CPSF6 promotes the Warburg effect and angiogenesis leading to tumor progression via c-Myc/ FBW7/ VEGF/ PD-L1 signaling axis with its siRNA synergistic potential with Sorafenib as a therapeutic target for liver cancer therapy.

Abstract

Background Though CPSF6 was known to act as an oncoprotein via glycolysis in several cancers, the oncogenic mechanism of CPSF6 was not explored in association with the Warburg effect and angiogenesis in hepatocellular carcinoma (HCC) to date. Methods MTT assay, colony formation assay, cell cycle analysis and microarray were conducted in CPSF6 depleted HCC cells. Expression of CPSF6 was evaluated in HCC tissues, cell lines and by The Cancer Genome Atlas (TCGA) analysis, while molecular mechanism of CPSF6 was assessed by Western blotting, Immunofluorescence and Immunoprecipitation. The growth of Hep3B cells was monitored in BALB/c orthotopic and xenograft tumor models with Immunohistochemistry analysis. Also, tube formation assay, CAM assay and ELISA were performed for angiogenesis by CPSF6. Results CPSF6 was overexpressed in HCC tissues with poor survival rates compared to normal tissues. Hence, CPSF6 depletion suppressed the viability and colonies, induced apoptosis via PARP cleavages and increased sub-G1 population in Hep3B and Huh7 cells. Also, CPSF6 enhanced the stability of c-Myc via their binding through nuclear colocalization as an upstream of c-Myc, mainly in nucleoplasm. Furthermore, CPSF6 depletion activated phosphorylation of c-Myc (T58) for c-Myc degradation and inactivated c-Myc (S62), which was disturbed by FBW7 depletion or proteosomal inhibitor MG132. Additionally, CPSF6 depletion suppressed the Warburg effect by inhibition of glucose, HK2, PKM2, LDH and lactate, showed synergistic effect with Sorafenib in Hep3B cells and also inhibited angiogenesis by tube formation and CAM assays along with decreased expression and production of VEGF. Notably, CPSF6 depletion attenuated PD-L1 along with the increase of CD4/CD8 cell percentage in the splenocytes of BALB/c nude mice bearing Hep3B cells. Consistently, CPSF6 depletion reduced the growth of Hep3B cells in BALB/c mice in orthotopic and xenograft tumor models with inhibition of tumor microenvironment associated proteins by immunohistochemistry. Conclusions These findings suggest that CPSF6 enhances the Warburg effect for immune escape and angiogenesis, leading to cancer progression via c-Myc/ FBW-7/ VEGF/PD-L1 signaling axis with its siRNA synergistic effect with Sorafenib as a molecular target of liver cancer therapy. Trial registration: retrospectively registered