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[Cancer Research 66, 2544-2552, March 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Cyclin D1 Is Transcriptionally Regulated by and Required for Transformation by Activated Signal Transducer and Activator of Transcription 3

Kenneth Leslie1, Cynthia Lang1, Geeta Devgan1, Janeen Azare1, Marjan Berishaj1, William Gerald2, Young Bae Kim3, Keren Paz3, James E. Darnell3, Christopher Albanese4, Toshiyuki Sakamaki4, Richard Pestell4 and Jacqueline Bromberg1

Departments of 1 Medicine and 2 Pathology, Memorial Sloan-Kettering Cancer Center; 3 The Rockefeller University, New York, New York; and 4 Department of Oncology and the Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia

Requests for reprints: Jacqueline Bromberg, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-8191; Fax: 646-422-2231; E-mail: bromberj{at}mskcc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that is aberrantly activated in many cancer cells. Constitutively activated STAT3 is oncogenic, presumably as a consequence of the genes that it differentially regulates. Activated STAT3 correlated with elevated cyclin D1 protein in primary breast tumors and breast cancer–derived cell lines. Cyclin D1 mRNA levels were increased in primary rat-, mouse-, and human-derived cell lines expressing either the oncogenic variant of STAT3 (STAT3-C) or vSrc, which constitutively phosphorylates STAT3. Mutagenesis of STAT3 binding sites within the cyclin D1 promoter and chromatin immunoprecipitation studies showed an association between STAT3 and the transcriptional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1–/– and cyclin D1–/+ fibroblasts led to anchorage-independent growth of only cyclin D1–/+ cells. Furthermore, knockdown of cyclin D1 in breast carcinoma cells led to a reduction in anchorage-independent growth. Phosphorylation of the retinoblastoma (Rb) protein [a target of the cyclin D1/cyclin-dependent kinase 4/6 (cdk4/6) holoenzyme] was delayed in the cyclin D1–/– cells relative to cyclin D1–/+ cells. The E7 oncogene, whose activity includes degradation of Rb and dissociation of Rb from E2F, did not confer anchorage-independent growth to the cyclin D1–/– cells but, in conjunction with vSrc, resulted in robust growth in soft agar. These results suggest both a cdk-dependent and cdk-independent role for cyclin D1 in modulating transformation by different oncogenes. (Cancer Res 2006; 66(5): 2544-52)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signal transducers and activators of transcription (STAT) proteins are a family of transcription factors whose activity is implicated in a wide variety of biological processes, including cell proliferation and carcinogenesis. STATs are latent transcription factors activated by phosphorylation of a tyrosine residue within their SH2 domain. Upon activation, STAT proteins dimerize, translocate to the nucleus, bind to DNA, and initiate transcription of a variety of target genes. STATs are dephosphorylated and exported back to the cytoplasm in their inactive state.

In contrast to normal cells where STAT3 is transiently tyrosine phosphorylated in response to growth factors, persistent tyrosine phosphorylation of STAT3 is found in a wide variety of human cancers (1). Constitutive STAT3 activation was first described in transformed cells as a consequence of the oncogenic tyrosine kinase vSrc (2). Furthermore, persistent STAT3 activity was shown to be a requirement for vSrc-mediated transformation of immortalized fibroblasts (3, 4). In addition, a constitutively active mutant form of STAT3, STAT3-C, which is dimerized by cysteine-cysteine residues instead of pY-SH2 interactions, can transform immortalized rodent fibroblasts and breast epithelial cells (5, 6). The mechanism of cellular transformation by activated STAT3 is likely to be a result of the genes that are transcriptionally regulated by STAT3.

Activated STAT3 can up-regulate the mRNA levels of many genes, including cyclin D1 (6). The transcriptional regulation of the cyclin D1 gene is complex and involves a number of proteins, including activator protein, Oct-1, ER, TCF/LEF, Ets, SP1, cyclic AMP–responsive element binding protein, and cyclin D1 itself (7–14). The D cyclins control cell cycle progression by assembling with the cyclin-dependent kinases 4/6 (cdk4/6) to form a holoenzyme (reviewed in ref. 15). Upon activation, the holoenzyme mediates progression of cells through the G1 phase of the cell cycle by phosphorylating substrates, such as the retinoblastoma protein (Rb). Phosphorylation of the Rb protein releases E2F transcription factors, leading to transcription and S-phase entry. Overexpression of cyclin D1 is found in many cancers and is sufficient to mediate mammary tumorigenesis (16, 17). Furthermore, cyclin D1–deficient animals are resistant to ras and neu-mediated skin and breast tumorigenesis, showing a requirement of cyclin D1 in promoting these kinds of tumors (18, 19).

