Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10061 RESEARCH ARTICLE Attenuation of Smad2 activity shows resistance to TGF-b signalling in mammary adenocarcinoma (MCF-7) cells Suman Sengupta 1,2 , Subhadip Kundu 1,2 and Arindam Bhattacharyya 1 * 1 Immunology Lab, Department of Zoology, University of Calcutta, Kolkata, West Bengal 700019, India 2 Department of Environmental Science, University of Kalyani, West Bengal 741235, India Abstract Transforming growth factor-b (TGF-b) is a potent inhibitor of the growth of normal mammary epithelial cells, and has a pleiotropic, context-dependent, concentration-dependent action. We found attenuation of TGF-b signalling in mammary adenoma carcinoma cells. Phosphorylation at the linker site of Smad2 occurred in a cooperative way during the attenuation of TGF-b signalling, and was associated with upregulation of CDK2 and cyclin D1. CDK2 inhibitor restored the anti-proliferative effect of TGF-b by upregulating p21, with inhibition of linker phosphorylation of Smad2. CDK2-mediated linker phosphorylation of Smad2 may be a plausible mechanism for the attenuation of TGF-b signalling in breast cancer. Keywords: breast cancer; CDK2; P 21 ; phospho linker Smad2; Smad2; TGF-b Introduction The transforming growth factor-b (TGF-b) family plays a pivotal role not only in the normal development of mammary glands, but also in the progression of human breast cancer (Chaudhury and Howe, 2009). It has a dual role which is completely time specific. At the early stage of cancer, the molecules are tumour suppressive, the effect completely reverses but gradually with the progression of the disease (Massague, 2008). In the later stages, it augments the growth- promoting effect involving enhancement of invasion, metastasis and angiogenesis. The effects of TGF-b can vary from being context-dependent, concentration-dependent, pleiotropic and can also indirectly have effects on different types of cells (Reiss, 1999). Despite the fact that TGF-b causes cell cycle arrest, it also aids cell proliferation in some cancer cell lines. The exact role and mode of action in cell cycle regulation in cancer cell line is emerging, but is not well understood. Smads are the most important downstream molecule which relay information from the cytoplasm to the nucleus in the TGF-b signalling pathway (Massagu e and Wotton, 2000; Moustakas et al., 2001). The diversification of TGF-b signalling depends upon phosphorylation taking place at different phosphorylation-prone sites on Smad (Wringhton et al., 2008). The mechanism involving the phosphorylation of Smad molecules is not clearly understood. For instance, phosphorylation at the c-terminal regions of Smad2 and Smad3 results in binding with Smad4 and eventually the complex undergoes a translocation into the nucleus, generating transcriptional regulatory complexes that ulti- mately demonstrate a cytostatic effect of TGF-b signalling (Massague, 2008). In contrast, phosphorylation on the linker region of the Smad molecule by Cyclin D1-CDK4 and Cyclin E-CDK2 (Zelivianski et al., 2010) causes proteasome- mediated degradation of Smad (Sapkota et al., 2007; Gao et al., 2009). Proteasome-mediated degradation reduces its cytoplasmic level, making cells unresponsive to TGF-b signalling (Kretzschmar et al., 1997, 1999; Grimm and Gurdon, 2002; Pera et al., 2003). Linker phosphorylation of Smad molecule by G1 cyclin-dependent kinases is evident during the cell cycle (Matsuura et al., 2004). A similar linkage is also exhibited by GSK3b complementing the action of MAPK (Fuentealba et al., 2007; Sapkota et al., 2007). Therefore, a more detailed study is required of the mechanism of sequential phosphorylation of Smads along with the preferential phosphorylation sites. It is mandatory to investigate whether the phosphorylation of the c-terminal or  Corresponding author: e-mail: arindam19@yahoo.com Abbreviations: TGF-b, transforming growth factor b; CDK2, cyclin-dependent kinase 2; MCF, 7 cells-mammary adenocarcinoma cells Cell Biol Int 37 (2013) 449–457 ß 2013 International Federation for Cell Biology 449