Cancer Stem Cells In Hepatocellular Carcinoma Research Articles

The Polycomb Gene Product BMI1 Contributes to the Maintenance of Tumor-Initiating Side Population Cells in Hepatocellular Carcinoma

Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4α gene

Previous studies have shown that hepatocyte nuclear factor-4alpha (HNF4alpha) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4alpha could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4alpha on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4alpha have not been reported. In this study, an adenovirus-mediated gene delivery system, which could efficiently transfer and express HNF4alpha, was generated to determine its effect on hepatoma cells (Hep3B and HepG2) in vitro and investigate the anti-tumor effect of HNF4alpha in mice. Our results demonstrated that forced re-expression of HNF4alpha induced the differentiation of hepatoma cells into hepatocytes, dramatically decreased "stemness" gene expression and the percentage of CD133(+) and CD90(+) cells, which are considered as cancer stem cells in HCC. Meanwhile, HNF4alpha reduced cell viability through inducing apparent apoptosis in Hep3B, while it induced cell cycle arrest and cellular senescence in HepG2. Moreover, infection of hepatoma cells by HNF4alpha abolished their tumorigenesis in mice. Most interestingly, systemic administration of adenovirus carrying the HNF4alpha gene protected mice from liver metastatic tumor formation, and intratumoral injection of HNF4alpha also displayed significant antitumor effects on transplanted tumor models. The striking suppression effect of HNF4alpha on tumorigenesis and tumor development is attained by inducing the differentiation of hepatoma cells--especially CSCs--into mature hepatocytes, suggesting that differentiation therapy with HNF4alpha may be an effective treatment for HCC patients. Our study also implies that differentiation therapy may present as one of the best strategies for cancer treatment through the induction of cell differentiation by key transcription factors.

Aldehyde Dehydrogenase Discriminates the CD133 Liver Cancer Stem Cell Populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+), yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) > CD133(+)ALDH(-) > CD133(-)ALDH(-). ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population.

CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway

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 responsible 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 chemotherapeutic agents (doxorubicin and fluorouracil) and the possible mechanistic pathway by which resistance may be regulated. Purified CD133+ 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 underlying mechanism of which required the preferential expression of survival proteins involved in the Akt/PKB and Bcl-2 pathway. Treatment of CD133+ HCC cells with an AKT1 inhibitor, specific to the Akt/PKB pathway, significantly reduced the expression of the survival proteins that was normally expressed endogenously. In addition, treatment of unsorted HCC cells with both anticancer drugs in vitro significantly enriched the CD133+ subpopulation. In conclusion, our results show that CD133+ HCC cells contribute to chemoresistance through preferential activation of Akt/PKB and Bcl-2 cell survival response. Targeting of this specific survival signaling pathway in CD133+ HCC CSCs may provide a novel therapeutic model for the disease.