Molecular and Cellular Pathobiology De fi nition of Smad 3 Phosphorylation Events That Affect Malignant and Metastatic Behaviors in Breast Cancer Cells

Smad3, amajor intracellular mediator of TGFb signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFb, the TGFb receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show thatmutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFb-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial– mesenchymal transition, and invasive activity. Moreover, all TGFb responses were completely lost onmutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. Cancer Res; 74(21); 1–11. 2014 AACR. Introduction TGFb is the prototype of a large family of pleiotropic polypeptide cytokines that regulate a multitude of physiologic functions such as cell proliferation, differentiation, apoptosis, and epithelial–mesenchymal transition (EMT; refs. 1–3). Functional aberrations of the TGFb signaling pathway underlie various forms of human cancer, fibrotic, and developmental disorders (3, 4). The ability of TGFb signaling to suppress proliferation of epithelial, neuronal, and hematopoietic cells, fromwhich amajority of human neoplasms originate, suggests a role of TGFb in tumor suppression (1–3, 5). However, TGFb has also been known to exert a multifaceted influence on gene expressions that are favorable to metastasis and cancer progression, depending on the clinical stages (1–5). TGFb signals are mediated through a multiplicity of pathways, including the predominant Smad signaling pathway, as well as MAPK, Rho-like GTPase, and PI3K pathways (6–8). In the canonical TGFb pathway, Smad2 and Smad3 are phosphorylated by the type I receptor kinase at the C-terminal SXS motif, form complexeswith Smad4, and then accumulate in the nucleus to regulate gene expression of target genes (6–8). Studies using mouse embryonic fibroblasts with a targeted deletion of either Smad2 or Smad3 showed that Smad3 is an essential mediator of TGFb-induced transcriptional responses (4, 9). The Smad proteins consist of conserved Nand Cterminal domains, separated by a divergent linker region. The Smad3 linker region containing four serine/threonine phosphorylation sites at Thr179, Ser204, Ser208, and Ser213 (10–18) can be phosphorylated by several kinases, such as the MAPK family and cyclin-dependent kinase (CDK) family (10–13, 15– 20). These residues except Ser213 are subject to phosphorylation in response to TGFb (11, 12, 14). Although phosphorylation of Smad3 C-tail SXS motifs by the TGFb type I receptor is required for the linker phosphorylation, the receptor itself does not phosphorylate the linker region (13). Interestingly, Smad linker phosphorylation has been associated with more aggressive disease in certain cancer and fibrosis models (18). Previous reports showed that mutation of the Smad3 linker CHA Cancer Research Institute, CHA University, Seoul, Korea. Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland. Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, Japan. Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, Korea. Genome Resource and Analysis Unit, RIKEN Center for Developmental Biology, Kobe, Japan. Department of Biology and Biomedical Science, Dong-A University, Busan, Korea. Department of Statistics, Seoul National University, Seoul, Korea. Center for Advanced Biotechnology and Medicine, Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, New Jersey. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). E. Bae and M. Sato contributed equally to this article. Current address for M. Sato: Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka 545–8585, Japan. Corresponding Authors: Akira Ooshima, CHA Cancer Institute, 605 Yeoksam-dong, Gangnam-gu, Seoul 135-081, Korea. Phone: 02-34682647; Fax: 02-3468-2651; E-mail: aooshima@cha.ac. kr; and Seong-Jin Kim, E-mail: kimsj@cha.ac. kr doi: 10.1158/0008-5472.CAN-14-0803 2014 American Association for Cancer Research. Cancer Research www.aacrjournals. org OF1 Research. on July 21, 2017. © 2014 American Association for Cancer cancerres.aacrjournals. org Downloaded from Published OnlineFirst September 9, 2014; DOI: 10.1158/0008-5472.CAN-14-0803