Insulin Receptor Substrate-1 Regulates the Transformed Phenotype of BT-20 Human Mammary Cancer Cells Ozlem Dalmizrak, 1 An Wu, 1 Jia Chen, 1 Hongzhi Sun, 1 Fransiscus E. Utama, 1 Diana Zambelli, 1,2 Thai H. Tran, 1 Hallgeir Rui, 1 and Renato Baserga 1 1 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania and 2 Istituti Ortopedici Rizzoli, Bologna, Italy Abstract Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblas- toma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin- like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell prolifer- ation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells. [Cancer Res 2007;67(5):2124–30] Introduction Insulin receptor substrate-1 (IRS-1) is a docking protein for both the insulin receptor and type I insulin-like growth factor receptor (IGF-IR). It binds to and activates phosphatidylinositol 3- kinase (PI3K) and, thus, plays an important role in signal transduction from the two receptors (1). IRS-1, especially when activated by the IGF-IR, sends a strong mitogenic, antiapoptotic, and antidifferentiation signal (1, 2). IRS-1 expression is often increased in human cancer (3), and overexpression or ectopic expression of IRS-1 causes cell transformation, including the ability to form colonies in soft agar and tumors in mice (4, 5). When IRS-1 levels are decreased by experimental procedures (antisense or small interfering RNA), cancer cells lose their transformed phenotype (6–8). IRS-1 levels are low or even absent in differentiating cells (2, 9, 10), and ectopic expression of IRS-1 inhibits differentiation (4, 10). IRS-1 translocates to nuclei and nucleoli (11, 12), where it binds the upstream binding factor 1 (UBF1; refs. 13, 14), a protein that participates in the regulation of rRNA synthesis (15). Nuclear translocation of IRS-1 has been reported in tissue sections of human breast cancer (16, 17) and human medulloblastoma (11). BT-20 cells originated from a human breast cancer, but they do notformcoloniesinsoftagarorformonlyafewsmallones(5,18). BT-20 cells do not express IRS-1 (5) although they do express IRS-2 (see below). The absence of IRS-1 expression makes BT-20 cells an attractive model to test the mechanisms by which IRS-1 promotes cell transformation in mammary cancer cells. We have compared parental BT-20 cells to three BT-20–derived cell lines, all expressing IRS-1 by stable transfection with plasmids or retroviruses. BT-20/ 103 cells express modest amounts of IRS-1; BT-20/159 cells express high levels; and BT-20/NLS/IRS1 express an IRS-1 with a nuclear localization signal (19). We have used as criteria for transformation formation of colonies in soft agar (20) and xenografts in mice. Using these criteria, we show that, regardless of levels or localization, expression of IRS-1 in BT-20 cells increases colony formation in soft agar and tumorigenicity in mice. The increased transformation is accompanied by IRS-1 occupancy of the rDNA and cyclin D1 promoters; the dramatic activation of the rDNA, cyclin D1, and c-myc promoters; and the displacement by IRS-1 of the retinoblastoma tumor suppressor protein (pRb) from the rDNA promoter. Materials and Methods Cell lines and cell culture. BT-20 breast cancer cells were obtained from Eva Surmacz (Temple University, Philadelphia, PA) and cultured routinely in DMEM/Nutrient Mixture F-12 (Ham) 1:1 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% calf serum, glutamine, and antibiotics at 37jC in a 10% CO 2 atmosphere. Media were supplemented with 2 Ag/mL ampicillin for BT-20/NLS/IRS1 cells, 20 Ag/mL hygromycin for BT-20/103 cells, and 0.5 Ag/mL puromycin for BT-20/159 cells. Stable transfection of BT-20 cells with the NLS-IRS-1 plasmid. Cells were transfected with pCMV/myc/nuc plasmid (Invitrogen, Carlsbad, CA) carrying a wild-type mouse IRS-1 cDNA at the Xho I/Not I restriction site by electroporation. Nucleofector device and solutions were used (Amaxa, Gaithersburg, MD) using the program recommended by the manufacturer for this cell type. Retroviral transduction of BT-20 cells with pGR103 and pGR159 plasmids. pGR103 and pGR159 plasmids were used for retroviral transduction of BT-20 cells. Plasmid pGR103 is a pMSCVhyg plasmid carrying wild-type mouse IRS-1 cDNA with its 3¶ untranslated region (3¶-UTR). Plasmid pGR159 is based on a self-inactivated form of the MSCV retroviralvectorsystemandcontainsaninternalcytomegaloviruspromoter, a puromycin resistance gene, and wild-type mouse IRS-1 cDNA lacking the 3¶-UTR (21). Transduction was carried out as previously described (21). Cell growth and colony formation in soft agar. Formonolayergrowth, quiescent BT-20, BT-20/NLS/IRS1, BT-20/103, and BT-20/159 cells were stimulated with serum or with 20 ng/mL IGF-I (Invitrogen). Cell numbers were determined in triplicate after 24, 48, and 72 h by counting only the cells able to exclude trypan blue. Anchorage-independent growth was determined as previously described (5). Xenografts. AnimalexperimentsweredoneunderapprovedInstitutional Animal Care and Use Committee protocols. Intact female NCR athymic Requests for reprints: Renato Baserga, Department of Cancer Research, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, 624 BLSB, Philadelphia, PA 19107. Phone: 215-503-4507; Fax: 215-923-0249; E-mail: b_lupo@ mail.jci.tju.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3954 Cancer Res 2007; 67: (5). March 1, 2007 2124 www.aacrjournals.org Research Article Research. on February 13, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from