Cancer Research recent issues

The History of Cancer Research: Introducing an AACR Centennial Series

Weinstein, I. B., Case, K. — 2008-08-28

Advances in Chemical Carcinogenesis: A Historical Review and Prospective

Loeb, L. A., Harris, C. C. — 2008-08-28

Pontin and Reptin, Two Related ATPases with Multiple Roles in Cancer

Huber, O., Menard, L., Haurie, V., Nicou, A., Taras, D., Rosenbaum, J. — 2008-08-28

Studies in model organisms or cultured human cells suggest potential implications in carcinogenesis for the AAA+ ATPases Pontin and Reptin. Both proteins are associated with several chromatin-remodeling complexes and have many functions including transcriptional regulation, DNA damage repair, and telomerase activity. They also interact with major oncogenic actors such as β-catenin and c-myc and regulate their oncogenic function. We only now begin to get insight into the role of Pontin and Reptin in human cancers. [Cancer Res 2008;68(17):6873–6]

Podoplanin Is a Novel Fos Target Gene in Skin Carcinogenesis

2008-08-28

Expression and function of the oncogenic transcription factor activator protein (AP-1; mainly composed of Jun and Fos proteins) is required for neoplastic transformation of keratinocytes in vitro and tumor promotion as well as malignant progression in vivo. Here, we describe the identification of 372 differentially expressed genes comparing skin tumor samples of K5-SOS-F transgenic mice (Fos^f/f SOS⁺) with samples derived from animals with a specific deletion of c-Fos in keratinocytes (Fos^ep SOS⁺). Fos-dependent transcription of selected genes was confirmed by quantitative real-time PCR analysis using tumor samples and mouse back skin treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). One of the most differentially expressed genes encodes the small mucin–like glycoprotein Podoplanin (Pdpn), whose expression correlates with malignant progression in mouse tumor model systems and human cancer. We found Pdpn and Fos expression in chemically induced mouse skin tumors, and detailed analysis of the Pdpn gene promoter revealed impaired activity in Fos-deficient mouse embryonic fibroblasts, which could be restored by ectopic Fos expression. Direct Fos protein binding to the Pdpn promoter was shown by chromatin immunoprecipitation and a TPA-induced complex at a TPA-responsive element–like motif in the proximal promoter was identified by electrophoretic mobility shift assays. In summary, we could define a Fos-dependent genetic program in a well-established model of skin tumors. Systematic analysis of these novel target genes will guide us in elucidating the molecular mechanisms of AP-1–regulated pathways that are critically implicated in neoplastic transformation and/or malignant progression. [Cancer Res 2008;68(17):6877–83]

BCR/ABL and Other Kinases from Chronic Myeloproliferative Disorders Stimulate Single-Strand Annealing, an Unfaithful DNA Double-Strand Break Repair

2008-08-28

Myeloproliferative disorders (MPD) are stem cell–derived clonal diseases arising as a consequence of acquired aberrations in c-ABL, Janus-activated kinase 2 (JAK2), and platelet-derived growth factor receptor (PDGFR) that generate oncogenic fusion tyrosine kinases (FTK), including BCR/ABL, TEL/ABL, TEL/JAK2, and TEL/PDGFβR. Here, we show that FTKs stimulate the formation of reactive oxygen species and DNA double-strand breaks (DSB) both in hematopoietic cell lines and in CD34⁺ leukemic stem/progenitor cells from patients with chronic myelogenous leukemia (CML). Single-strand annealing (SSA) represents a relatively rare but very unfaithful DSB repair mechanism causing chromosomal aberrations. Using a specific reporter cassette integrated into genomic DNA, we found that BCR/ABL and other FTKs stimulated SSA activity. Imatinib-mediated inhibition of BCR/ABL abrogated this effect, implicating a kinase-dependent mechanism. Y253F, E255K, T315I, and H396P mutants of BCR/ABL that confer imatinib resistance also stimulated SSA. Increased expression of either nonmutated or mutated BCR/ABL kinase, as is typical of blast phase cells and very primitive chronic phase CML cells, was associated with higher SSA activity. BCR/ABL-mediated stimulation of SSA was accompanied by enhanced nuclear colocalization of RAD52 and ERCC1, which play a key role in the repair. Taken together, these findings suggest a role of FTKs in causing disease progression in MPDs by inducing chromosomal instability through the production of DSBs and stimulation of SSA repair. [Cancer Res 2008;68(17):6884–8]

Efficient Cross-presentation Depends on Autophagy in Tumor Cells

Li, Y., Wang, L.-X., Yang, G., Hao, F., Urba, W. J., Hu, H.-M. — 2008-08-28

Cross-presentation of antigens is critical for the induction of adaptive immunity against tumor cells and infectious pathogens. Currently, it is not known how cross-presentation of tumor antigens is regulated by autophagy. Using both HEK 293T cells that expressed the model antigen OVA and melanoma cells as antigen donors, we show that macroautophagy in tumor cells is essential for cross-presentation by dendritic cells both in vitro and in vivo. Inhibition of autophagy abolished cross-presentation almost completely, whereas induction of autophagy dramatically enhanced the cross-presentation of tumor antigens. Moreover, purified autophagosomes were found to be efficient antigen carriers for cross-presentation. Our findings not only identified a novel role for autophagy as an active process in antigen sequestration and delivery to dendritic cells for cross-presentation, but also suggested, for the first time, that isolated autophagosomes may have potential as potent vaccines for immunotherapy against cancer and infectious diseases. [Cancer Res 2008;68(17):6889–95]

