Hepatocellular carcinoma (HCC) exhibits cellular heterogeneity and embryonic stem‐cell–related genes are preferentially overexpressed in a fraction of cancer cells of poorly differentiated tumors. However, it is not known whether or how these cancer cells contribute to tumor initiation and progression. Here, our data showed that increased expression of pluripotency transcription factor Nanog in cancer cells correlates with a worse clinical outcome in HCC. Using the Nanog promoter as a reporter system, we could successfully isolate a small subpopulation of Nanog‐positive cells. We demonstrate that Nanog‐positive cells exhibited enhanced ability of self‐renewal, clonogenicity, and initiation of tumors, which are consistent with crucial hallmarks in the definition of cancer stem cells (CSCs). NanogPos CSCs could differentiate into mature cancer cells in in vitro and in vivo conditions. In addition, we found that NanogPos CSCs exhibited resistance to therapeutic agents (e.g., sorafenib and cisplatin) and have a high capacity for tumor invasion and metastasis. Knock‐down expression of Nanog in NanogPos CSCs could decrease self‐renewal accompanied with decreased expression of stem‐cell–related genes and increased expression of mature hepatocyte‐related genes. Overexpression of Nanog in NanogNeg cells could restore self‐renewal. Furthermore, we found that insulin‐like growth factor (IGF)2 and IGF receptor (IGF1R) were up‐regulated in NanogPos CSCs. Knock‐down expression of Nanog in NanogPos CSCs inhibited the expression of IGF1R, and overexpression of Nanog in NanogNeg cells increased the expression of IGF1R. A specific inhibitor of IGF1R signaling could significantly inhibit self‐renewal and Nanog expression, indicating that IGF1R signaling participated in Nanog‐mediated self‐renewal. Conclusion: These data indicate that Nanog could be a novel biomarker for CSCs in HCC, and that Nanog could play a crucial role in maintaining the self‐renewal of CSCs through the IGF1R‐signaling pathway. (HEPATOLOGY 2012;56:1004–1014)

BackgroundHigh invasion and metastasis are the primary factors causing poor prognosis of patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. In this study, we investigate the molecular mechanism by which hypoxia promotes HCC invasion and metastasis through inducing epithelial-mesenchymal transition (EMT).MethodsThe expression of EMT markers was analyzed by immunohistochemistry. Effect of hypoxia on induction of EMT and ability of cell migration and invasion were performed. Luciferase reporter system was used for evaluation of Snail regulation by hypoxia-inducible factor -1α (HIF-1α).ResultsWe found that overexpression of HIF-1α was observed in HCC liver tissues and was related to poor prognosis of HCC patients. HIF-1α expression profile was correlated with the expression levels of SNAI1, E-cadherin, N-cadherin and Vimentin. Hypoxia was able to induce EMT and enhance ability of invasion and migration in HCC cells. The same phenomena were also observed in CoCl2-treated cells. The shRNA-mediated HIF-1α suppression abrogated CoCl2-induced EMT and reduced ability of migration and invasion in HCC cells. Luciferase assay showed that HIF-1α transcriptional regulated the expression of SNAI1 based on two hypoxia response elements (HREs) in SNAI1 promoter.ConclusionsWe demonstrated that hypoxia-stabilized HIF1α promoted EMT through increasing SNAI1 transcription in HCC cells. This data provided a potential therapeutic target for HCC treatment.

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DNA methylation is an important epigenetic modification and is frequently altered in cancer. Convert of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation (TET) family enzymes plays important biological functions in embryonic stem cells, development, aging and disease. Recent reports showed that level of 5 hmC was altered in various types of cancers. However, the change of 5 hmC level in hepatocellular carcinoma (HCC) and association with clinical outcome were not well defined. Here, we reported that level of 5 hmC was decreased in HCC tissues, as compared with non-tumor tissues. Clincopathological analysis showed the decreased level of 5 hmC in HCC was associated with tumor size, AFP level and poor overall survival. We also found that the decreased level of 5 hmC in non-tumor tissues was associated with tumor recurrence in the first year after surgical resection. In an animal model with carcinogen DEN-induced HCC, we found that the level of 5 hmC was gradually decreased in the livers during the period of induction. There was further reduction of 5 hmC in tumor tissues when tumors were developed. In contrast, level of 5 mC was increased in HCC tissues and the increased 5 mC level was associated with capsular invasion, vascular thrombosis, tumor recurrence and overall survival. Furthermore, our data showed that expression of TET1, but not TET2 and TET3, was downregulated in HCC. Taken together, our data indicated 5 hmC may be served as a prognostic marker for HCC and the decreased expression of TET1 is likely one of the mechanisms underlying 5 hmC loss in HCC.

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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...