Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs) that participate in tumor propagation, resistance to conventional therapy, and promotion of tumor recurrence, causing poor patient outcomes. The protein SRY (sex determining region Y)-box 9 (Sox9) is a transcription factor expressed in some solid tumors, including HCC. However, the molecular mechanisms underlying Sox9 function in liver CSCs remain unclear. Here, we show that Sox9 is highly expressed in liver CSCs and that high levels of Sox9 predict a decreased probability of survival in HCC patients. We demonstrate that Sox9 is required for maintaining proliferation, self-renewal, and tumorigenicity in liver CSCs. Overexpression of exogenous Sox9 in liver non-CSCs restored self-renewal capacity. Additionally, a reduction in the asymmetrical cell division of spheroid-cultured liver CSCs was observed when compared with differentiated cancer cells or liver CSCs with inhibited Notch signaling. Furthermore, we demonstrate that Sox9 is responsible for the asymmetrical-to-symmetrical cell division switch in liver CSCs. Sox9 also negatively regulates Numb expression, contributing to a feedback circuit that maintains Notch activity and directs symmetrical cell division. Clinical analyses revealed that the Sox9 High Numb Low profile is associated with poor prognosis in human HCC patients. Conclusion: These findings demonstrate that Sox9 plays a critical role in self-renewal and tumor propagation of liver CSCs and identify the molecular mechanisms regulated by Sox9 that link tumor initiation and cell division. (HEPATOLOGY 2016;64:117-129) H epatocellular carcinoma (HCC) is one of the most common cancers in the world. Development of HCC can be driven by a small heterogeneous population of tumor-derived cancer stem cells (CSCs) or tumor-initiating cells. (1) Understanding how liver CSCs regulate tumor growth and development is of key importance for future treatment strategies.The SRY-box family proteins are well-established regulators of cell fate decisions during development, and dysregulation of Sox factors has been further implicated in diseases including cancer. (2) During tumorigenesis, the SoxE group member SRY (sex determining region Y)-box 9 (Sox9) is up-regulated in a number of tumors and plays an essential for cancer progression as an oncogene. (3)(4)(5) In addition, high levels of Sox9 expression enhanced the tumorigenic and metastasis-seeding abilities of human breast cancer cells in a transplant model, (6) and epithelial-mesenchymal transition conferred on the cells many of the properties associated with CSCs. (6,7) A mouse model of basal cell carcinoma has revealed that Abbreviations: BrdU, 5-bromo-2 0 -deoxyuridine; CSC, cancer stem cell; DAPT, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; DMEM, Dulbecco's modified Eagle's medium; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; IHC, immunohistochemis...

Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor containing cancer stem cells (CSCs), which participate in tumor invasion, therapeutic resistance, and tumor relapse leading to poor outcome and limited therapeutic options. Histone deacetylatase sirtuin 1 (SIRT1) has been shown to be up-regulated in human cancers; however, its role in liver CSCs is unknown. In this study, we explored the biological functions of SIRT1 in liver CSCs. Our data show that SIRT1 is highly expressed in liver CSCs and decreases during differentiation. In addition, high levels of SIRT1 predict a decreased probability of survival in patients with HCC. SIRT1 is responsible for the maintenance of self-renewal and tumorigenicity of liver CSCs, and overexpression of exogenous SIRT1 can restore self-renewal of non-CSCs. We demonstrated that SOX2 is a main downstream regulator of SIRT1-mediated self-renewal and tumorigenicity potential of liver CSCs. Mechanistically, SIRT1 regulates transcription of the SOX2 gene by way of chromatin-based epigenetic changes, which are dependent on DNA methylation. This effect is achieved by alternation of histone modification and interaction with DNA methyltransferase 3A, resulting in hypermethylation of SOX2 promoter. Furthermore, we demonstrated that insulin growth factor signaling plays an important role in maintaining SIRT1 expression through increased SIRT1 protein stability. Conclusions: These findings highlight the importance of SIRT1 in the biology of liver CSCs and suggest that SIRT1 may serve as a molecular target for HCC therapy. (HEPATOLOGY 2016;64:814-827)

Cancer stem-like cells (CSC) in hepatocellular carcinoma (HCC) are thought to mediate therapeutic resistance and poor survival outcomes, but their intrinsic and extrinsic control is not well understood. In this study, we found that the chromatin modification factor LSD1 is highly expressed in HCC CSC where it decreases during differentiation. LSD1 was responsible for maintaining CSC self-renewal and tumorigenicity in HCC, and its overexpression was sufficient to drive self-renewal of non-CSC. Levels of acetylated LSD1 were low in CSC with high LSD1 activity, and these CSC were capable of self-renewal. Notch signaling activated LSD1 through induction of the sirtuin SIRT1, leading to deacetylation and activation of LSD1 and CSC self-renewal. Notably, we found that LSD1 expression was increased in cancer-associated fibroblasts (CAF) as an upstream driver of Notch3-mediated CSC self-renewal. In clinical specimens of HCC, the presence of CAF, LSD1, and Notch3 strongly associated with poor patient survival. Overall, our results reveal that CAF-induced expression of Notch3 is responsible for LSD1 activation in CSC, driving their self-renewal in HCC. These seminal findings illuminate a complex pathway in the tissue microenvironment of liver cancer, which is responsible for orchestrating the self-renewal of stem-like cancer cells, with potential implications to improve therapy and limit relapses. .

Introduction Combining lenvatinib with a programmed cell death protein-1 (PD-1) inhibitor has been explored for the treatment of un-resectable hepatocellular carcinoma (uHCC). This study aimed to investigate the real-world efficacy of and prognostic factors for survival associated with lenvatinib plus PD-1 inhibitor treatment in a large cohort of Asian uHCC patients even the global LEAP-002 study failed to achieve the primary endpoints. Methods Patients with uHCC treated with lenvatinib and PD-1 inhibitors were included. The primary endpoints were overall survival (OS) and progression-free survival (PFS), and the secondary endpoints were the objective response rate (ORR) and adverse events (AEs). Prognostic factors for survival were also analyzed. Results A total of 378 uHCC patients from two medical centers in China were assessed retrospectively. The median patient age was 55 years, and 86.5% of patients were male. Hepatitis B virus (HBV) infection (89.9%) was the dominant etiology of uHCC. The median OS was 17.8 (95% confidence interval (CI) 14.0–21.6) months. The median PFS was 6.9 (95% CI 6.0–7.9) months. The best ORR and disease control rate (DCR) were 19.6% and 73.5%, respectively. In multivariate analysis, Child‒Pugh grade, Barcelona Clinic Liver Cancer stage, Eastern Cooperative Oncology Group performance status score, involved organs, tumor burden score, and combination with local therapy were independent prognostic factors for OS. A total of 100% and 57.9% of patients experienced all-grade and grade 3/4 treatment-emergent AEs, respectively. Conclusion This real-world study of lenvatinib plus PD-1 inhibitor treatment demonstrated long survival and considerable ORRs and DCRs in uHCC patients in China. The tolerability of combination therapy was acceptable but must be monitored closely.