDT

The transformation of a healthy cell into a malignant neoplasm involves numerous genetic mutations and aberrations in gene expression. As few of these changes are shared between individuals or types of cancer, the best source for eliciting broad-spectrum tumor immunity remains each patient's own tumor. Previously, we have shown that combining blockade of the T-cell–negative costimulatory molecule CTL-associated antigen 4 (CTLA-4) and vaccination with irradiated B16 tumor expressing granulocyte macrophage colony-stimulating factor (GM-CSF; Gvax) promotes rejection of established murine melanomas. Here we show that, like GM-CSF, the cytokine Flt3 ligand (Flt3L) expressed in B16 and coupled with CTLA-4 blockade promotes both prophylactic and therapeutic rejection of B16. When administered at the site of growing tumor, Gvax fails to prevent tumor outgrowth in any mice, whereas the B16-Flt3L vaccine (Fl3vax) induces the rejection of 75% of melanomas implanted 3 days before vaccination. Relative to Gvax, Fl3vax promotes greater infiltration of both the vaccine site and the tumor site by CD8⁺ T cells and "sentinel" and plasmacytoid dendritic cells. Gvax and Fl3vax did not synergize when used in combination in treating B16 melanoma even in the context of CD25⁺ regulatory T-cell depletion. Further, we show that a combination of Flt3L expression and CTLA-4 blockade can also promote the rejection of established TRAMP prostate adenocarcinomas, proving that the utility of this treatment extends beyond melanoma. Engineering Flt3L to be constitutively secreted and attaching an IgG2a tail yielded a B16 vaccine that, when combined with CTLA-4 blockade, prevented the outgrowth of significantly more 5-day implanted B16-BL6 tumors than did Gvax. [Cancer Res 2009;69(19):7747–55]

Tumor Apoptotic Bodies Inhibit CTL Responses and Antitumor Immunity via Membrane-Bound Transforming Growth Factor-{beta}1 Inducing CD8+ T-Cell Anergy and CD4+ Tr1 Cell Responses

Thu, 01 Oct 2009 11:26:05 PDT

Tumor cell apoptosis induced by radiation therapy results in apoptotic tumor cells and apparition of membrane blebs termed apoptotic bodies (APB). The immune responses induced by apoptotic tumor cells have been extensively studied. However, the role of APB in modulation of tumor immune responses is elusive. In this study, we induced apoptosis in 90% ovabumin-expressing EG7 tumor cells by in vitro irradiation (9,000 rad) of tumor cells with a subsequent cell culture for 9 hours. APB purified from irradiation-induced apoptotic EG7 cell culture supernatant by differential ultracentrifugation were vesicles with 50 to 90 nm in diameter and expressed apoptosis-specific Annexin V, 14-3-3, and Histone H3. We then investigated its potential modulation in DC_OVA–induced T-cell responses and antitumor immunity. We found that EG7-derived APB were tolerogenic and capable of suppressing DC_OVA-stimulated CD8⁺ CTL responses and antitumor immunity via its induction of CD8⁺ T-cell anergy and type 1 regulatory CD4⁺ T-cell responses. Analysis of apoptotic tumor cells and APB revealed the expression of membrane-bound transforming growth factor (TGF)-β1 associated with irradiation-induced apoptosis formation, which is a result from activation of transcriptional factor NF-AT specific for TGF-β1 promoters. Our data further elucidate that it is the membrane-bound TGF-β1 expression on APB that contributes to its in vitro antiproliferative effect as shown by using neutralizing TGF-β1–specific antibody. Administration of anti–TGF-β1 antibody in vivo also blocked APB-mediated immune suppression of CD8⁺ CTL responses and antitumor immunity. Therefore, our study may have great impact in designing a combined radiation therapy with immunotherapy of cancer. [Cancer Res 2009;69(19):7756–66]

Antitumor Immunity Can Be Uncoupled from Autoimmunity following Heat Shock Protein 70-Mediated Inflammatory Killing of Normal Pancreas

