Role Of PRDX2in Regulating "Stemness" Of Colorectal Cancer Cells And Its Underlying Mechanism

PART Ⅰ Role of PRDX2in Acquiring Stemness and its Effect onStem-like Phenotype in Colorectal Caner CellsObjective: To explore the roles of PRDX2in acquiring stem-likeproperties and its effects on expressing stem cell characteristics incolorectal cancer stem-like cells.Methods: We used serum-free medium suspension method to culture,enrich, screen and identify human colorectal cancer stem-like cells in vitro,and builded the colorectal cancer stem cells model. Overexpression or genesilencing of PRDX2were made for human colorectal cancer cell lineSW480and SW620using lentivirus-mediated gene transfer system. SW480and SW620stem-like spheroid cells in transfected group and control groupwere cultured through floating culture in serum free medium. Variationsincluding sphere forming, cell growth and proliferation, resistance tochemotherapy, acquistion of stemness and tumorigenesis in vivo wereexamined after up-regulating or down-regulating PRDX2expression incolorectal cancer cells. After silencing PRDX2gene expression using PRDX2-shRNA lentiviral vector, the expression of stem markers such asCD133, CD44, Bmi-1, Lgr5were detected in SW480and SW620stem-likecells by qRT-PCR and western blotting. Other stemness variationsincluding self-renewal capacity, continuous colony forming sphere ability,resistance to chemotherapy and subcutaneous xenotransplantedtumorigenesis abilities were also observed. Effects of PRDX2ontumorigenesis were evaluated in vivo using xenograft tumor model.Results: SW480and SW620stem-like spheres have higher PRDX2expression compared to adherent cells. SW480and SW620stem-likespheres cells have obvious stem cell characteristics, and both the mRNAand protein expression of CD133, CD44, Bmi-1and Lgr5were increasedsignificantly when compared with those of parental cells, which reachedthe highest in the second generation sphere cells. In tumor sphere cellsoverexpressing PRDX2from SW480and SW620, the amount and qualityof tumor spheres formation, second-forming spheres capability, resistanceto chemotherapy, self-renewal capacity and subcutaneous xenotransplantedtumorigenesis were obviously enhanced, while the results were reversedafter the expression of PRDX2was silenced by RNA interference.Conclusion: PRDX2is involved in the acquisition of stem-like propertiesand the expression of stemness in colorectal cancer stem-like spheres cells.PRDX2is likely to be required in acquisition and maintenance of stem-like characteristic in colorectal cancer stem-like spheres cells. PART Ⅱ The Molecular Mechanism of PRDX2In Regulating“Stemness” of Colorectal Stem-Like CellsObjective: To explore the role of PRDX2in regulating stemness incolorectal cancer stem-like cells and the involving molecular mechanism.Methods: Cancer stem cell-specific markers and P53expression in tumorspheroid cells were examined after cultured with serum-free medium.Expression of stem markers such as Nanog, Sox2, Oct4, Lin28, c-Myc,KIf4, Bmi-1were detected by RT-PCR and western blot in colorectalcancer stem-like cells overexpressing or silencing PRDX2. Interactionbetween PRDX2and c-Myc was explored by co-immunoprecipitation,immunocytochemistry and Immunohistochemistry. The role of PRDX2inthe process of epithelial-mesenchymal transition (EMT), invasion andmetastasis of colorectal cancer cells was examined using qPT-PCR, westernblot and immunocytochemistry. Western blot, qRT-PCR and xenotrans-planted mice model were used to futher explore whether PRDX2modulatesthe acquisition and maintenance of stemness characteristics throughPI3K/AKT pathway in colorectal cancer cells.Results: Higher expression of stemness markers and typical stem cellcharacteristics were observed in colorectal cancer stem-like sphere cellscompared to adherent cells after they were enriched in suspension culturefrom SW480and SW620. Colorectal cancer stem-like sphere cells have highly P53protein expression, which accompany with mesenchymal-epithelial transition (MET) occuring in the process of enrichment culture.Variation of PRDX2expression in sphere cells caused changes of essentialtranscription factors such as Sox2, Oct4, c-Myc. Moreover，the expressionof intestinal stem cell markers such as Bmi-1and Lgr5were increased ordecreased accordingly. The expression of PRDX2in colorectal cancer cellswas positive correlated with c-Myc expression. However, it was negativelycorrelated with p-c-Myc expression, and their interaction can also bedemonstrated preliminarily. Our findings showed that PRDX2activatedp-AKT (Ser-473) without altering AKT expression, which indicated thatPRDX2played an important role in regulating stemness of the tumor viaPI3K/AKT pathway. In addition, PRDX2inhibited TGF-β1-induced EMTin colorectal cancer cells and promoted MET process, contributing to theinhibition of invasion and metastasis of colorectal cancer cells. PRDX2hasprotective effect on colorectal cancer cells against apoptosis by modulatingBcl2expression.Conclusion: PRDX2regulates the acquisition and maintenance ofstem-like characteristics in colorectal cancer cells via PI3K/AKT pathway.