Abstract 1043: Distinctive roles of Smad3 C-tail and linker phosphorylation between cancer progression and suppression in TGF-beta1/Smad3 signal

Abstract

Abstract Transforming growth factor B1 (TGF-B) is the prototype of a large family of pleiotropic polypeptide cytokines that regulate a multitude of physiological functions. The ability of TGF-B1/Smad3 signal to inhibit proliferation of epithelial, neuronal and hematopoietic cells from which most human neoplasms originate suggests a role of TGF-B in tumor suppression. Smad3, a key transcription factor in TGF-B signaling, functions as both a positive and negative regulator in carcinogenesis. TGF-B induces phosphorylation of Smad3 C-terminal SXS motif in its canonical pathway as well as the serine/threonine residues in the linker region. The linker region has been known to contain multiple phosphorylation sites for proline-directed kinases such as CDK, c-Jun, p38 MAPK, Erk and GSK-3B. Here we present that the blockade of Smad3 C-terminal phosphorylation by adenoviral dominant-negative mutant promotes tumorigenesis, but markedly inhibits metastasis to the lung as induced by tail-vein injection and orthotopic transplant of human and mouse breast cancer cell lines. Conversely, mutation of Smad3 linker phosphorylation sites greatly suppresses tumorigenesis, while it enhances metastatic dissemination to the lung. In vitro study revealed that the Smad3 linker region is constitutively phosphorylated in the cancer cells, and that the mutation of Smad3 linker phosphorylation sites enhances the TGF-B1-induced EMT, growth arrest and apoptosis, suggesting that the canonical TGF-B1 signal mediated through C-tail phosphorylation is modulated by negative inputs from the linker phosphorylation. Furthermore, the blockade of linker phosphorylation reduces the stem cell population following suppression of mammosphere formation and downregulation of embryonic transcription factors including Oct4, NANOG and Sox-2, and induces apoptosis by repressing an antiapoptotic protein Bcl-2, suggesting that the Smad3 linker phosphorylation retains function for the maintenance of cancer stemness and survival. These results indicate that oncogenic and cancer-suppressive TGF-B1/Smad3 signals are regulated by the site-specific phosphorylation of Smad3 protein, imparting a new aspect to anti-cancer therapies that target the TGF-B signal pathway. This work was supported by a National Research Foundation grant of Korea (2009-0081756) funded by the Korea government. Citation Format: Eunjin Bae, Akira Ooshima, Seong-Jin Kim. Distinctive roles of Smad3 C-tail and linker phosphorylation between cancer progression and suppression in TGF-beta1/Smad3 signal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2014-1043