Inflammatory Cytokines Induce Phosphorylation and Ubiquitination of Prostate Suppressor Protein NKX3.1

Markowski, M. C., Bowen, C., Gelmann, E. P. — 2008-08-28

Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)- and interleukin-1β accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF- is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF- on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention. [Cancer Res 2008;68(17):6896–901]

Inhibition of Activated Fibroblast Growth Factor Receptor 2 in Endometrial Cancer Cells Induces Cell Death Despite PTEN Abrogation

2008-08-28

KRAS activation and PTEN inactivation are frequent events in endometrial tumorigenesis, occurring in 10% to 30% and 26% to 80% of endometrial cancers, respectively. Because we have recently shown activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 16% of endometrioid endometrial cancers, we sought to determine the genetic context in which FGFR2 mutations occur. Analysis of 116 primary endometrioid endometrial cancers revealed that FGFR2 and KRAS mutations were mutually exclusive, whereas FGFR2 mutations were seen concomitantly with PTEN mutations. Here, we show that shRNA knockdown of FGFR2 or treatment with a pan-FGFR inhibitor, PD173074, resulted in cell cycle arrest and induction of cell death in endometrial cancer cells with activating mutations in FGFR2. This cell death in response to FGFR2 inhibition occurred within the context of loss-of-function mutations in PTEN and constitutive AKT phosphorylation, and was associated with a marked reduction in extracellular signal-regulated kinase 1/2 activation. Together, these data suggest that inhibition of FGFR2 may be a viable therapeutic option in endometrial tumors possessing activating mutations in FGFR2, despite the frequent abrogation of PTEN in this cancer type. [Cancer Res 2008;68(17):6902–7]

PTEN Acetylation Modulates Its Interaction with PDZ Domain

Ikenoue, T., Inoki, K., Zhao, B., Guan, K.-L. — 2008-08-28

The PTEN tumor suppressor gene is frequently inactivated in human cancer. As a major tumor suppressor, PTEN function must be tightly regulated. Both phosphorylation and membrane association have been reported to regulate PTEN activity. In addition, the COOH terminus of PTEN has a typical PDZ domain-binding motif that interacts with several PDZ domain-containing proteins. In this report, we show that PTEN is acetylated on Lys⁴⁰², which is in the COOH-terminal PDZ domain-binding motif. We show that CBP plays a major role in PTEN acetylation, whereas the SIRT1 deacetylase is mainly responsible for PTEN deacetylation. Interestingly, Lys⁴⁰² acetylation modulates PTEN interaction with PDZ domain-containing proteins, indicating a potential role of acetylation in regulating PTEN function. [Cancer Res 2008;68(17):6908–12]

PIK3CA Mutations and Copy Number Gains in Human Lung Cancers

2008-08-28

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance. [Cancer Res 2008;68(17):6913–21]

Semaphorin-3B Is an Angiogenesis Inhibitor That Is Inactivated by Furin-Like Pro-Protein Convertases

2008-08-28

Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic factor that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and 22-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m–expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial growth factor–induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic fibroblast growth factor–induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B. [Cancer Res 2008;68(17):6922–31]

A Stochastic Model for Cancer Stem Cell Origin in Metastatic Colon Cancer

2008-08-28

Human cancers have been found to include transformed stem cells that may drive cancer progression to metastasis. Here, we report that metastatic colon cancer contains clonally derived tumor cells with all of the critical properties expected of stem cells, including self-renewal and the ability to differentiate into mature colon cells. Additionally, when injected into mice, these cells initiated tumors that closely resemble human cancer. Karyotype analyses of parental and clonally derived tumor cells expressed many consistent (clonal) along with unique chromosomal aberrations, suggesting the presence of chromosomal instability in the cancer stem cells. Thus, this new model for cancer origin and metastatic progression includes features of both the hierarchical model for cancerous stem cells and the stochastic model, driven by the observation of chromosomal instability. [Cancer Res 2008;68(17):6932–41]

Combined Lysophosphatidic Acid/Platelet-Derived Growth Factor Signaling Triggers Glioma Cell Migration in a Tenascin-C Microenvironment

2008-08-28

The antiadhesive extracellular matrix molecule tenascin-C abrogates cell spreading on fibronectin through competitive inhibition of syndecan-4, thereby preventing focal adhesion kinase (FAK) activation and triggering enhanced proteolytic degradation of both RhoA and tropomyosin 1 (TM1). Here, we show that simultaneous signaling by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) initiates glioma cell spreading and migration through syndecan-4–independent activation of paxillin and FAK and by stabilizing expression of RhoA, TM1, TM2, and TM3. By using gene silencing methods, we show that paxillin, TM1, TM2, and TM3 are essential for LPA/PDGF-induced cell spreading on a fibronectin/tenascin-C (FN/TN) substratum. LPA/PDGF-induced cell spreading and migration on FN/TN depends on phosphatidylinositol 3-kinase, RhoKinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 but is independent of phospholipase C and Jun kinase. RNA microarray data reveal expression of tenascin-C, PDGFs, LPA, and the respective receptors in several types of cancer, suggesting that the TN/LPA/PDGF axis exists in malignant tumors. These findings may in turn be relevant for diagnostic or therapeutic applications targeting cancer. [Cancer Res 2008;68(17):6942–52]

BRCA1-Associated Protein-1 Is a Tumor Suppressor that Requires Deubiquitinating Activity and Nuclear Localization