Here, we have shown that activated STAT3 interacts with the cyclin D1 promoter and regulates its expression. Furthermore, we have determined that cyclin D1 is required for STAT3-C–mediated and vSrc-mediated anchorage-independent growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids constructs, transfections, luciferase assays, and protein and RNA analysis. Murine STAT3-C in RcCMV, pBabe/vSrc, and RcCMV/vsrc constructs and pBabe vectors were previously described (5, 20). pBPSTR cyclin D1 and the human cyclin D1 luciferase 1745 promoter construct were described previously (9). Cyclin D1 small interfering RNA (siRNA) constructs were generated by Qiagen (Valencia, CA): forward, 5'-CAAGCUCAAGUGGAACCUGdTdT-3'; reverse, 5'-CAGGUUCCACUUGAGCUUGdTdT-3'; and nonsilencing control: forward, 5'-UUCUCCGAACGUGUCACGUdTdT-3'; reverse, 5'-ACGUGACACGUUCGGAGAAdTdT-3' were transfected into cells using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. pBabe/Ras was a gift from P. Sicinski (Dana-Farber Cancer Institute; ref. 19). Truncations of the 1745 promoter were generated by introduction of restriction sites at the indicated sites (9). Mutant constructs of the cyclin D1 luciferase 1745 promoter were generated by site directed mutagenesis, whereby each STAT binding site was converted from TTNNNNNAA to AANNNNNAA using Quick Change Mutagenesis according to the manufacturer's instructions (Stratagene, La Jolla, CA). The pBabe E7 human papillomavirus construct was a gift from Denise Galloway (Fred Hutchinson Cancer Center, Seattle, WA). Luciferase assays were carried out according to the manufacturer's instructions (Promega, Fitchburg Center, WI) by cotransfecting cDNA expression plasmids, reporter genes, and Renilla luciferase (Dual-Luciferase Reporter Assay) to normalize for transfection efficiency. Nuclear and cytoplasmic extracts were prepared as previously described (21). Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA), and Western blot analysis was carried out by standard methods (22). RNA was isolated using the Rneasy kit (Qiagen).

Antibodies, Western blotting, Northern blotting, and electrophoretic mobility shift assay. Antibodies for Western blots: cyclin D1 (sc-718, 1:1,000), cyclinA (sc-239, 1:1,000), cyclin E (sc-48, 1:1,000), STAT3 (sc-482X), and STAT1(sc-346X) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); cyclin D2 (Ab-4) and cyclin D3 (Ab-1) were from NeoMarkers (Lab Vison Corp., Fremont, CA); FLAG, M2 (F3165), and tubulin (T5168) were from Sigma (St. Louis, MO); STAT3 (9132) and Tyr705 STAT3 (9131; 1:1,000) were from Cell Signaling (Beverly, MA); anti-Rb (G3-245) was from PharMingen (San Jose, CA). vSrc antibody was a gift from Marilyn Resh (Cell Biology, Sloan Kettering Institute, New York, NY). Northern Blots were carried out as previously described (20). Electrophoretic mobility shift assay (EMSA) assays were carried out as previously described (20) using the anti-FLAG (F3165), anti-STAT3(sc-482x), and anti-STAT1(sc-346x) for supershifting.

Cell culture, Janus-activated kinase inhibitor, retroviral infections, and soft agar assay/anchorage-independent growth. NIH 3T3 and 293T cells (American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 10% cosmic calf serum (HyClone, Logan, UT). 3Y1 cells (provided by H. Hanafusa, Osaka Bioscience Institute, Osaka, Japan) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). 2fTGH cells (provided by G. Stark, Lerner Research Institute, Cleveland, OH) were grown in DMEM containing 10% cosmic calf serum. STAT3-C expressing human mammary epithelial cells (HMEC) were grown in MEGM (Cambrex, Walkersville, MD; ref. 5). MDA-MB-468 and MDA-MB-435 cell lines (American Type Culture Collection) were grown in DMEM-HG-F12-NEAA-Na Pyruvate supplemented with 10% FCS. Cells were treated with 0.5 µmol/L Janus-activated kinase (JAK) inhibitor I (Calbiochem, San Diego, CA; refs. 23, 24). The cyclin D1–deficient and matched wild-type mouse embryonic fibroblasts (MEF) were prepared as previously described (25) from the mating of cyclin D1+/– animals (a gift from P. Sicinski). Immortalization of the MEFs was carried out using the NIH3T3 protocol: MEFs were passaged every 3 days (1:3) until the cells senesced (after 25-30 passages), cells which grew following crisis were subcloned, and a minimum of 10 different subclones from each genetic background was isolated. Immortalized MEFs were grown in DMEM containing 10% FBS. All transfections were carried out using SuperFect (Qiagen). Retroviral infections were done using the RetroMax Retroviral Expression System pCL-Eco (Imgenex, Sorrento Valley, CA) according to the manufacturer's directions. Clonal selection was carried out using puromycin (2 µg/mL; Sigma), G418 (800 µg/mL; Sigma), or hygromycin (200 µg/mL; Sigma). A minimum of three independent clones were examined for equivalent expression of either STAT3-C, vSrc, ras, or E7 in the various genetic backgrounds. Soft agar assays were done as previously described (3).

In vitro kinase assay. vSrc kinase assays were done as described previously (26). Radioimmunoprecipitation assay extracts were isolated and vSrc was immunoprecipitated from 1 mg of protein (1:200 anti-vSrc). Incorporation of [{gamma}-32P]ATP into enolase or vSrc was visualized by autoradiography. The same blot was probed with anti-vSrc antibody.

