Abstract A35: A role for drug resistance in hepatocellular carcinoma: Upregulatation of CD133, activation of IGF-1R signaling, and IGF-1R nuclear translocation

Abstract

Abstract Introduction: Hepatocellular carcinoma (HCC) is an aggressive neoplasm that is resistant to chemotherapy with mechanisms underlying this drug resistance not well understood. CD133 is a known cancer stem cell (CSC) marker whose high expression is associated with aggressive tumor behavior and poor prognosis. The insulin-like growth factor 1 receptor (IGF-1R) signaling pathway has been shown to play a significant role in tumor behavior. To examine possible mechanisms of drug resistance in HCC, we investigated the role of CD133 expression and IGF-1R signaling activation using geftinib. Methods: We evaluated and separated five different human HCC cell lines (Mahlavu, Huh7, HepG2, Hep3B, PLC/PRF/5) using based on CD133 expression. We subjected these cells to geftinib at different doses, and determined IGF-1R signaling pathway activation using immunoblotting against relevant proteins in the pathway. CD133 mRNA level was examined using quantitative real-time PCR and its expression level was detected using immunofluorescence imaging and flow cytometry after drug treatment. Cell viability and sensitivity to drug treatment were measured using the MTT assay. Results: HCC cell lines showed variable percentages of total cell populations expressing CD133: Mahlavu 0.12 %, HepG2 46 %, Huh7 80.2 %, Hep3B 98.2 %, and PLC/PRF/5 83.5 %. Mahlavu and PLC/PRF/5 were found to be the most resistant cell lines even at higher doses (17.5 uM). Since Mahlavu cells displayed reduced CD133 expression, experiments were focused on this cell line to evaluate the CD133 expression changes after geftinib treatment. After three rounds (9 days) of high dose geftinib treatment, CD133 expression was increased in a dose dependent manner; the percentages of total cell populations expressing CD133 increased to 43.9 % at 10 uM concentration and 52.9 % at 15 uM concentration as compared to 0.12 % in untreated cells. In addition, we found a significant increase in IGF-1R and AKT phosphorylation as well as IGFBP3 expression in those geftinib treated cells. Furthermore, after cell sorting of Huh7 cells by CD133 expression using flow cytometry, we observed that CD133 positive cells showed more resistant to geftinib treatment. Immunofluorescence imaging analysis demonstrated that increased IGF-1R protein translocated to nuclei after geftinib treatment. Conclusion: Geftinib treatment enhanced CD133 expression level and activated IGF-1R signaling in HCC Mahlavu cells, one of the possible mechanism could be the nuclear translocation of IGF-1R. Our findings underscore the importance of a combined therapeutic approach with geftinib and IGF-1R blockage in the treatment of human HCC. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A35.