2008-08-28

BRCA1-associated protein-1 (BAP1), a deubiquitinating enzyme of unknown cellular function, is mutated in breast and lung cancers. In this study, we have shown for the first time that BAP1 has tumor suppressor activity in vivo by showing that BAP1 can suppress tumorigenicity of lung cancer cells in athymic nude mice. We show that BAP1 fulfills another criterion of a genuine tumor suppressor because cancer-associated BAP1 mutants are deficient in deubiquitinating activity. We show for the first time that one of the two predicted nuclear targeting motifs is required for nuclear localization of BAP1 and that a truncation mutant found in a lung cancer cell line results in BAP1 that fails to localize to the nucleus. Furthermore, we show that deubiquitinating activity and nuclear localization are both required for BAP1-mediated tumor suppression in nude mice. We show that BAP1 exerts its tumor suppressor functions by affecting the cell cycle, speeding the progression through the G₁-S checkpoint, and inducing cell death via a process that has characteristics of both apoptosis and necrosis. Surprisingly, BAP1-mediated growth suppression is independent of wild-type BRCA1. Because deubiquitinating enzymes are components of the ubiquitin proteasome system, this pathway has emerged as an important target for anticancer drugs. The identification of the deubiquitinating enzyme BAP1 as a tumor suppressor may lead to further understanding of how the ubiquitin proteasome system contributes to cancer and aid in the identification of new targets for cancer therapy. [Cancer Res 2008;68(17):6953–62]

EBV Is Necessary for Proliferation of Dually Infected Primary Effusion Lymphoma Cells

Mack, A. A., Sugden, B. — 2008-08-28

Epstein Barr virus (EBV) and Kaposi's sarcoma–associated herpesvirus (KSHV) are found together in ~80% of primary effusion lymphomas (PEL), but their contribution to these cancers is unclear. We found that dominant-negative derivatives of EBNA1 inhibited EBV-positive PEL cells from forming colonies. Those rare PEL cells that proliferated after expression of the dominant-negative derivatives usually expressed these derivatives at low or undetectable levels and continued to maintain their EBV genomes. Those proliferating cells expressing higher levels of the derivatives expressed mutant derivatives that could not bind DNA. These findings indicate that EBV is required to sustain proliferation, as measured by colony formation of dually infected PEL cells. The dominant-negative derivatives of EBNA1 had no effect on the colony-forming ability of five EBV-negative, KSHV-negative hematopoietic cell lines. Surprisingly, they did inhibit the colony-forming ability of EBV-negative, KSHV-positive PEL cells. The small fraction of cells that continued to proliferate expressed only mutants of the EBNA1 derivatives that could no longer bind DNA. These findings indicate that the site-specific DNA-binding activity of EBNA1 or its derivatives when expressed efficiently in EBV-negative, KSHV-positive PEL cells inhibits their colony formation possibly through their binding to the KSHV genome. [Cancer Res 2008;68(17):6963–8]

Illegitimate WNT Pathway Activation by {beta}-Catenin Mutation or Autocrine Stimulation in T-Cell Malignancies

2008-08-28

Recent studies in mice have shown a role for the canonical WNT pathway in lymphocyte development. Because cancers often arise as a result of aberrant activation of signaling cascades that normally promote the self-renewal and expansion of their progenitor cells, we hypothesized that activation of the WNT pathway might contribute to the pathogenesis of lymphoproliferative disease. Therefore, we screened a large panel (n = 162) of non–Hodgkin lymphomas (NHL), including all major WHO categories, for nuclear expression of β-catenin, a hallmark of "active" WNT signaling. In 16 lymphomas, mostly of T-lineage origin, nuclear localization of β-catenin was detected. Interestingly, some of these tumors contained established gain-of-function mutations in the gene encoding β-catenin (CTNNB1); however, in the majority, mutations in either CTNNB1 or APC were not detected. Functional analysis of WNT signaling in precursor T-lymphoblastic lymphomas/leukemias, the NHL subset in which β-catenin accumulation was most prevalent (33% positive), revealed a constitutively activated, but still responsive, WNT pathway, which controlled T-cell factor–mediated gene transcription and cell growth. Our data indicate that activation of the WNT pathway, either by CTNNB1 mutation or autocrine stimulation, plays a role in the pathogenesis of a subset of NHLs, in particular, those of T-cell origin. [Cancer Res 2008;68(17):6969–77]

Nonsteroidal Anti-inflammatory Drugs Suppress Glioma via 15-Hydroxyprostaglandin Dehydrogenase

2008-08-28

Studies have conjectured that nonsteroidal anti-inflammatory drugs (NSAID) inhibit growth of various malignancies by inhibiting cyclooxygenase-2 (COX-2) enzyme activity. Yet, several lines of evidence indicate that a COX-2–independent mechanism may also be involved in their antitumor effects. Here, we report that NSAIDs may inhibit the growth of glioblastoma multiforme (GBM) cells through COX-2–independent mechanisms, including up-regulation of both 15-hydroxyprostaglandin dehydrogenase (15-PGDH, the key prostaglandin catabolic enzyme) and the cell cycle inhibitor p21. Using Western blot and real-time PCR analysis in various GBM cell lines, we observed up-regulation of 15-PGDH and p21 after NSAIDs treatment. To elucidate the role of 15-PGDH in GBM, transfection assays were conducted using the T98G GBM cell line. Overexpression of 15-PGDH suppressed cell growth and was associated with increased expression of p21. In an attempt to investigate the roles of COX-2, 15-PGDH, and p21 in the inhibition of growth of GBM, small interfering RNA (siRNA) against each of these proteins was transfected into T98G cells. Inhibition of growth mediated by NSAIDs was partially reversed after knockdown of either 15-PGDH or p21, but not after COX-2 knockdown. Moreover, expression level of p21 was not affected in COX-2 siRNA transfected cells. Our studies provide evidence that the up-regulation of 15-PGDH induced by NSAIDs has the potential to inhibit growth of GBM, in part, by up-regulation of p21 possibly independent from COX-2 enzymatic function. [Cancer Res 2008;68(17):6978–86]

