Pyruvate Kinase M2Affects Liver Cancer Cell Behavior Through Up-Regulation Of HIF-1α And Bcl-xL In Culture

Background and aims:Cancer cells consume large amounts of glucose to produce lactate, even in the presence of ample oxygen. This phenomenon is known as the Warburg effect. The pyruvate kinase promotes aerobic glycolysis, and the M2isoform (PKM2) is highly expressed in many cancer cells. Although the Warburg effect is a hallmark of cancer, the mechanism by which PKM2contributes to the Warburg effect, and its role in tumor growth remains to be defined. In this study, we investigate the expression of PKM2in hepatocellular carcinoma, and whether PKM2promote hepatocarcinogenesis as well as the underlying mechanism.Methods:We compared mRNA and protein expressions of PKM2in primary liver cancer tissues and adjacent normal tissues by Real-time PCR and immunohistochem istry. Interfered and overexpressed PKM2cell lines of HepG2were constructed to analyze the role of PKM2on proliferation of liver cancer cells by cell proliferation and anchorage-dependent colony formation assays. Western blot were performed to investigate the expression of PKM2, STAT3, phosphorylated STAT3, HIF-1α,p65, phosphorylated p65, and Bcl-xL in HepG2cells. We investigated whether the reduced cell growth mediated by PKM2knockdown was caused by apoptosis.Results:1) The expression of PKM2in hepatocellular carcinoma tissues and adjacent normal tissuesa)The mRNA levels of PKM2in hepatocellular carcinoma tissues and adjacent normal tissues relative to GAPDH were determined by qRT-PCR,(n=115). b)The pattern of PKM2protein in hepatocellular carcinoma tissues and adjacent normal tissues detected by immunohistochemistry.2) Effect of aberrant PKM2on the growth of HepG2cells in vitroa) HepG2cells were transduced with PKM2-shRNA (PKM2-shRNAl or PKM2-shRNA2) or control-shRNA (vector1) PKM2expression in HepG2cells was analyzed by qRT-PCR (top) and Western Blot (bottom).b) PKM2expression in whole cell lysates (WCL) of HepG2wild-type cells (Control) and HepG2cells transfected with lentivirus containing pLL3.7(vector1), pLV-GFP (vector2), pLL3.7-PKM2-shRNA2(PKM2-shRNA) and pLV-GFP-PKM2(PKM2) by Western Blot.c) Proliferation of HepG2cells was measured at24,48,72,96, and120h. The results from triplicate experiments are shown.d) Anchorage-dependent colony formation assay in monolayer culture. HepG2cells were plated in a10cm dish. After two weeks of incubation, the cells were stained with crystal violet. Data are represented as means±SD from three independent experiments. P<0.05.3) Nuclear PKM2activates transcription of HIF-la by phosphorylating STAT3at Y705a)The expression levels of HIF-la in hepatocellular carcinoma tissues and adjacent normal tissues relative to GAPDH were determined by qRT-PCR, n=115,P<0.05.b)The expression pattern of HIF-la protein in hepatocellular carcinoma tissues and adjacent normal tissues was measured by immunohistochemistry.c)Immunohistochemical staining of HIF-la in tumor and adjacent tissue. Data are represented as mean±SEM. P<0.05.d)HepG2cells were transfected with PKM2-shRNA and PKM2, and HIF-la expression was analyzed by qRT-PCR.e) Western Blot analysis of the expression of STAT3, p-STAT3(S727and Y705), HIF-1α, p65, p-p65, Bcl-xL in whole cell lysates (WCL), PKM2in whole cell lysates (WCL) and nuclear extracts (NE) of HepG2cells. GAPDH was used as a loading control. Data are represented as means±SD from three independent experiments. P<0.05.4) PKM2may regulate the expression of Bcl-xL gene by alternating the activity of p65a)The expression levels of Bcl-xL in hepatocellular carcinoma tissues and adjacent normal tissues relative to GAPDH were determined by qRT-PCR,(n=115).b)HepG2cells were transfected with PKM2-shRNA and PKM2, and Bcl-xL expression was analyzed by qRT-PCR using total RNA from whole cell lysates (WCL).c)HepG2cells were treated with20or40μM PS1145. Bcl-xL expression was analyzed by Western Blot.d) Alexa Fluor-647(APC)/7-ADD assay of HepG2cell apoptosis detected by flow cytometry. The PKM2-shRNA group had more cell death. Data are represented as means±SD from three independent experiments.Conclusions:The current findings demonstrate that PKM2is overexpressed in hepatocellular carcinoma tissues and promotes liver cancer cell proliferation. PKM2may function by up-regulating HIF-la and Bcl-xL expression in the liver.