Cyclin D1-Smad2/3-Smad4 signaling promotes liver CSC self-renewal and Smad inhibitor induces CSC differentiation and chemosensitization

Abstract

 Hepatocellular carcinoma (HCC), is the fifth most prevalent cancer and the third leading most common cause of all cancer-related deaths worldwide. Also HCC is one of the well-informed cancers that resistant to conventional chemotherapeutic drugs resulting in low response rate and high relapse. HCC is badly in need of novel therapeutic modalities. Cancer stem cells (CSCs) represent a distinct population that can be isolated from tumor tissues and have long-term clonal repopulation and self-renewal capacity. Liver cancer cells are equipped with apoptotic block, high drug transporter expression, efficient DNA repair and quiescence, which are the mechanisms of therapy resistance. Interestingly, these mechanisms are possessed by CSCs. Despite these known mechanisms, recent evidences from experimental and clinical studies suggest that the stemness properties of a cancer cell are the core contributors linking to clinical therapy failure and recurrences. Thus therapeutic targeting of determinants of self-renewal machinery, such as stem cell maintenance pathways, could be effective means to eliminate CSCs. Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-Smad2/3-Smad4 pathway in hepatocellular carcinoma (HCC) cancer CSCs and in HCC primary tumors. The primary biological function of cyclin D1 is the promotion of cellular proliferation. Cyclin D1 is also a well-established human oncogene and is important for the development and progression of several cancers. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties to liver cancer cells by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing expression of stem cell-associated genes Nanog, Oct4, and Nordal/Activin, improving mobility, and increasing chemoresistance. The interaction of cyclin D1 with both Smad4 and Smad2/3 has been revealed, and cyclin D1 precipitated more Smad4 and Smad2/3 proteins in cyclin D1-overexpressing cells. These results suggested that cyclin D1 interacts with and activates Smad2/3 and Smad4 to issue in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. HCC The cyclin D1-Smad2/3-Smad4 pathway is also found in HCC patients and predicts disease progression. Afterwards, we overexpressed Smad 2 and Smad4 on HCC cell lines and result in promotion of stemness features and EMT. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization of cancer spherical cells. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-Smad2/3-Smad4 axis is an important signaling pathway in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequent significant chemosensitization, which could be an effective strategy to target CSCs.