Glycogen Synthase Kinase 3{beta} Regulates Cell Death Induced by Synthetic Triterpenoids

Vene, R., Larghero, P., Arena, G., Sporn, M. B., Albini, A., Tosetti, F. — 2008-08-28

The induction of programmed cell death in premalignant or malignant cancer cells by chemopreventive agents could be a valuable tool to control prostate cancer initiation and progression. In this work, we present evidence that the C-28 methyl ester of the synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) induces cell death in androgen-responsive and unresponsive human prostate cancer cell lines at nanomolar and low micromolar concentrations. CDDO-Me induced caspase-3, caspase-8, and caspase-9 activation; poly(ADP-ribose) polymerase cleavage; internucleosomal DNA fragmentation; and loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction in PC3 and DU145 cells. However, caspase-3 and caspase-8 inhibition by Z-DEVD-fmk and Z-IETD-fmk, respectively, or general caspase inhibition by BOC-D-fmk or Z-VAD-fmk did not rescue loss of cell viability induced by CDDO-Me, suggesting the activation of additional caspase-independent mechanisms. Interestingly, CDDO-Me induced inactivating phosphorylation at Ser⁹ of glycogen synthase kinase 3β (GSK3β), a multifunctional kinase that mediates essential events promoting prostate cancer development and acquisition of androgen independence. The GSK3 inhibitor lithium chloride and, more effectively, GSK3 gene silencing sensitized PC3 and DU145 prostate cancer cells to CDDO-Me cytotoxicity. These data suggest that modulation of GSK3β activation is involved in the cell death pathway engaged by CDDO-Me in prostate cancer cells. [Cancer Res 2008;68(17):6987–96]

EBV Latent Membrane Protein 1 Effects on Plakoglobin, Cell Growth, and Migration

Shair, K. H.Y., Schnegg, C. I., Raab-Traub, N. — 2008-08-28

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-B (NF-B) signaling. Inhibitors of PI3K, Akt, and NF-B signaling dramatically reduced these enhanced properties. An IB super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-B activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-B–activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-B and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration. [Cancer Res 2008;68(17):6997–7005]

The Relative Contribution of Point Mutations and Genomic Rearrangements in BRCA1 and BRCA2 in High-Risk Breast Cancer Families

2008-08-28

The demand for BRCA1 and BRCA2 mutation screening is increasing as their identification will affect medical management. However, both the contribution of different mutation types in BRCA1 and BRCA2 and whom should be offered testing for large genomic rearrangements have not been well established in the U.S. high-risk population. We define the prevalence and spectrum of point mutations and genomic rearrangements in BRCA genes in a large U.S. high-risk clinic population of both non-Ashkenazi and Ashkenazi Jewish descent, using a sample set representative of the U.S. genetic testing population. Two hundred fifty-one probands ascertained through the University of Pennsylvania high-risk clinic, all with commercial testing for BRCA1 and BRCA2, with an estimated prevalence of BRCA mutation ≥10% using the Myriad II model and a DNA sample available, were studied. Individuals without deleterious point mutations were screened for genomic rearrangements in BRCA1 and BRCA2. In the 136 non-Ashkenazi Jewish probands, 36 (26%) BRCA point mutations and 8 (6%) genomic rearrangements (7 in BRCA1 and 1 in BRCA2) were identified. Forty-seven of the 115 (40%) Ashkenazi Jewish probands had point mutations; no genomic rearrangements were identified in the group without mutations. In the non-Ashkenazi Jewish probands, genomic rearrangements constituted 18% of all identified BRCA mutations; estimated mutation prevalence (Myriad II model) was not predictive of their presence. Whereas these findings should be confirmed in larger sample sets, our data suggest that genomic rearrangement testing be considered in all non-Ashkenazi Jewish women with an estimated mutation prevalence ≥10%. [Cancer Res 2008;68(17):7006–14]

HEXIM1 Regulates 17{beta}-Estradiol/Estrogen Receptor-{alpha}-Mediated Expression of Cyclin D1 in Mammary Cells via Modulation of P-TEFb

Ogba, N., Chaplin, L. J., Doughman, Y. Q., Fujinaga, K., Montano, M. M. — 2008-08-28

Estrogen receptor (ER) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ER and bridges a functional interaction between ER and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ER-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-7 cells led to a decrease in estrogen-induced cyclin D1 expression, whereas down-regulation of HEXIM1 expression led to an enhancement of estrogen-induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of ER, P-TEFb, and S2P RNAP II to promoter and coding regions of ER-responsive genes pS2 and CCND1 with increased HEXIM1 expression in MCF-7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced P-TEFb activity. Whereas there have been previous reports on HEXIM1 inhibition of P-TEFb activity, our studies add a new dimension by showing that E₂/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells. Together, these studies provide novel insight into the role of HEXIM1 and ER in mammary epithelial cell function. [Cancer Res 2008;68(17):7015–24]

