Cyclin D1 Is Transcriptionally Regulated by and Required for Transformation by Activated Signal Transducer and Activator of Transcription 3 Kenneth Leslie, 1 Cynthia Lang, 1 Geeta Devgan, 1 Janeen Azare, 1 Marjan Berishaj, 1 William Gerald, 2 Young Bae Kim, 3 Keren Paz, 3 James E. Darnell, 3 Christopher Albanese, 4 Toshiyuki Sakamaki, 4 Richard Pestell, 4 and Jacqueline Bromberg 1 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 Abstract 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 differen- tially 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 transcrip- tional regulation of the human cyclin D1 gene. Introduction of STAT3-C and vSrc into immortalized cyclin D1 À/À and cyclin D1 À/+ fibroblasts led to anchorage-independent growth of only cyclin D1 À/+ 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 À/À cells relative to cyclin D1 À/+ 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 À/À 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 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 transcrip- tion 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 dephos- phorylated 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 G 1 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 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, 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@mskcc.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2203 Cancer Res 2006; 66: (5). March 1, 2006 2544 www.aacrjournals.org Research Article Research. on February 20, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from