162 CD133-Targeted Oncolytic Adenovirus to Attack Colon Cancer Stem Cells

Introduction: There exists a highly tumorigenic subset of hepatocellular carcinoma (HCC) cells defined by high expression of CD44 and CD133, and that subset has been reported to contain cancer stem-like cells (CSCs) in HCC. Kruppel-like factor 5 (KLF5) is a transcription factor containing 3 zinc finger domains and one transactivation domain. KLF5 regulates many factors related to cell cycle, migration, inflammation, angiogenesis and stemness and has cancer promoting effects in some cancers. Furthermore, some reports have indicated that KLF5 might have important roles in regulation of CSCs. However, the function of KLF5 in HCC remains to be elucidated. Functional roles of KLF5 in regulation of CSCs in HCC were examined in the present study. Methods: HCC and hepatoblastoma cell lines, Huh7, Alexander and HepG2 cells were analyzed. Anti-CD44 and anti-CD133 antibodies were used to detect CSCs in HCC by fluorescence-activated cell sorting (FACS). Indicated cell population was sorted by FACS Aria III (Becton Dickinson). MTS assay was carried out to determine sensitivity to chemotherapeutic reagents, 5FU and CDDP. RNA sequencing was performed by next generation sequencer, IonTM PGM (Lifetechnologies). Retroviral-mediated gene transfer was used to make a stable cell line overexpressing KLF5. Two independent sequences of siRNA against KLF5 were used to knock-down KLF5. Tumorigenicity was determined by soft-agar colony formation assay and xenograft experiments using athymic nude mice. Results: FACS showed heterogeneous cell population in several HCC cell lines. Sorted CD44High/CD133High cells were significantly more resistant to anti-cancer drugs, 5FU and CDDP, and had more colony forming capacity than CD44Low/CD133Low cells as reported previously. RNA sequencing revealed completely different gene expression profiles between CD44High/CD133High and CD44Low/CD133Low cells, and identified over 500 mRNAs, including KLF5, significantly up-regulated in the sorted CD44High/CD133High cells. Overexpression of KLF5 increased the ratio of CD44High/CD133High cells, and consistent with the upregulation of CD44High/CD133High cells, the KLF5 overexpressing cells were more resistant to the anti-cancer drugs and had more colony forming capacity. By contrast, knock-down of KLF5 by siRNA diminished CD44High/CD133High cell population. Finally, KLF5 overexpressing cells formed tumors more frequently in nude mice. Conclusion: Those data provide a novel mechanistic insight that KLF5 might promote tumorigenicity of HCC by upregulation of CSCs and could be a therapeutic target against CSCs in HCC.