Chromatin immunoprecipitation. Chromatin immunoprecipitation assays were done on 2fTGH cells expressing STAT3-C or control vector by using the chromatin immunoprecipitation assay kit (Upstate Biotechnology, Waltham, MA). STAT3-DNA and STAT3-C/DNA complexes were precipitated by using anti-STAT3 antibody (9132, Cell Signaling) or M2 antibody (Sigma). Polyclonal IgG antibody was used as a negative control. Precipitated DNA was amplified by radioactive PCR using primers flanking the gamma-activated sites (GAS) site at –984 and primers surrounding GAS sites at –568 and –475. Primers used for PCR were as follows: 5'-GCACCAAAGAGACAGAAC-3' (–1295) and 5'-TTAACCGGGAGAAACACACC-3' (–891), or 5'-AACTTGCACAGGGGTTGTGT-3' (–627) and 5'-GAGACCACGAGAAGGGGTGACTG-3' (–405). Primers used for PCR in the NIH3T3 chromatin immunoprecipitation experiments were as follows: 5'-GGGGGAACACCACCACCCTC-3' (–1157) and 5'-GCAACAGCTCAAGATGGTGGCC-3' (–845).

Immunohistochemistry. Multitissue blocks of formalin-fixed, paraffin-embedded breast cancer tissue (containing four representative 0.6-mm cores) were prepared by using a tissue arrayer, and immunohistochemistry for phospho-STAT3 was done as described (5). Cyclin D1 rabbit monoclonal antibody (Lab Vision) was used at a 1:50 dilution. Scoring of the tissue microarray was done by two independent observers (J.F.B. and W.G.). For a tumor to be considered positive for either phospho-STAT3 or D1, all four replicates in the tissue array had to have a similar staining intensity; otherwise, it was excluded. Statistical analyses were done by using STATVIEW (SAS Institute, Cary, NC). The correlation between the scores of both scorers and the relationship between that of phospho-STAT3 and cyclin D1 were measured by using the {chi}2 test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activated STAT3 correlates with elevated cyclin D1 protein in primary breast tumors and breast cancer–derived cell lines. High levels of activated STAT3 correlate positively with elevated cyclin D1 mRNA and protein expression in both cell lines and tumors (20, 27–32). We and others previously reported that by immunohistochemical analysis of tissue microarrays (TMA) of primary breast cancer specimens (36 tumors and 8 normal), 28% contain high levels (+++) of nuclear phospho-STAT3 (pYSTAT3), 33% contain moderate levels of pYstat3 (++), and 39% contain no to little pYSTAT3 (5, 33). Sequential sections of these TMAs (36 primary breast carcinomas) were examined for cyclin D1 expression by immunohistochemistry and a positive correlation (P < 0.001) between high to moderate levels of tyrosine phospho-STAT3 and elevated cyclin D1 protein was observed (Fig. 1A). MDA-MB-468 and MDA-MB-435 breast cancer–derived cell lines express constitutively activated STAT3. Treatment of these cell lines with either a JAK2 inhibitor or with a novel anti-STAT3 compound led to a decrease in STAT3 activity with subsequent growth arrest and a concomitant decrease in cyclin D1 levels (32, 34). Similarly, we treated these cell lines with a JAK inhibitor, which led to both a reduction in phospho-STAT3 and a decrease in cyclin D1 levels (Fig. 1B). These observations suggest that activated STAT3 may play a role in regulating the abundance of cyclin D1.


Figure 1
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  		Figure 1. Activated STAT3 correlates with elevated cyclin D1 protein in primary breast tumors and breast cancer–derived cell lines and leads to increased cyclin D1 mRNA levels in rat-, murine-, and human-derived cell lines. A, immunohistochemistry was done on sequential sections of 36 primary breast cancer TMAs with antiphospho-STAT3 (pYStat3) and anti-cyclin D1 antibodies. A schematic overview of the tissue arrays and a summary of the immunohistochemistry results. Representative sections of strong staining (+++ and black), moderate staining (++ and gray), and weak to no staining as (0 and white). A positive correlation was observed between (+++/++) staining for phospho-STAT3 and cyclin D1 (P < 0.001) by {chi}2 test. B, MDA-MB-468 and MDA-MB-435 cells were grown in the presence of DMSO (C) or 0.5 µmol/L JAK inhibitor (JI) for 6 hours. Extracts were isolated and analyzed by Western blot for tyrosine phospho-STAT3 (pYStat3), total STAT3, cyclin D1, and tubulin. C, RNA from rat 3Y1 fibroblasts, (D) mouse NIH3T3 fibroblasts, and (E) human 2fTGH fibrosarcoma-derived cell lines expressing vector control (Rc), STAT3-C (3-C), or v-Src (vsrc) and HMECs expressing vector control (pB) and STAT3-C (3-C) were isolated, and cyclin D1 mRNA levels were determined by Northern blot analysis and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). F, cyclin D1 protein levels were determined in the 2fTGH-derived cell lines by Western blot and normalized to tubulin.