Functional Genetic Variations in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Multiple Types of Cancer

2008-08-28

Antitumor T lymphocytes play a pivotal role in immunosurveillance of malignancy. The CTL antigen 4 (CTLA-4) is a vital negative regulator of T-cell activation and proliferation. This study examined whether genetic polymorphisms in CTLA-4 are associated with cancer susceptibility. A two-stage investigation using haplotype-tagging single nucleotide polymorphism approach and multiple independent case-control analyses was performed to assess the association between CTLA-4 genotypes and cancer risk. Functional relevance of the polymorphisms was examined by biochemical assays. We found that the 49G>A polymorphism in the CTLA-4 leading sequence causing ¹⁷Ala to ¹⁷Thr amino acid substitution is associated with increased susceptibility to multiple cancers, including lung, breast, esophagus, and gastric cardia cancers. Genotyping in 5,832 individuals with cancer and 5,831 control subjects in northern and southern Chinese populations showed that the CTLA-4 49AA genotype had an odds ratio of 1.72 (95% confidence interval, 1.50–2.10; P = 3.4 x 10^–7) for developing cancer compared with the 49GG genotype. Biochemical analyses showed that CTLA-4–¹⁷Thr had higher capability to bind B7.1 and stronger inhibitory effect on T-cell activation compared with CTLA-4–¹⁷Ala. T cells carrying the 49AA genotype had significantly lower activation and proliferation rates compared with T cells carrying the 49GG genotype upon stimulation. These results are consistent with our hypothesis and indicate that genetic polymorphisms influencing T-cell activation modify cancer susceptibility. [Cancer Res 2008;68(17):7025–34]

Molecular Basis for the Induction of an Angiogenesis Inhibitor, Thrombospondin-1, by 5-Fluorouracil

2008-08-28

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein. [Cancer Res 2008;68(17):7035–41]

Vessel Fractions in Tumor Xenografts Depicted by Flow- or Contrast-Sensitive Three-Dimensional High-Frequency Doppler Ultrasound Respond Differently to Antiangiogenic Treatment

2008-08-28

High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors. [Cancer Res 2008;68(17):7042–9]

Gemcitabine and Cytosine Arabinoside Cytotoxicity: Association with Lymphoblastoid Cell Expression

2008-08-28

Two cytidine analogues, gemcitabine (dFdC) and 1-β-d-arabinofuranosylcytosine (AraC), show significant therapeutic effect in a variety of cancers. However, response to these drugs varies widely. Evidence from tumor biopsy samples shows that expression levels for genes involved in the cytidine transport, metabolism, and bioactivation pathway contribute to this variation in response. In the present study, we set out to test the hypothesis that variation in gene expression both within and outside of this "pathway" might influence sensitivity to gemcitabine and AraC. Specifically, Affymetrix U133 Plus 2.0 GeneChip and cytotoxicity assays were performed to obtain basal mRNA expression and IC₅₀ values for both drugs in 197 ethnically defined Human Variation Panel lymphoblastoid cell lines. Genes with a high degree of association with IC₅₀ values were involved mainly in cell death, cancer, cell cycle, and nucleic acid metabolism pathways. We validated selected significant genes by performing real-time quantitative reverse transcription-PCR and selected two representative candidates, NT5C3 (within the pathway) and FKBP5 (outside of the pathway), for functional validation. Those studies showed that down-regulation of NT5C3 and FKBP5 altered tumor cell sensitivity to both drugs. Our results suggest that cell-based model system studies, when combined with complementary functional characterization, may help to identify biomarkers for response to chemotherapy with these cytidine analogues. [Cancer Res 2008;68(17):7050–8]

ATM Acts Downstream of ATR in the DNA Damage Response Signaling of Bystander Cells

Burdak-Rothkamm, S., Rothkamm, K., Prise, K. M. — 2008-08-28

This study identifies ataxia-telangiectasia mutated (ATM) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, ATM, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/ATM/DNA-PK–proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and ATM but not DNA-PK–deficient bystander cells. ATM activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, ATM-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to H2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation. [Cancer Res 2008;68(17):7059–65]

Chemoprevention and Treatment of Experimental Cowden's Disease by mTOR Inhibition with Rapamycin

Squarize, C. H., Castilho, R. M., Gutkind, J. S. — 2008-08-28

Cowden's disease is an autosomal dominant disorder characterized by the development of multiple mucocutaneous lesions and benign tumors, and enhanced cancer predisposition. Most Cowden's disease patients harbor inactivating mutations in the PTEN tumor suppressor gene which encodes a lipid phosphatase, PTEN, which restrains the phosphatidylinositol 3-kinase–Akt signaling pathway. We observed that the epithelial-specific deletion of Pten in mice causes multiple hyperproliferative and tumor lesions that strikingly resemble Cowden's disease. This animal model system provided an opportunity to explore novel therapeutic approaches in Cowden's disease. Indeed, we show here that rapamycin administration, which inhibits a key downstream target of Akt, mammalian target of rapamycin (mTOR), promotes the rapid regression of advanced mucocutaneous lesions. Furthermore, when administered before disease manifestation, rapamycin can halt the development of Cowden's disease–like lesions, thereby prolonging animal survival. These findings suggest that mTOR inhibition with rapamycin may represent a suitable therapeutic option for the chemoprevention and treatment of Cowden disease patients and others tumor syndromes that involve defective PTEN function. [Cancer Res 2008;68(17):7066–72]

