Cyclin D1-Smad2/3-Smad4 signaling promotes liver CSC self-renewal and CSC differentiation by Smad inhibitor is a key stage for chemosesitization

Abstract

Targeting cancer stemness and induction of differentiation are the new directions for cancer therapy. We investigated the role of cyclin D1-SMAD2/3-SMAD4 signaling pathway in liver cancer stem cells (CSCs) and its potential as therapeutic target. Cyclin D1 dependent phosphorylation of SMAD2/3 and enhanced expression of SMAD4 were detected in HCC spherical cells as well as in over 50% of primary HCC tumor tissues. HCC patients with high levels of cyclin D1, pSMAD2/3, and SMAD4 together displayed a poor overall survival. Overexpression of cyclin D1 conferred liver cancer cells CSC features such as capacity of single sphere formation, increased CD90+ and EpCAM+ liver CSC population, enhanced pluripotency-associated gene and metabolic glycolysis gene expression, which was via activation of SMAD2/3 and SMAD4. Cyclin D1-SMAD2/3-SMAD4 signaling-regulated cancer spheres also showed enhanced epithelial-mesenchymal transition (EMT) phenotype and highly resistant to chemotherapy. Application of TGF-b/SMAD inhibitor (SB431542) inhibited CSC sphere growth moderately although the inhibition was specifically in cyclin D1-expressing but not in parental spheres (a further proof of cyclin D1-mediated activation of SMADs). However, pre-treated with low dose of SMAD inhibitor induced cancer spheres differentiation leading to significant chemosensitization. The same strategy (pre-treatment with low dose of SB431542 followed by cisplatin regularly) significantly decreased tumorigenicity in cyclin D1 sphere-derived xenograft tumor model, with 8 out of 14 (57%) cell injections were tumor free at endpoint, and the formed tumor was only 24% of tumor size of vehicle or cisplatin groups. Interestingly, xenograft tumor was unaffected when applying SMAD inhibitor and cisplatin simultaneously. Mechanistically, SMAD inhibitor reduced CD90+, EpCAN+, and CD133+ liver CSC populations; reduced stemness gene and glycolytic gene expression; reversed EMT phenotype; all these resulting in impaired self-renewal and induced differentiation, which is an effective step for reversing chemoresistance. Together, cyclin D1-SMAD2/3-SMAD4 signaling pathway promotes liver cancer CSC self-renewal. The induction of CSC differentiation by TGF-b/SMAD inhibitor followed by chemotherapy provides a new path for combined therapy.