 
Activated STAT3 leads to increased cyclin D1 mRNA levels in rat-, mouse-, and human-derived cell lines. We presume that activated STAT3 transcriptionally regulates cyclin D1. Thus, cyclin D1 mRNA levels were examined in rat-derived (3Y1), mouse-derived (NIH3T3), and human-derived (2fTGH and HMECs) cell lines expressing a constitutively activated STAT3 (STAT3-C). Similarly, vSrc, a tyrosine kinase that constitutively phosphorylates STAT3, was introduced into the same three cell lines. Northern blot analysis revealed elevated levels of cyclin D1 RNA in STAT3-C– and in vSrc-expressing rat-, mouse-, and human-derived cell lines compared with vector control–expressing cell lines (Fig. 1C-E). In addition, cyclin D1 protein levels were increased in the 2fTGH cells expressing either STAT3-C or v-Src compared with vector containing 2fTGH cells (Fig. 1F). Thus, in three different mammalian species, we observed an increase in cyclin D1 mRNA levels in cells expressing constitutively activated STAT3.

Cyclin D1 promoter exhibits significant identity between species and contains multiple STAT binding sites. Given the observed increase in cyclin D1 mRNA by activated STAT3 across three species, we compared the cyclin D1 promoter sequences among the rat, mouse, and human genes. A 1-kbp upstream region from the putative transcriptional start site of cyclin D1 was used in pairwise Basic Local Alignment Search Tool (BLAST) searches to ascertain the percentage of identity in this region between organisms. The regions of maximal identity between the rat and mouse promoters and between the mouse and human promoters are outlined in Fig. 2A. The percentage of identity between the rat and mouse cyclin D1 promoter sequences was ≥86% (indicated by black bars), whereas an identity of ≥78% was found comparing the mouse to human sequences (indicated by grey bars). STAT binding recognition sites, also known as GAS sites (TTN5AA), were found throughout each of the promoter sequences analyzed and are indicated by shaded triangles. The rat, mouse, and human promoters contain four putative GAS sites conserved in relative location to one another. These can be found at positions –983/–971/–984, –530/–521/–568, –286/–278/–239, and –27/–27/–27 in the rat, mouse, and human promoters, respectively (Fig. 2A).


Figure 2
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  		Figure 2. Cyclin D1 is a direct transcriptional target of STAT3. A, 1 kb of rat, mouse, and human cyclin D1 promoter sequence upstream of the start site of transcription were compared using pairwise BLAST alignment analysis. Sequences of >86% identity between rat and mouse (black) and sequences of >78% identity between mouse and human (gray). GAS sites (TTN5AA; {blacktriangledown}). B, a human cyclin D1 luciferase-driven promoter construct was truncated from –1,745 to –495, –168, or –61 bp bases from the start site of transcription. These promoter luciferase constructs were transiently transfected into 293T cells together with the internal control Renilla luciferase reporter (dual luciferase reporter system) and (C) STAT3-C or with (D) vSrc coupled with STAT3{alpha} or STAT3ß. Luciferase activities normalized against the internal control. Columns, mean of three experiments; bars, SD. E, GAS sites at positions –984-568, –475, and –239 of the human cyclin D1 promoter were used as EMSA gel shift probes to assess their capacity for binding activated STAT3. Anti-sera to STAT3 (s3), STAT1 (s1), high-affinity m67 binding site (c), or a scrambled m67 site (sp) were added to the reaction mixture to examine specificity. Site-directed mutagenesis of the GAS sites at –984 (GAS1), –568 (GAS2), and –475 (GAS3) within the 1745 (FL) human cyclin D1 luciferase reporter construct was done. These constructs were transiently transfected into 293T cells together with Renilla luciferase reporter and (F) STAT3-C or (G) vSrc. Luciferase activities normalized against the internal control. Columns, mean of three experiments; bars, SD. H, 2fTGH cells expressing control vector (–) or STAT3-C (+) were subjected to a chromatin immunoprecipitation assay using antibodies to STAT3, Flag (M2), or IgG as a negative control. Coprecipitated DNA was amplified by PCR using primers flanking the GAS site at –984 and primers surrounding GAS sites at –568 and –475.

 
STAT3 can be found associated with the cyclin D1 promoter, and luciferase constructs incorporating the promoter region are sensitive to mutational disruption of these GAS sites. To investigate the role of STAT3 in the transcriptional regulation of the cyclin D1 gene, a human cyclin D1 promoter luciferase construct was used. A 1,745-bp length promoter construct was truncated to exclude sequential 5' regions of the promoter to examine the effects of GAS site exclusion on luciferase activity. Specifically, the full-length 1,745-bp cyclin D1 promoter luciferase construct was truncated to –495, –168, and –61 bp from the 3' end of the promoter (Fig. 2B). STAT3-dependent regulation of these luciferase constructs was determined by cotransfecting the cyclin D1 luciferase constructs with a STAT3-C expression plasmid into 293T cells (Fig. 2C). STAT3-C–mediated expression of the cyclin D1 luciferase constructs decreased by more than half when two putative STAT3 binding sites were eliminated. The loss of two more putative sites (–168) led to a further decrease in activity. To principally evaluate activated STAT3 on cyclin D1 luciferase expression, vSrc was cotransfected with either wild-type STAT3 or with a dominant-negative form of STAT3 (STAT3ß). An almost linear decrease in luciferase activity was observed in the truncated reporters (Fig. 2D).

Four putative GAS sites (locations –984, –568, –475, and –239) within the human cyclin D1 promoter were examined by EMSA. All four sites bound activated STAT3 with variable affinities (Fig. 2E). Specificity was shown by disruption of the STAT3 binding-complex with either {alpha}-STAT3 antibody or competing for the binding complex with a high-affinity binding site (m67) for STAT3. No disruption of binding was observed using {alpha}-STAT1 antibody or a mutant m67 (Fig. 2E for the –984 GAS; data not shown).

To further confirm the ability of STAT3 to bind and regulate transcription of the cyclin D1 promoter luciferase construct, the GAS sites at –984, –568, –475, and –239 were mutated from TTN5AA to AAN5AA. These mutations were introduced singularly, as well as in combination to elucidate the level of synergy between GAS sites. The mutant cyclin D1 luciferase constructs were cotransfected with STAT3-C (Fig. 2F) or with vSrc (Fig. 2G). Each mutated luciferase construct resulted in at least a 3-fold or 2-fold decrease in expression when compared with the intact full-length 1,745-bp construct for STAT3-C– and vSrc-expressing cells, respectively. The combinatorial mutant constructs exhibited a further decrease in luciferase activity below the single mutant levels. When v-Src was cotransfected with the luciferase constructs, the mutations had a less dramatic effect but nonetheless showed decreased reporter activity in all mutants. In both cases, the –568 mutation displayed the greatest change in reporter activity.

Cross-linking chromatin immunoprecipitation assays were carried out to determine whether the potential GAS sites within the cyclin D1 promoter-bound STAT3. Primer sets were used to target the regions of the human cyclin D1 promoter corresponding to the GAS site found at –984, or the GAS sites found at –568, and –475. Cell extracts from 2fTGH cells expressing either Flag-tagged STAT3-C or RcCMV control were subjected to chromatin immunoprecipitation analysis with antibodies to STAT3, Flag, or IgG (negative control). Amplification by PCR of both promoter targets was observed in the samples expressing STAT3-C when either anti-Flag or anti-STAT3 antisera was used to immunoprecipitate the protein-DNA complexes (Fig. 2H). These data indicate that activated STAT3 can be found associated with the human cyclin D1 promoter. Chromatin immunoprecipitation assays were done on mouse NIH 3T3 cells expressing STAT3-C, which was found to bind to the GAS site at position –976 on the mouse cyclin D1 promoter (data not shown; K.P. and J.E.D). The other potential STAT3 binding sites were not examined.

Reduction of cyclin D1 leads to a G1 arrest and a marked reduction in anchorage-independent growth of MDA-MB-435 cells. Activated STAT3 is capable of inducing cellular transformation of immortalized cell lines (5, 6). Transformation is likely to be a consequence of the genes that are transcriptionally regulated by STAT3. We hypothesized that expression of cyclin D1 was critical for the growth of the MDA-MB 435 breast cancer derived cell line (see Fig. 1B). siRNA to cyclin D1 was transfected into this cell line and a reduction in cyclin D1 protein was observed over a 7-day period compared with control siRNA (Fig. 3A). Cell cycle analysis of these cells after 48 hours revealed a block in G1 similar to that of cells, which were serum starved (Fig. 3B; data not shown). Transfected cells were placed in soft agar to examine anchorage-independent growth, and after 7 days, colonies were counted and found to be significantly decreased in the cells expressing less cyclin D1 (Fig. 3C). Thus, cyclin D1 seems to be required for cell cycle progression and anchorage-independent growth of MDA-MB-435 cells.


Figure 3
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  		Figure 3. Reduction of cyclin D1 leads to a G1 arrest and a marked reduction in anchorage-independent growth of MDA-MB-435 cells. STAT3-C induced anchorage-independent growth of WT but not D1–/– cells. A, MDA-MB-435 cells were transfected with siRNA to D1 (D1) or control siRNA (C), and after 2, 5, and 7 days, cell extracts were analyzed by Western blot for cyclin D1 expression and normalized using tubulin. B, after 2 days, a fraction of these transfected cells were analyzed by fluorescence-activated cell sorting for cell cycle distribution and displayed as % G1, S, G2. C, 5,000 cells/3 mL were plated into soft agar 2 days after transfection, and colony number was determined after 7 days. This experiment was done in triplicate. Bars, SD. STAT3-C (3-C) and pBabe (pB) control vector were introduced into immortalized WT and D1–/– MEFs. Nuclear extracts from STAT3-C– and pBabe-expressing cell lines were examined for expression of Flag-tagged STAT3-C by (D) Western blot analysis and (E) EMSA gel shift. Specificity of binding was determined by supershifting the STAT3-C complex using an M2 antibody. F, STAT3-C–expressing D1–/– and WT cells were plated in soft agar, and number of colonies formed were scored after 2 weeks. All experiments were done in triplicate. Bars, SD.