Isoaspartate-Glycine-Arginine: A New Tumor Vasculature-Targeting Motif

2008-08-28

Asparagine deamidation in peptides or in fibronectin fragments containing the asparagine-glycine-arginine sequence generates isoaspartate-glycine-arginine (isoDGR), a new vβ3 integrin-binding motif. Because vβ3 is expressed in angiogenic vessels, we hypothesized that isoDGR-containing peptides could be exploited as ligands for targeted delivery of drugs to tumor neovasculature. We found that a cyclic CisoDGRC peptide coupled to fluorescent nanoparticles (quantum dots) could bind vβ3 integrin and colocalize with anti-CD31, anti-vβ3, and anti-5β1 antibodies in human renal cell carcinoma tissue sections, indicating that this peptide could efficiently recognize endothelial cells of angiogenic vessels. Using CisoDGRC fused to tumor necrosis factor (TNF) we observed that ultralow doses (1–10 pg) of this product (called isoDGR-TNF), but not of TNF or CDGRC-TNF fusion protein, were sufficient to induce antitumor effects when administered alone or in combination with chemotherapy to tumor-bearing mice. The antitumor activity of isoDGR-TNF was efficiently inhibited by coadministration with an excess of free CisoDGRC, as expected for ligand-directed targeting mechanisms. These results suggest that isoDGR is a novel tumor vasculature–targeting motif. Peptides containing isoDGR could be exploited as ligands for targeted delivery of drugs, imaging agents, or other compounds to tumor vasculature. [Cancer Res 2008;68(17):7073–82]

Synergistic Inhibition with a Dual Epidermal Growth Factor Receptor/HER-2/neu Tyrosine Kinase Inhibitor and a Disintegrin and Metalloprotease Inhibitor

2008-08-28

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu–transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu–positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points. [Cancer Res 2008;68(17):7083–9]

Thrombospondin 1 Promotes Tumor Macrophage Recruitment and Enhances Tumor Cell Cytotoxicity of Differentiated U937 Cells

2008-08-28

Inhibition of tumor growth by thrombospondin (TSP) 1 is generally attributed to its antiangiogenic activity, but effects on tumor immunity should also be considered. We show that overexpression of TSP1 in melanoma cells increases macrophage recruitment into xenograft tumors grown in nude or beige/nude mice. In vitro, TSP1 acutely induces expression of plasminogen activator inhibitor-1 (PAI-1) by monocytic cells, suggesting that TSP1-induced macrophage recruitment is at least partially mediated by PAI-1. Tumor-associated macrophages (TAM) can either promote or limit tumor progression. The percentage of M1-polarized macrophages expressing inducible nitric oxide synthase is increased in TSP1-expressing tumors. Furthermore, soluble TSP1 stimulates killing of breast carcinoma and melanoma cells by IFN-–differentiated U937 cells in vitro via release of reactive oxygen species. TSP1 causes a significant increase in phorbol ester–mediated superoxide generation from differentiated monocytes by interaction with ₆β₁ integrin through its NH₂-terminal region. The NH₂-terminal domain of TSP2 also stimulates monocyte superoxide production. Extracellular calcium is required for the TSP1-induced macrophage respiratory burst. Thus, TSP1 may play an important role in antitumor immunity by enhancing recruitment and activation of M1 TAMs, which provides an additional selective pressure for loss of TSP1 and TSP2 expression during tumor progression. [Cancer Res 2008;68(17):7090–10]

EWS-FLI1 Suppresses NOTCH-Activated p53 in Ewing's Sarcoma

2008-08-28

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21^WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21^WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21^WAF1/CIP1. These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53. [Cancer Res 2008;68(17):7100–9]

Tissue Prostate-Specific Antigen Facilitates Refractory Prostate Tumor Progression via Enhancing ARA70-Regulated Androgen Receptor Transactivation

2008-08-28

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G₁ phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer. [Cancer Res 2008;68(17):7110–9]

Malignant Pleural Mesothelioma-Targeted CREBBP/EP300 Inhibitory Protein 1 Promoter System for Gene Therapy and Virotherapy

Fukazawa, T., Matsuoka, J., Naomoto, Y., Maeda, Y., Durbin, M. L., Tanaka, N. — 2008-08-28

Gene therapy and virotherapy are one of the approaches used to treat malignant pleural mesothelioma. To improve the efficiency of targeting malignant mesothelioma cells, we designed a novel system using the promoter of the CREBBP/EP300 inhibitory protein 1 (CRI1), a gene specifically expressed in malignant pleural mesothelioma. Four tandem repeats of the CRI1 promoter (CRI1^{–138 4x}) caused significantly high promoter activity in malignant pleural mesothelioma cells but little promoter activity in normal mesothelial cells and normal fibroblasts. The recombinant adenoviral vector expressing proapoptotic BH3-interacting death agonist or early region 1A driven by the CRI1^{–138 4x} promoter induced cell death in malignant mesothelioma cells but not in normal cells. Moreover, these viruses showed antitumor effects in a mesothelioma xenograft mouse model. Here, we describe a novel strategy to target malignant mesothelioma using the CRI1^{–138 4x} promoter system. [Cancer Res 2008;68(17):7120–9]

