S100A2, an EF hand calcium-binding protein, is a potential biomarker in several cancers and is also a TGF-β (transforming growth factor-β)-regulated gene in melanoma and lung cancer cells. However, the mechanism of S100A2 regulation by TGF-β and its significance in cancer progression remains largely unknown. In the present study we report the mechanism of S100A2 regulation by TGF-β and its possible role in TGF-β-mediated tumour promotion. Characterization of the S100A2 promoter revealed an AP-1 (activator protein-1) element at positions -1161 to -1151 as being the most critical factor for the TGF-β1 response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays confirmed the functional binding of the AP-1 complex, predominantly JunB, to the S100A2 promoter in response to TGF-β1 in HaCaT keratinocytes. JunB overexpression markedly stimulated the S100A2 promoter which was blocked by the dominant-negative JunB and MEK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1] inhibitor, PD98059. Intriguingly, despite the presence of a putative SMAD-binding element, S100A2 regulation by TGF-β1 was found to be SMAD3 independent. Interestingly, p53 protein and TGF-β1 show synergistic regulation of the S100A2 promoter. Finally, knockdown of S100A2 expression compromised TGF-β1-induced cell migration and invasion of Hep3B cells. Together our findings highlight an important link between the TGF-β1-induced MAPK and p53 signalling pathways in the regulation of S100A2 expression and pro-tumorigenic actions.

S100 family of calcium-binding proteins is commonly upregulated in a variety of tumor types and is often associated with tumor progression. Among several S100 members, altered expression of S100A2 is a potential diagnostic and prognostic marker in cancer. Several reports suggest a role for S100A2 in metastasis. Earlier, our studies established regulation of S100A2 by transforming growth factor-β (TGF-β) and its involvement in TGF-β-mediated cancer cell invasion and migration. However, the molecular mechanisms of S100A2 protumorigenic actions remain unexplored. In the present study, we demonstrate that overexpression of S100A2 in A549 lung cancer cells induced epithelial-mesenchymal transition (EMT) followed by increased invasion, loose colony morphology in soft agar and enhanced Akt phosphorylation (Ser-473). Furthermore, overexpression of S100A2 led to increased tumor growth in immunocompromised mice. In agreement, immunohistochemical examination of resected xenograft tumors established inverse correlation between S100A2 and E-cadherin expression together with activated Akt signaling. Interestingly, our study demonstrates a strong dependence of S100A2 and Smad3 in TGF-β-induced Hep3B cell EMT and invasion. Most importantly, we demonstrate that these effects of S100A2 are manifested through functional interaction with Smad3, which is enhanced in the presence of high calcium and TGF-β. S100A2 stabilizes Smad3 and binds to its C-terminal MH2 domain. Additionally, loss of S100A2 attenuates the transcription of TGF-β/Smad3 target genes involved in tumor promotion, such as PA1-1 and vimentin. Collectively, our findings present the first mechanistic details of S100A2 protumorigenic actions and its involvement in TGF-β-mediated cancer cell invasion and EMT.