 
Activated STAT3 transforms wild-type but not cyclin D1–deficient cells. To investigate a dependency of cyclin D1 expression in STAT3-mediated transformation, both wild-type (WT) and cyclin D1–deficient (D1–/–) MEFs from littermates were generated by crossing cyclin D1 heterozygotes (25). Primary MEFs were immortalized using the NIH 3T3 method (35). These cell lines were retrovirally infected with either pBabe vector control or STAT3-C. A minimum of five independent cell lines expressing STAT3-C or control vector were studied for the remaining analyses. Expression levels of STAT3-C were approximately equal in both cell lines as determined by Western blot analysis (Fig. 3D) and EMSA (Fig. 3E). Both WT and D1–/– cells, expressing equivalent amounts of STAT3-C, were grown in soft agar to determine their capacity for anchorage-independent growth. D1–/– lines expressing STAT3-C did not form colonies in soft agar, in contrast to the STAT3-C–expressing WT cells (Fig. 3F).

To investigate whether the above results were specific to STAT3-C, we retrovirally infected WT and D1–/– cells with vSrc, and cell lines were established. Extracts were isolated from the D1–/– or WT cell lines containing vSrc and vSrc activity, and expression was determined to be equal in these cell lines by immunoprecipitation-kinase assays and Western blot analysis. Phospho-vSrc (Fig. 4A) or phospho-enolase (Fig. 4B) levels were equivalent in these cell lines. Immunoprecipitation efficiency and vSrc expression was determined for each cell line using Western blot analysis to detect vSrc protein (Fig. 4C). We also examined the in vivo capacity of vSrc to phosphorylate STAT3 by EMSA gel shift analysis and Western blot revealing equivalent levels of phospho-STAT3 and binding in both the D1–/– and WT cell lines (Fig. 4D). Next, we determined the ability of the vSrc expressing cells to grow in an anchorage-independent manner. WT MEFs expressing vSrc formed >500 colonies per 20,000 plated, whereas the vSrc expressing D1–/– cells formed only 5 to 10 colonies per 20,000 plated (Fig. 4E). pBabe control containing cell lines did not form colonies in agar. Introduction of activated Ras has previously been shown to induce transformation of cyclin D1–/– fibroblasts (19). To determine whether the immortalized D1–/– cells could be transformed, activated Ras was introduced and expressed in the D1–/– cells, and these cells were observed to grow in an anchorage-independent manner (Fig. 4E). The levels of the other G1 cyclins (cyclins D2, D3, and E) were similar in the WT and D1–/– cells and vSrc expression led to an increase in cyclin E levels in both cell lines (Fig. 4F). Thus, the lack of cyclin D1 conferred resistance to growth in soft agar by vSrc and STAT3-C but not by Ras.


Figure 4
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  		Figure 4. vSrc induced anchorage-independent growth of WT but not D1–/– cells. vSrc (vs) and pBabe (pB) control vector were introduced into immortalized WT and D1–/– MEFs. Radioimmunoprecipitation assay extracts were isolated from these cell lines, and vSrc activity and expression was determined by an immunoprecipitation kinase assay for (A) phospho-src, (B) phospho-enolase, and (C) vSrc Western blot analysis. Nuclear extracts from these cell lines were isolated and examined by EMSA for (D) STAT3 binding activity and Western blot analysis for phosphorylated STAT3 (pStat3) and total STAT3 (Stat3). E, pBabe (pB) and vSrc (vs) expressing D1–/– and WT cells were plated in soft agar, and the number of colonies formed were scored after 2 weeks. All experiments were done in triplicate. Bars, SD. F, extracts were isolated from pBabe (pB) and vSrc (vs) containing cell lines and examined for the expression of cyclins D1, D2, D3, and E and tubulin.

 
We next examined whether the independent process of immortalization of the WT and D1–/– MEFs may have led to other genetic changes, which could account for the lack of transformation by STAT3-C or vSrc in the D1–/– cells. The immortalized D1–/– cells were infected with a cyclin D1 retroviral expression construct. Extracts were isolated from the cyclin D1–reconstituted cells (D1+), WT cells, and D1–/– cells, and expression of cyclins D1, D2, D3, and E was determined by Western blot analysis (Fig. 5A). Cyclin D1 levels were similar in the reconstituted cells to that of WT cells as were the other G1 cyclin proteins. The D1–/– and D1+ cells were infected with pBabe vector control, vSrc, and STAT3-C retroviral constructs. Expression and subsequent STAT3 activity was determined by EMSA gel shift analysis (Fig. 5B). The specificity of STAT3-C binding was confirmed by supershifting the binding complex with an M2 antibody. We compared the ability of these cells to grow in an anchorage-independent manner. As was observed in Fig. 3C and Fig. 4E, expression of STAT3-C or vSrc in the D1–/– cells led to no or minimal colony formation (Fig. 5C and D). In contrast, the D1+ cells expressing either STAT3-C or vSrc grew in soft agar showing a requirement for cyclin D1 in mediating anchorage-independent growth by activated STAT3 (Fig. 5C and D).


Figure 5
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  		Figure 5. Reintroduction of cyclin D1 into cyclin D1–/– fibroblasts restored anchorage-independent growth by STAT3-C and vSrc. cyclin D1–/– cells were infected with pBPSTR cyclin D1, and cell lines were isolated. A, extracts from D1–/–, D1+, and WT cell lines were isolated, and expression of cyclins D1, D2, D3, and E and tubulin was determined by Western blot analysis. D1+ and D1–/– cells were infected with pBabe (pB), vSrc, or STAT3-C (3-C), and cell lines were established. B, nuclear extracts from these cell lines were examined by EMSA for expression of activated STAT3. M2 antibody was used to supershift (SS) the STAT3-C containing complex. C, STAT3-C (3-C) or vSrc (vs) expressing D1–/– or D1+ cells were plated in soft agar, and the number of colonies formed were scored after 2 weeks. All experiments were done in triplicate. Bars, SD.