Thu, 01 Oct 2009 11:26:05 PDT

We have a long-term interest in the connectivity between autoimmunity and tumor rejection. However, outside of the melanocyte/melanoma paradigm, little is known about whether autoimmune responses to normal tissue can induce rejection of tumors of the same histologic type. Here, we induced direct, pathogen-like cytotoxicity to the normal pancreas in association with the immune adjuvant heat shock protein 70. In sharp contrast to our studies with a similar approach for the treatment of prostate cancer, inflammatory killing of the normal pancreas induced a Th1-like, anti-self-response to pancreatic antigens, which was rapidly suppressed by a concomitant suppressive regulatory T cell (Treg) response. Interestingly, even when Treg cells were depleted, the Th1-like response was insufficient to induce significant ongoing autoimmunity. However, the Th1-like response to antigens expressed in the pancreas at the time of damage was sufficient to induce rejection of tumors expressing either a foreign (ova) antigen or fully syngeneic tumor antigens (on Panc02 tumor cells), provided that Treg were depleted before inflammatory killing of the normal pancreas. Taken together, these data indicate that profound differences exist between the immunoprotective mechanisms in place between different tissues (pancreas and prostate) in their response to pathogen-like damage. Moreover, they also show that, although multiple layers of immunologic safeguards are in place to prevent the development of severe autoimmune consequences in the pancreas (in contrast to the prostate), tumor rejection responses can still be decoupled from pathologic autoimmune responses in vivo, which may provide novel insights into the immunotherapeutic treatment of pancreatic cancer. [Cancer Res 2009;69(19):7767–74]

Platelet-Derived Transforming Growth Factor-{beta} Down-Regulates NKG2D Thereby Inhibiting Natural Killer Cell Antitumor Reactivity

Kopp, H.-G., Placke, T., Salih, H. R. — Thu, 01 Oct 2009 11:26:05 PDT

Natural killer (NK) cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines, which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells such as dendritic cells. In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion, suggesting that platelets are an important additional player in NK cell-tumor interaction. Moreover, it has been shown that metastasizing tumor cells do not travel through the blood alone but are rapidly coated by platelets. However, the knowledge about the molecular mechanisms by which platelets influence NK cells is fragmentary at best. Here we show that platelet-derived soluble factors, secreted on coating of tumor cells or after stimulation with classic platelet agonists, impair NK cell antitumor reactivity resulting in diminished granule mobilization, cytotoxicity, and IFN- production. The impaired NK cell reactivity was not due to induction of apoptosis but mediated by down-regulation of the activating immunoreceptor natural killer group 2, member D (NKG2D) on NK cells by platelet-derived transforming growth factor β (TGF-β). Neutralization of TGF-β in platelet releasate not only prevented NKG2D down-regulation but also restored NK cell antitumor reactivity. Thus, our data elucidate the molecular basis of the previously described influence of platelets on NK cell antitumor reactivity and suggest that therapeutic intervention in tumor cell-platelet interaction and the resulting TGF-β release by platelets may serve to enhance antitumor immunity. [Cancer Res 2009;69(19):7775–83]

Design of Agonistic Altered Peptides for the Robust Induction of CTL Directed towards H-2Db in Complex with the Melanoma-Associated Epitope gp100

Thu, 01 Oct 2009 11:26:05 PDT

Immunogenicity of tumor-associated antigens (TAA) is often weak because many TAA are autoantigens for which the T-cell repertoire is sculpted by tolerance mechanisms. Substitutions at main anchor positions to increase the complementarity between the peptide and the MHC class I (MHC-I) binding cleft constitute a common procedure to improve binding capacity and immunogenicity of TAA. However, such alterations are tailored for each MHC-I allele and may recruit different CTL specificities through conformational changes in the targeted peptides. Comparative analysis of substituted melanoma-differentiation antigen gp100 in complex with H-2D^b revealed that combined introduction of glycine and proline residues at the nonanchor positions 2 and 3, respectively, resulted in an agonistic altered peptide with dramatically enhanced binding affinity, stability, and immunogenicity of this TAA. Peptide vaccination using the p2Gp3P-altered peptide version of gp100 induced high frequencies of melanoma-specific CTL in the endogenous CD8⁺ repertoire. Crystal structure analysis of MHC/peptide complexes revealed that the conformation of the modified p2Gp3P-peptide was similar to the wild-type peptide, and indicated that this mimotope was stabilized through interactions between peptide residue p3P and the tyrosine residue Y159 that is conserved among most known MHC-I molecules throughout mammalian species. Our results may provide an alternative approach to enhance MHC stabilization capacity and immunogenicity of low-affinity peptides for induction of robust tumor-specific CTL. [Cancer Res 2009;69(19):7784–92]

Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer

Thu, 01 Oct 2009 11:26:05 PDT

Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an overrepresentation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH, we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome 21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer. [Cancer Res 2009;69(19):7793–802]

MYC Activity Mitigates Response to Rapamycin in Prostate Cancer through Eukaryotic Initiation Factor 4E-Binding Protein 1-Mediated Inhibition of Autophagy

Thu, 01 Oct 2009 11:26:05 PDT

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E–binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy. [Cancer Res 2009;69(19):7803–10]

An A13 Repeat within the 3'-Untranslated Region of Epidermal Growth Factor Receptor (EGFR) Is Frequently Mutated in Microsatellite Instability Colon Cancers and Is Associated with Increased EGFR Expression

Thu, 01 Oct 2009 11:26:05 PDT

Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3'-untranslated region (3'-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3'-UTR mutant MSI cell lines. Cell lines with an EGFR 3'-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3'-UTR mutations were more sensitive to EGFR inhibition than EGFR 3'-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors. [Cancer Res 2009;69(19):7811–8]

Role of SV40 Integration Site at Chromosomal Interval 1q21.1 in Immortalized CRL2504 Cells

Thu, 01 Oct 2009 11:26:05 PDT

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by β-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies. [Cancer Res 2009;69(19):7819–25]

Immutable Functional Attributes of Histologic Grade Revealed by Context-Independent Gene Expression in Primary Breast Cancer Cells

Thu, 01 Oct 2009 11:26:05 PDT

Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a "paracrine-independent expression of grade-associated" (PEGA) gene signature. PEGA members extended beyond "proliferation-driven" signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed. [Cancer Res 2009;69(19):7826–34]

Extracellular Signal-Regulated Kinase Positively Regulates the Oncogenic Activity of MCT-1 in Diffuse Large B-Cell Lymphoma

Thu, 01 Oct 2009 11:26:05 PDT

The MCT-1 oncogene was originally identified from lymphoma cell lines. Herein we establish that MCT-1 is highly expressed in 85% of human diffuse large B-cell lymphomas (DLBCL) and that knocking down MCT-1 by a specific short hairpin RNA in DLBCL cells induces apoptosis, supporting a critical role for MCT-1 in DLBCL cell survival. However, the mechanism underlying MCT-1 regulation is largely unknown. We find that MCT-1 is phosphorylated and up-regulated by extracellular signal-regulated kinase (ERK). Furthermore, by using a small inhibitory molecule targeting ERK, we interrupted MCT-1 phosphorylation and stability. Significantly, cells with distinct levels of MCT-1 protein displayed differential sensitivity to ERK inhibitor–induced apoptosis. Treatment with the ERK inhibitor showed marked in vivo antitumor activity in a human DLBCL xenograft model. Our findings establish a functional molecular interaction between MCT-1 and the MEK/ERK signaling pathway and suggest that the activation of MCT-1 function by its upstream kinase ERK plays an important role in lymphomagenesis. [Cancer Res 2009;69(19):7835–43]

Haplotype and Cell Proliferation Analyses of Candidate Lung Cancer Susceptibility Genes on Chromosome 15q24-25.1

Thu, 01 Oct 2009 11:26:05 PDT

Recent genome-wide association studies have linked the chromosome 15q24-25.1 locus to nicotine addiction and lung cancer susceptibility. To refine the 15q24-25.1 locus, we performed a haplotype-based association analysis of 194 familial lung cases and 219 cancer-free controls from the Genetic Epidemiology of Lung Cancer Consortium (GELCC) collection, and used proliferation and apoptosis analyses to determine which gene(s) in the 15q24-25.1 locus mediates effects on lung cancer cell growth in vitro. We identified two distinct subregions, hapL (P = 3.20 x 10^–6) and hapN (P = 1.51 x 10^–6), which were significantly associated with familial lung cancer. hapL encompasses IREB2, LOC123688, and PSMA4, and hapN encompasses the three nicotinic acetylcholine receptor subunit genes CHRNA5, CHRNA3, and CHRNB4. Examination of the genes around hapL revealed that PSMA4 plays a role in promoting cancer cell proliferation. PSMA4 mRNA levels were increased in lung tumors compared with normal lung tissues. Down-regulation of PSMA4 expression decreased proteasome activity and induced apoptosis. Proteasome dysfunction leads to many diseases including cancer, and drugs that inhibit proteasome activity show promise as a form of cancer treatment. Genes around hapN were also investigated, but did not show any direct effect on lung cancer cell proliferation. We concluded that PSMA4 is a strong candidate mediator of lung cancer cell growth, and may directly affect lung cancer susceptibility through its modulation of cell proliferation and apoptosis. [Cancer Res 2009;69(19):7844–50]

TrkBT1 Induces Liver Metastasis of Pancreatic Cancer Cells by Sequestering Rho GDP Dissociation Inhibitor and Promoting RhoA Activation

Thu, 01 Oct 2009 11:26:05 PDT

Many genetic and molecular alterations, such as K-ras mutation and NF-B activation, have been identified in pancreatic cancer. However, the mechanisms by which pancreatic cancer metastasizes still remain to be determined. Although we previously showed that the tropomyosin-related kinase B (TrkB) was significantly correlated with the development of liver metastasis, its function in pancreatic cancer metastasis remained unresolved. In the present study, we showed that overexpressed TrkB is an alternatively spliced transcript variant of TrkB (TrkBT1) with a unique COOH-terminal 12–amino acid sequence and is mainly localized in the cytoplasm. Our results showed that overexpression of Flag-tagged TrkBT1 but not a Flag-tagged TrkBT1 COOH-terminal deletion mutant (Flag-TrkBT1C) in nonmetastatic pancreatic cancer cells enhanced cell proliferation, promoted formation of colonies in soft agar, stimulated tumor cell invasion, and induced liver metastasis in an orthotopic xenograft mouse model of pancreatic cancer. TrkBT1 interacted with Rho GDP dissociation inhibitor (GDI) in vivo, but Flag-TrkBT1C did not. Furthermore, overexpression of Flag-TrkBT1 and knockdown of RhoGDI expression by RhoGDI short hairpin RNAs promoted RhoA activation, but Flag-TrkBT1C overexpression did not. Therefore, our results showed that TrkBT1 overexpression induces liver metastasis of pancreatic cancer and uncovered a unique signaling mechanism by which TrkBT1 sequesters GDI and activates RhoA signaling. [Cancer Res 2009;69(19):7851–9]

Circulating Tumor Cells Are Transcriptionally Similar to the Primary Tumor in a Murine Prostate Model

Helzer, K. T., Barnes, H. E., Day, L., Harvey, J., Billings, P. R., Forsyth, A. — Thu, 01 Oct 2009 11:26:05 PDT

The abundance of circulating tumor cells (CTC) indicates patient prognosis. Molecular characterization of CTCs may add additional information about a patient's disease. However, currently available methods are limited by contamination with blood cells. We describe a study using a modified CTC-chip to capture CTCs from an orthotopic xenograft model. Using laser capture microscopy to collect CTCs from the chip, we compared transcripts from purified CTCs with those from primary and metastatic tissue. Transcriptional profiles showed strong concordance among primary, metastatic, and CTC sources. Moreover, cells captured on the chip were viable and could be expanded in culture. We conclude that the CTC-chip is a useful tool to further characterize animal models of cancer and that viable CTCs can be isolated and show transcriptional similarity to solid tumors. [Cancer Res 2009;69(19):7860–6]

Adipocytes Impair Leukemia Treatment in Mice

Thu, 01 Oct 2009 11:26:05 PDT

Obesity is associated with increased cancer incidence and mortality. We have previously found that obesity in children is associated with a 50% increased recurrence of acute lymphoblastic leukemia (ALL) in high-risk patients. We have therefore developed novel in vivo and in vitro preclinical models to study the mechanism(s) of this association. Obesity increased relapse after monotherapy with vincristine (P = 0.03) in obese mice injected with syngeneic ALL cells. This occurred although the drug was dosed proportionally to body weight, equalizing blood and tissue drug levels. In coculture, 3T3-L1 adipocytes significantly impaired the antileukemia efficacy of vincristine, as well as three other chemotherapies (P < 0.05). Interestingly, this protection was independent of cell-cell contact, and it extended to human leukemia cell lines as well. Adipocytes prevented chemotherapy-induced apoptosis, and this was associated with increased expression of the two prosurvival signals Bcl-2 and Pim-2. These findings highlight the role of the adipocyte in fostering leukemia chemotherapy resistance, and may help explain the increased leukemia relapse rate in obese children and adults. Given the growing prevalence of obesity worldwide, these effects are likely to have increasing importance to cancer treatment. [Cancer Res 2009;69(19):7867–74]

Vascular Adhesion Protein-1 Enhances Tumor Growth by Supporting Recruitment of Gr-1+CD11b+ Myeloid Cells into Tumors

Marttila-Ichihara, F., Auvinen, K., Elima, K., Jalkanen, S., Salmi, M. — Thu, 01 Oct 2009 11:26:05 PDT

Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1–deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1⁺CD11b⁺ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1⁺CD11b⁺ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1⁺CD11b⁺ myeloid cells. [Cancer Res 2009;69(19):7875–83]

Blockade of a Chemokine, CCL2, Reduces Chronic Colitis-Associated Carcinogenesis in Mice

Thu, 01 Oct 2009 11:26:06 PDT

Accumulating evidence indicates the crucial contribution of chronic inflammation to various types of carcinogenesis, including colon carcinoma associated with ulcerative colitis and asbestosis-induced malignant mesothelioma. Ulcerative colitis–associated colon carcinogenesis can be recapitulated in mice by azoxymethane administration followed by repetitive dextran sulfate sodium ingestion. In the course of this carcinogenesis process, the expression of a macrophage-tropic chemokine, CCL2, was enhanced together with intracolonic massive infiltration of macrophages, which were a major source of cyclooxygenase (COX)-2, a crucial mediator of colon carcinogenesis. Mice deficient in CCL2-specific receptor, CCR2, exhibited less macrophage infiltration and lower tumor numbers with attenuated COX-2 expression. Moreover, CCL2 antagonists decreased intracolonic macrophage infiltration and COX-2 expression, attenuated neovascularization, and eventually reduced the numbers and size of colon tumors, even when given after multiple colon tumors have developed. These observations identify CCL2 as a crucial mediator of the initiation and progression of chronic colitis–associated colon carcinogenesis and suggest that targeting CCL2 may be useful in treating colon cancers, particularly those associated with chronic inflammation. [Cancer Res 2009;69(19):7884–92]

Comment Re: MDA-MB-435 and M14 Cell Lines: Identical but not M14 Melanoma?

Hollestelle, A., Schutte, M. — Thu, 01 Oct 2009 11:26:06 PDT

Correction: Article on Breast Cancer Tumor Progression

Thu, 01 Oct 2009 11:26:06 PDT

Correction: Review Article on Clonal Mutations in the Cancer-Associated Fibroblasts

Thu, 01 Oct 2009 11:26:06 PDT

Correction: Article on PTHrP Stabilizes AR and Enhances Prostate Cancer Growth

Thu, 01 Oct 2009 11:26:06 PDT