Nitric Oxide Is a Key Component in Inflammation-Accelerated Tumorigenesis

2008-08-28

Nitric oxide (NO^•), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO^• and the cellular microenvironment influences the role of NO^• in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO^• accelerates spontaneous tumor development. C. parvum–induced inflammation and increased NO^• synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53–/–NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53–/–NOS2–/– mice did not show any difference in tumor latency between C. parvum–treated and control groups. In C. parvum–treated p53–/–NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser⁴⁷³ (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum–treated p53–/–NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80⁺, CD86⁺, CD40⁺, and CD83⁺ on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum–treated p53–/–NOS2+/+ mice indicates a role of NO^• in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO^• can accelerate tumor development. [Cancer Res 2008;68(17):7130–6]

CXCL13 Is Highly Produced by Sezary Cells and Enhances Their Migratory Ability via a Synergistic Mechanism Involving CCL19 and CCL21 Chemokines

2008-08-28

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance. [Cancer Res 2008;68(17):7137–46]

LRRC3B, Encoding a Leucine-Rich Repeat-Containing Protein, Is a Putative Tumor Suppressor Gene in Gastric Cancer

2008-08-28

Leucine-rich repeat-containing 3B (LRRC3B) is an evolutionarily highly conserved leucine-rich repeat-containing protein, but its biological significance is unknown. Using restriction landmark genomic scanning and pyrosequencing, we found that the promoter region of LRRC3B was aberrantly methylated in gastric cancer. Gastric cancer cell lines displayed epigenetic silencing of LRRC3B, but treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased LRRC3B expression in gastric cancer cell lines. Real-time reverse transcription-PCR analysis of 96 paired primary gastric tumors and normal adjacent tissues showed that LRRC3B expression was reduced in 88.5% of gastric tumors compared with normal adjacent tissues. Pyrosequencing analysis of the promoter region revealed that LRRC3B was significantly hypermethylated in gastric tumors. Stable transfection of LRRC3B in SNU-601 cells, a gastric cancer cell line, inhibited anchorage-dependent and anchorage-independent colony formation, and LRRC3B expression suppressed tumorigenesis in nude mice. Microarray analysis of LRRC3B-expressing xenograft tumors showed induction of immune response–related genes and IFN signaling genes. H&E-stained sections of LRRC3B-expressing xenograft tumors showed lymphocyte infiltration in the region. We suggest that LRRC3B is a putative tumor suppressor gene that is silenced in gastric cancers by epigenetic mechanisms and that LRRC3B silencing in cancer may play an important role in tumor escape from immune surveillance. [Cancer Res 2008;68(17):7147–55]

CREB3L2-PPAR{gamma} Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

2008-08-28

The discovery of gene fusion mutations, particularly in leukemia, has consistently identified new cancer pathways and led to molecular diagnostic assays and molecular-targeted chemotherapies for cancer patients. Here, we report our discovery of a novel CREB3L2-PPAR fusion mutation in thyroid carcinoma with t(3;7)(p25;q34), showing that a family of somatic PPAR fusion mutations exist in thyroid cancer. The CREB3L2-PPAR fusion encodes a CREB3L2-PPAR fusion protein that is composed of the transactivation domain of CREB3L2 and all functional domains of PPAR1. CREB3L2-PPAR was detected in <3% of thyroid follicular carcinomas. Engineered overexpression of CREB3L2-PPAR induced proliferation by 40% to 45% in primary human thyroid cells, consistent with a dominant oncogenic mechanism. Wild-type CREB3L2 was expressed in the thyroid as a bZIP transcription factor with a transmembrane domain that has flanking S1P and S2P proteolytic cleavage sites. Native CREB3L2 was cleaved to nuclear CREB3L2 by regulated intramembrane proteolysis in normal thyroid cells that expressed the S1P and S2P proteases. Nuclear CREB3L2 stimulated transcription 8-fold from the EVX1 cyclic AMP (cAMP) response element in the absence of cAMP, whereas CREB3L2-PPAR inhibited transcription 6-fold from EVX1 in the same experiments. CREB3L2-PPAR also inhibited 4-fold the expression of thyroglobulin, a native cAMP-responsive gene, in primary thyroid cells treated with thyroid-stimulating hormone. Our findings identify a novel CREB3L2-PPAR gene fusion mutation in thyroid carcinoma and reveal a thyroid signaling pathway that is regulated by intramembrane proteolysis and disrupted in cancer. [Cancer Res 2008;68(17):7156–64]

Characterization of the Recurrent 8p11-12 Amplicon Identifies PPAPDC1B, a Phosphatase Protein, as a New Therapeutic Target in Breast Cancer

2008-08-28

The 8p11-12 chromosome region is one of the regions most frequently amplified in breast carcinoma (10–15% of cases). Several genes within this region have been identified as candidate oncogenes, as they are both amplified and overexpressed. However, very few studies have explored the role of these genes in cell transformation, with the aim of identifying valuable therapeutic targets. An analysis of comparative genomic hybridization array and expression profiling data for a series of 152 ductal breast carcinomas and 21 cell lines identified five genes (LSM1, BAG4, DDHD2, PPAPDC1B, and WHSC1L1) within the amplified region as consistently overexpressed due to an increased gene copy number. The use of small interfering RNA to knock down the expression of each of these genes showed the major role played by two genes, PPAPDC1B and WHSC1L1, in regulating the survival and transformation of two different cell lines harboring the 8p amplicon. The role of these two genes in cell survival and cell transformation was also confirmed by long-term knockdown expression studies using short hairpin RNAs. The potential of PPAPDC1B, which encodes a transmembrane phosphatase, as a therapeutic target was further shown by the strong inhibition of growth of breast tumor xenografts displaying 8p11-12 amplification induced by the silencing of PPAPDC1B. The oncogenic properties of PPAPDC1B were further shown by its ability to transform NIH-3T3 fibroblasts, inducing their anchorage-independent growth. Finally, microarray experiments on PPAPDC1B knockdown indicated that this gene interfered with multiple cell signaling pathways, including the Janus-activated kinase-signal transducer and activator of transcription, mitogen-activated protein kinase, and protein kinase C pathways. PPAPDC1B may also potentiate the estrogen receptor pathway by down-regulating DUSP22. [Cancer Res 2008;68(17):7165–75]

