Nanog regulates self-renewal of cancer stem cells through the insulin-like growth factor pathway in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) exhibits cellular heterogeneity and embryonic stem-cellrelated 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-cellrelated 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;56:10041014)