The Histone Methyltransferase G9a Promotes Cholangiocarcinogenesis through Regulation of the Hippo Pathway Kinase LATS2 and YAP Signaling Pathway

Abstract

BackgroundCholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. A better understanding of the molecular mechanisms of cholangiocarcinogenesis is of paramount importance to develop more effective target therapy.MethodsqRT‐PCR and Western blotting analyses were used to determine the expression of specific mRNAs and proteins. ChIP‐PCR was performed to analyze the enrichment of G9a and H3K9me2 in the promoter region of LATS2 gene. Dual‐Luciferase reporter assay system was used to evaluate YAP luciferase activity. CCA xenograft was developed by inoculation of human CCA cells with or without G9a depletion into SCID mice. CCA tumor induction was developed by SB transposase‐mediated integration of oncogenes (NICD/Akt) to mice via hydrodynamic tail vein (HDTV) injection.ResultsWe present novel evidence that the histone‐lysine methyltransferase G9a is upregulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase LATS2 and YAP signaling pathway. Kaplan–Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by siRNA/shRNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0638) significantly inhibited human CCA cell growth in vitro and in SCID mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of NICD and myr‐Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt‐injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a‐derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2 and this effect led to subsequent activation of oncogenic YAP. Accordingly, out data showed that G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition.ConclusionOur findings provide novel evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2‐YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.