 
Oncoprotein E7 can obviate the requirement for cyclin D1 in vSrc-mediated anchorage-independent growth. We attempted to determine a mechanism by which cyclin D1 was required for vSrc-mediated transformation of immortalized MEFs. A principal activity of cyclin D1 is to regulate the activity of cdk4/6 who in turn phosphorylates and inactivates the Rb protein (36). The Rb protein mediates progression through the G1-S phase of the cell cycle via its interactions with the E2F transcription factor. Upon phosphorylation of Rb by the cyclin D1/cdk4/6 complex, pRb releases the E2F factor, allowing transcriptional activity, entry into the S phase of the cell cycle, and subsequent cell proliferation (37). The phosphorylation status of the Rb protein in the D1–/–, D1+, and WT cell lines was examined by Western blot analysis using a phospho-Rb antibody (Fig. 6A and B). These cell lines were serum starved for 16 hours, leading to partial cell cycle arrest, and following the addition of serum, extracts were isolated at the indicated times (Fig. 6A and B). Upon release from serum starvation, a slower migrating hyperphosphorylated form of Rb (pp-Rb) appeared by 6 hours in the D1+ and WT cells, whereas ppRb levels were observed 16 hours after the addition of serum in the D1–/– cells. The rates of proliferation for these cells was determined by tritiated-thymidine uptake. D1–/– cells grew at approximately the same rate as WT and D1+ cells, which suggested that the delay in Rb phosphorylation in the D1–/– cells did not significantly affect the rate of growth of the D1–/– cells (data not shown; ref. 19).


Figure 6
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  		Figure 6. Rb phosphorylation is delayed in the D1–/– cells, and introduction of the E7 oncoprotein restores anchorage-independent growth of vSrc expressing D1–/– cells. A, D1–/–and D1+ reconstituted cells were serum starved overnight leading to partial cell cycle synchronization and released with serum containing medium. Extracts were isolated at the indicated times and analyzed for phospho-Rb (pRb) protein by Western blot analysis. B, phospho-Rb levels were also examined in WT versus D1–/– cells following release from serum starvation (C and D). D1–/– and D1+ cell lines expressing either pBabe (pB) control or vSrc were infected with a retrovirus encoding the E7 oncoprotein. D1–/– and D1+ cells expressing either vSrc (S), E7, or both vSrc and E7 (S/E7) were plated in soft agar and assayed for colony number after 2 weeks. Increasing numbers of cells were plated for each cell type and ranged from 5 x 103 to 40 x 103 cells per well. All experiments were done in triplicate. Bars, SD.

 
To determine whether the delay in Rb phosphorylation could account for the resistance of D1–/– cells to transformation by vSrc, the human papilloma E7 oncogene was introduced by retroviral infection into the D1–/– and D1+ cell lines. The E7 oncoprotein can inhibit the interaction between Rb and the E2F proteins as well as target Rb for degradation (38). Thus, expression of the E7 protein should obviate a need for Rb phosphorylation. Expression of E7 alone induced anchorage-independent growth of D1+ cells as did vSrc and expression of both was synergistic (Fig. 6C). In contrast, expression of either vSrc or E7 individually failed to promote anchorage-independent growth in the D1–/– cells, whereas expression of both E7 and vSrc resulted in robust colony formation (Fig. 6D). Thus, a potential mechanism of resistance to transformation by vSrc in the D1–/– cells is through differential Rb phosphorylation, which is obviated by E7.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activated STAT3 is found in a large number of malignancies. A causal role of activated STAT3 in promoting cellular transformation has been suggested by studies which inhibited STAT3 function both in vitro and in vivo (39, 40). Here, we show a positive correlation between elevated levels of tyrosine phospho-STAT3 and cyclin D1 in primary breast tumors (Fig. 1). In addition, JAK kinase inhibition resulted in a reduction in tyrosine phospho-STAT3 in breast cancer–derived cell lines, which led to a reduction in cyclin D1 protein (Fig. 1; refs. 32, 34). Furthermore, introduction of STAT3-C into a number of cell types has led to cellular transformation (5, 6). These observations suggested that activated STAT3 may play a critical role in tumorigenesis. Deregulated expression of transcription factors can mediate oncogenesis through differential gene expression (41). Thus, identifying STAT3 target genes and determining their relative importance to cellular transformation guides us in understanding mechanisms of transformation (30). A number of transcripts have been described to be increased in cancer cells following STAT3 activation, including cyclin D1, BclXL, survivin, Mcl-1, c-fos, vascular endothelial growth factor (VEGF), c-Myc, schlaefen, proliferin, and matrix metalloproteinase-9 (5, 6, 30, 42–44). With respect to transformation of cells, one of the more interesting transcripts increased in cells expressing activated STAT3 is cyclin D1.

