ORIGINAL ARTICLE CD133 þ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway S Ma 1,2 , TK Lee 3 , B-J Zheng 4 , KW Chan 1 and X-Y Guan 2 1 Department of Pathology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; 2 Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; 3 Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China and 4 Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China The recent discovery of cancer stem cells (CSCs) has played a pivotal role in changing our view of carcinogenesis and chemotherapy. Based on this concept, CSCs are res- ponsible for the formation and growth of neoplastic tissue and are naturally resistant to chemotherapy, explaining why traditional chemotherapies can initially shrink a tumor but fails to eradicate it in full, allowing eventual recurrence. Recently, we identified a CSC population in hepatocellular carcinoma (HCC) characterized by their CD133 phenotype. However, the molecular mechanism by which it escapes conventional therapies remains unknown. Here, we examined the sensitivity of these cells to chemo- therapeutic agents (doxorubicin and fluorouracil) and the possible mechanistic pathway by which resistance may be regulated. Purified CD133 þ HCC cells isolated from human HCC cell line and xenograft mouse models survived chemotherapy in increased proportions relative to most tumor cells which lack the CD133 phenotype; the under- lying mechanism of which required the preferential expres- sion of survival proteins involved in the Akt/PKB and Bcl-2 pathway. Treatment of CD133 þ HCC cells with an AKT1 inhibitor, specific to the Akt/PKB pathway, signi- ficantly reduced the expression of the survival proteins that was normally expressed endogenously. In addition, treat- ment of unsorted HCC cells with both anticancer drugs in vitro significantly enriched the CD133 þ subpopulation. In conclusion, our results show that CD133 þ HCC cells contribute to chemoresistance through preferential activa- tion of Akt/PKB and Bcl-2 cell survival response. Target- ing of this specific survival signaling pathway in CD133 þ HCC CSCs may provide a novel therapeutic model for the disease. Oncogene (2008) 27, 1749–1758; doi:10.1038/sj.onc.1210811; published online 24 September 2007 Keywords: HCC; CD133; survival; cancer stem cells; chemoresistance Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide affecting one million indivi- duals annually (Thorgeirsson and Grisham, 2002). Liver resection or transplantation is available for early-stage HCC; however, most patients with the disease are usually diagnosed at an advanced stage, resulting in the limited efficacy of these potentially curative therapies. Further, the fact that HCC is resistant to conventional chemotherapy or radiotherapy leaves this disease with no effective therapeutic options and a very poor prognosis. The ‘stem cell model’ of carcinogenesis suggests that cancers originate and are maintained by a rare fraction of cells called cancer stem cells (CSCs) (Reya et al., 2001; Pardal et al., 2003). These cells not only possess uni- que survival mechanisms but also harbor distinctive stem/progenitor cell properties including the ability to self-renew, differentiate and proliferate following a prolonged period of quiescence. If tumor growth and metastasis are really driven by CSCs, this can explain why current chemotherapies, developed largely against the bulk tumor mass, are only transiently, if at all, able to shrink the primary tumor, but are unable to provide a lasting cure for the disease. It is likely that these residual CSCs, due to their resistance ability, are able to survive in a dormant state for many years after remission and result in tumor relapse, with renewed resilience and aggression. To date, the existence of CSCs has been proven in the context of many cancers, including those of leukemia, breast cancer, glioblastoma, prostate cancer, pancreatic cancer and colon cancer (Lapidot et al., 1994; Bonnet and Dick, 1997; Al-Hajj et al., 2003; Hemmati et al., 2003; Singh et al., 2003, 2004; Collins et al., 2005; Ponti et al., 2005; Haraguchi et al., 2006; Patrawala et al., 2006; Li et al., 2007; O’Brien et al., 2007; Ricci-Vitiani et al., 2007). Recently, we have determined that HCC is hierarchically organized and originates from a primitive stem/progenitor group of cells for which CD133 þ precursors constitute one of the most immature stage (Ma et al., 2007). CD133 þ HCC cells isolated from human HCC cell lines and xenograft tumors not only possess a greater colony-forming efficiency, higher proliferative output and greater ability to form tumor Received 21 June 2007; revised 6 August 2007; accepted 21 August 2007; published online 24 September 2007 Correspondence: Professor X-Y Guan, Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, Laboratory of Cancer Genetics, The University of Hong Kong, Room L10-56, 10/F, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong, China. E-mail: xyguan@hkucc.hku.hk Oncogene (2008) 27, 1749–1758 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc