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)

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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A positive margin results in a significantly worse outcome for pT1-2, pN0-1, and I-II stage gastric cancer patients who undergo D2/D3 lymphadenectomy, but not for those in pT3-4, pN2-3, and III-IV stage.