RET/PTC Rearrangements Preferentially Occurred in Papillary Thyroid Cancer among Atomic Bomb Survivors Exposed to High Radiation Dose

2008-08-28

A major early event in papillary thyroid carcinogenesis is constitutive activation of the mitogen-activated protein kinase signaling pathway caused by alterations of a single gene, typically rearrangements of the RET and NTRK1 genes or point mutations in the BRAF and RAS genes. In childhood papillary thyroid cancer, regardless of history of radiation exposure, RET/PTC rearrangements are a major event. Conversely, in adult-onset papillary thyroid cancer among the general population, the most common molecular event is BRAF^V600E point mutation, not RET/PTC rearrangements. To clarify which gene alteration, chromosome aberration, or point mutation preferentially occurs in radiation-associated adult-onset papillary thyroid cancer, we have performed molecular analyses on RET/PTC rearrangements and BRAF^V600E mutation in 71 papillary thyroid cancer cases among atomic bomb survivors (including 21 cases not exposed to atomic bomb radiation), in relation to radiation dose as well as time elapsed since atomic bomb radiation exposure. RET/PTC rearrangements showed significantly increased frequency with increased radiation dose (P_trend = 0.002). In contrast, BRAF^V600E mutation was less frequent in cases exposed to higher radiation dose (P_trend < 0.001). Papillary thyroid cancer subjects harboring RET/PTC rearrangements developed this cancer earlier than did cases with BRAF^V600E mutation (P = 0.03). These findings were confirmed by multivariate logistic regression analysis. These results suggest that RET/PTC rearrangements play an important role in radiation-associated thyroid carcinogenesis. [Cancer Res 2008;68(17):7176–82]

Manipulation of [11C]-5-Hydroxytryptophan and 6-[18F]Fluoro-3,4-Dihydroxy-L-Phenylalanine Accumulation in Neuroendocrine Tumor Cells

2008-08-28

[¹¹C]-5-Hydroxytryptophan ([¹¹C]HTP) and 6-[¹⁸F]fluoro-3,4-dihydroxy-l-phenylalanine ([¹⁸F]FDOPA) are used to image neuroendocrine tumors with positron emission tomography. The precise mechanism by which these tracers accumulate in tumor cells is unknown. We aimed to study tracer uptake via large amino acid transporters, peripheral decarboxylation (inhibited by carbidopa), and intracellular breakdown by monoamine oxidase (MAO). [¹¹C]HTP and [¹⁸F]FDOPA tracer accumulation was assessed in a human neuroendocrine tumor cell line, BON. The carbidopa experiments were done in a tumor-bearing mouse model. Intracellular [¹¹C]HTP accumulation was 2-fold higher than that of [¹⁸F]FDOPA. Cellular transport of both tracers was inhibited by amino-2-norbornanecarboxylic acid. The MAO inhibitors clorgyline and pargyline increased tracer accumulation in vitro. Carbidopa did not influence tracer accumulation in vitro but improved tumor imaging in vivo. Despite lower accumulation in vitro, visualization of [¹⁸F]FDOPA is superior to [¹¹C]HTP in the neuroendocrine pancreatic tumor xenograft model. This could be a consequence of the serotonin saturation of BON cells in the in vivo model. [Cancer Res 2008;68(17):7183–90]

Chondroitin Sulfate E Fragments Enhance CD44 Cleavage and CD44-Dependent Motility in Tumor Cells

2008-08-28

During tumor cell invasion, certain extracellular matrix (ECM) components such as hyaluronan (HA) are degraded into small oligosaccharides, which are detected in patients. We previously reported that such HA oligosaccharides induce the proteolytic cleavage of an ECM-binding molecule CD44 from tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we report that chondroitin sulfate E (CSE), another component of the tumor ECM, strongly enhances CD44 cleavage and tumor cell motility when degraded into oligosaccharides. CSE and its degradation products were detected in pancreatic ductal adenocarcinoma. In CD44-expressing pancreatic tumor cells, degraded forms of CSE but not intact CSE enhanced CD44 cleavage; enzymatic digestion of such low-molecular weight CSE (LMW-CSE) abrogated this enhancement. Among the LMW-CSE preparations examined, 3-kDa CSE most potently induced CD44 cleavage. Nuclear magnetic resonance analysis showed that the 3-kDa-CSE bound to CD44, and that blocking such binding abrogated the CD44 cleavage induction. LMW-CSE also induced prominent filopodia formation and cytoskeletal changes in tumor cells; these effects were also abrogated by blocking the LMW-CSE binding to CD44. Chemically synthesized CSE hexasaccharides also enhanced the CD44 cleavage and tumor cell motility in a CD44-dependent manner. We conclude that the degraded forms of CSE modulate cell adhesion and migration by interacting with tumor-cell CD44, suggesting that the degradation products of tumor-associated ECMs that interact with CD44 play a significant role in CD44-mediated tumor progression. [Cancer Res 2008;68(17):7191–9]