We have found that cyclin D1 mRNA and protein levels are increased as a function of activated STAT3 in rat-, mouse-, and human-derived cell lines. The promoter of the cyclin D1 gene in all three species contains conserved GAS sites. Disruption of these GAS sites in the human promoter led to a decrease in STAT3-mediated transcriptional activation. Furthermore, activated STAT3 could interact with both the human and mouse cyclin D1 promoter by chromatin immunoprecipitation analysis. These data show that STAT3 can directly regulate transcription of the cyclin D1 gene. However, the presence of activated STAT3 does not necessarily result in increased expression of cyclin D1 in all cells. For example, in normal rat fetal liver cells, STAT3 activation by cytokines or introduction of STAT3-C led to a decrease in cyclin D1 mRNA levels (27). These data suggest that activated STAT3 can initiate cyclin D1 transcription in conjunction with other transcription factors, which may not be found in certain normal cells. For example, STAT3 in association with c-jun can activate the rat {alpha}-2 M gene and repress transcription of Fas (45, 46). Additionally, SP1 in conjunction with STAT3 can activate the VEGF gene or C/EBP{delta} gene (47, 48).

Cyclin D1 has been recognized as a critical mediator of tumorigenesis. More than 30% of human mammary carcinomas overexpress cyclin D1 protein as do a number of other malignancies (49). Transgenic mice that overexpress cyclin D1 in the mammary glands develop breast cancer, showing a sufficiency of enhanced cyclin D1 expression in mediating tumorigenesis (17). Cyclin D1–deficient mice were resistant to breast cancers induced by the ras and neu oncogenes (19). In contrast, immortalized MEFs from cyclin D1–deficient mice were fully transformed by the neu and ras oncogenes (19). It was suggested that the differential dependence of cyclin D1 in ras induced transformation was due to the presence of compensatory cyclin D2 in MEFs, which was lacking in mammary epithelial cells (19). However, cyclin D1–deficient keratinocytes express cyclins D2, D3, and E, yet ras-mediated tumorigenesis of these cells was markedly reduced (18). Here, we show that vSrc- and STAT3-C–mediated anchorage-independent growth of immortalized MEFs is dependent upon cyclin D1 expression, whereas introduction of ras into cyclin D1–deficient MEFs led to robust growth in soft agar. It is striking that despite the myriad of genes that are regulated by STAT3-C or vSrc, that a dependence upon cyclin D1 for anchorage-independent growth was observed (30). Defining those genes that are regulated by ras, for example, and not by src may elucidate those pathways that can obviate a need for cyclin D1. The mechanism(s) by which different oncogenes are differentially dependent upon cyclin D1 for transformation is not clear but is likely related to the different functions of cyclin D1.

Cyclin D1 functions in part as a regulator of the cdk4/6 holoenzyme, which inactivates the Rb protein promoting progression through the cell cycle. We observed a delay in Rb phosphorylation in the D1–/– cells compared with the D1+ or WT cells (Fig. 6A and B), which might explain their resistance to vSrc-mediated transformation. The E7 oncoprotein that functions in part by targeting the Rb protein for degradation, as well as disrupting the Rb-E2F association, was introduced into vSrc-expressing D1+ and D1–/– cell lines. E7 expression led to growth in soft agar of D1+ cells and in conjunction with vSrc led to robust anchorage-independent growth in D1–/– cells, whereas E7 alone did not lead to transformation of D1–/– cells (Fig. 6D; refs. 50, 51). The inability of E7 to transform D1–/– cells indicates a cdk-independent function of cyclin D1 in anchorage-independent growth.

These data suggest that both the cdk-dependent and cdk-independent roles of cyclin D1 function in promoting anchorage-independent growth. Cyclin D1's cdk-independent functions include chromatin remodeling by associating with histone deacetylases and p300 (52). Furthermore, cyclin D1 can directly associate with STAT3 and can modulate its transcriptional activity (in a cdk-independent manner; ref. 53). It remains to be determined which functions of cyclin D1 are required in modulating transformation by different oncogenes. Many tumors, including breast, head and neck, and lung cancers, express constitutively activated STAT3 in part as a consequence of abnormal src signaling (34, 54, 55). We speculate that these tumors might display a dependency on cyclin D1 for growth and transformation, perhaps as a consequence of differential Rb phosphorylation. Furthermore, we predict that tumors mutant for Rb would not be dependent upon cyclin D1 expression. Nonetheless, we believe that certain tumors will undoubtedly be dependent upon the cdk-regulatory function of cyclin D1, and it would be interesting to determine whether these tumors are also dependent upon STAT3 or possibly src for their malignant phenotype.


   Acknowledgments
 
Grant support: NIH grant R01 CA87637, McDonnell S. Foundation Award, Charles E. Culpeper Scholarship Award, Breast Cancer Alliance Award, Lerner and Leigh Awards (J.F. Bromberg).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Marilyn Resh for the vSrc antibody, H. Hanafusa for 3Y1 cells and the vSrc construct, Denise Galloway for the pBabe/E7 construct, and Peter Sicinski for the pBabe/Ras construct and for the D1-deficient animals.

Received 6/23/05. Revised 1/ 3/06. Accepted 1/18/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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