It may act as a downstream molecule to maintain HCC stemness and serve as a good marker for HCC initiating cells. CD133 or CD90 have been used to identify potential hepatic CSCs35,42. of EpCAM, a Wnt/-catenin signaling target, attenuated the activities of these cells. == Conclusions == Taken together, our results suggest that HCC growth and invasiveness is dictated by a subset of EpCAM+cells, opening a new avenue for HCC cancer cell eradication by targeting Wnt/-catenin signaling components such as EpCAM. == INTRODUCTION == Tumors originate from normal cells as a result of accumulated genetic/epigenetic changes. Although ANGPT2 considered monoclonal in origin, tumor cells are heterogeneous in their morphology, clinical behavior, and molecular profiles1,2. Tumor cell heterogeneity has previously been explained by the clonal evolution model3, however, recent evidence has suggested that heterogeneity may be due to derivation from endogenous stem/progenitor cells4or de-differentiation of a transformed cell5. This hypothesis supports an early proposal that cancers represent blocked ontogeny6and a derivative that cancers are transformed stem cells7. This renaissance of stem cells as targets of malignant transformation has led to realizations about the similarities between cancer cells and normal stem cells in their capacity to self-renew, produce heterogeneous progenies, and limitlessly divide8. The cancer stem cell (CSC) (or Tumor Initiating Cell) concept is that a subset of cancer cells bears stem cell features that are indispensable for a tumor. Accumulating evidence suggests the involvement of CSCs in the perpetuation of various cancers including leukemia, breast cancer, brain cancer, prostate cancer and colon cancer9-13. Experimentally, putative CSCs have been isolated using cell surface markers specific for normal stem cells. Stem cell-like features of CSC have been confirmed by functionalin vitroclonogenicity andin vivotumorigenicity assays. For example, leukemia-initiating cells in NOD/SCID mice are CD34++CD3811. Breast cancer CSCs are CD44+CD24/lowcells while tumor initiating cells of the brain, colon and prostate are CD133+10,12,13. CSCs are considered more metastatic and drug/radiation resistant than non-CSCs in the tumor, and are responsible for cancer relapse. These findings warrant the development of treatment strategies that can specifically eradicate CSCs14,15. Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide16. Although the cellular origin of HCC is unclear17,18, HCC has heterogeneous pathologies and genetic/genomic profiles19, suggesting that HCC can initiate in different cell lineages20. The liver is considered as a maturational lineage system similar to that in the bone marrow21. Experimental evidence indicates that certain forms of hepatic stem cells (HpSC), present in human livers of all donor ages, are multipotent and can give rise to hepatoblasts (HB)22,23, which are, in turn, bipotent progenitor cells that can progress either into the hepatocytic or biliary lineages22,24. Alpha-fetoprotein (AFP) is one of the earliest markers detected in the liver bud specified from the ventral foregut25,26, but its expression has only been found in HB and to a lesser extent in committed hepatocytic progenitors, not in later lineages nor RPH-2823 in normal human HpSC22. Recent studies also indicate that EpCAM is a biomarker for HpSC as it is expressed in RPH-2823 HpSCs and HBs22-24. We recently identified a novel HCC classification system based on EpCAM and AFP status27. Gene expression profiles revealed that EpCAM+AFP+HCC (referred to as Hepatic Stem Cell-like HCC; HpSC-HCC) has progenitor features with poor prognosis, whereas EpCAMAFPHCC (referred to as Mature Hepatocyte-like HCC; MH-HCC) have adult hepatocyte features with good prognosis. Wnt/-catenin signaling, a critical player for maintaining embryonic stem cells28, is activated in EpCAM+AFP+HCC, and EpCAM is a direct transcriptional target of Wnt/-catenin signaling29. Moreover, EpCAM+AFP+HCC cells are more sensitive to -catenin inhibitors than EpCAMHCC cellsin vitro29. Interestingly, a heterogeneous expression of EpCAM and AFP was observed in clinical tissues, a feature that may be attributed to the presence of a subset of CSCs. In this study, we have confirmed that EpCAM+HCC cells are highly invasive and tumorigenic, and have activated Wnt/-catenin signaling. We also demonstrate a crucial role of EpCAM in the maintenance of hepatic CSCs. Our data shed new light on the pathogenesis of HCC and may open new avenues for therapeutic interventions for targeting hepatic CSCs. == Materials and Methods == == Clinical Specimens == HCC samples were obtained with informed consent from patients who underwent radical resection at the Liver Cancer Institute of Fudan University or college, Eastern Hepatobiliary Surgery Institute and the Liver Disease Center of Kanazawa University or college Hospital, and the study was authorized by the Institutional Review RPH-2823 Table of the respective Institutes. The microarray data from medical specimens are publicly available (GEO.