Cyclin D1 in Regulation of Liver Cancer Stem Cells

Abstract

Cancer stem cells (CSCs) are capable of self-renewal, multipotency, in vivo tumorigenicity, and driving metastasis, leading to recurrence of tumor. Thus, novel therapies capable of eliminating CSCs are needed. Cyclin D1 is deregulated in many types of cancers. Beyond being a cell cycle regulator and oncogene, cyclin D1 also promotes stem cell (SC) self-renewal and induced pluripotent stem cells (iPSCs) reprogramming efficiency. Yet, the role of cyclin D1 in regulation of CSC is not well defined. In this study, we investigated potential role of cyclin D1 in regulation of liver cancer stem cell properties. In cyclin D1 overexpressed two lines of liver cancer cells, anchorage-independent spherical colony (spheres) formation was enriched. From dissected single spherical cells, the capacity of the secondary and the third sphere formation was significantly higher in cyclin D1-expressing liver cancer cells compared to parental cells, suggesting an enhanced self-renewal capacity. Cyclin D1 expression conferred liver cancer cells more CSC and SC properties, shown by the increased CD90+ and EpCAM+ liver CSC populations and the enhanced expression of SC-associated genes Nanog, Oct4 and Nodal. At longer culturing time, cyclin D1-expressing spheres maintained a good spherical morphology, whereas parental spheres became more differentiated. Interestingly, Smad2/3, a TGF-β signaling effector, was highly phosphorylated in cyclin D1-expressing spherical cancer cells compared to spherical cells without cyclin D1. Application of TGF-β/Smad inhibitor (SB431542) significantly reduced the CSC sphere formation in cyclin D1-expressing cells but had no effects on parental cell sphere formation. In addition, Erk inhibitor (UO126) showed no inhibiting effects, a further proof of the cyclin D1- mediated activation of Smad2/3. Our preliminary data also indicated the effects of SB431542 in induction of CSC sphere differentiation and reduction of tumorigenicity. Thus, TGF-β/Smad inhibitor might have therapeutic potential for targeting liver CSCs. Liver cells are known being highly resistance to chemotherapeutic drugs. We further investigated the sphere formation capacity of cyclin D1-expressing cells under the treatment of chemotherapeutic agents in combination with Smad inhibitor and found that there was an sensitised effect in low dose and synergistic effect in high dose of SB431542. In summary, our results suggest that cyclin D1 might promote liver CSC proliferation and self-renewal via activation of TGF-β/Smad signaling pathway, with underlining mechanism is under investigation.