15‐hydroxyprostaglandin Dehydrogenase is Regulated by Histone Methyltransferase G9a in Cholangiocarcinoma
Authors: Zhang Jinqiang , Chen Weina , Han Chang , Song Kyoungsub , Ma Wenbo , Wu Tong 
Published: June 2021

Cholangiocarcinoma (CCA) is a lethal malignancy without effective treatment options. We analyzed public available microarray dataset GSE76297 (which includes gene‐expression profiling information of 91 CCA tissues and 92 non‐tumor tissues), and observed that 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) expression levels in CCA tissues were decreased by approximately 2.5‐folds compared to paired normal liver tissues. Using a mouse intrahepatic CCA model induced by hydrodynamic tail vein (HDTV) injection of transposase‐based plasmids expressing consistently activated form of Notch (NICD) and AKT, we have shown that HDTV injection of 15‐PGDH expressing plasmids decreased CCA development. Further in vitro experiments showed that 15‐PGDH overexpression reduced cell growth and colony formation in the cholangiocarcinoma cell line, CCLP1. Next, we analyzed the levels of 54 histone lysine methyltransferases (HMTs) in dataset GSE76297, and observed that G9a is one of the most significantly increased HMT in patients. Gene co‐expression analyses demonstrated that 15‐PGDH and G9a expressions were inversely correlated in patients' CCA tissues. According, we observed that the NICD/AKT‐induced CCA in mice showed increased G9a with decreased 15‐PGDH. Furthermore, inhibition of G9a by shRNA or pharmacological inhibitors UNC0642 (5μM) or UNC0631 (1μM) led to restore of 15‐PGDH expression in CCLP1 cells. Our data indicated that G9a knockdown or inhibitor treatments inhibited CCLP1 cell proliferation and colony‐forming capability. These results suggest that G9a suppresses the expression of 15‐PGDH in CCA. To further explore the mechanism of 15‐PGDH regulation by G9a, we carried out ChIP assays using anti‐H3K9me2, anti‐G9a or IgG control in CCLP1 cells. We observed that anti‐H3K9me2 IP led to a >150‐time enrichment of the promoter region (−2k to 0) of 15‐PGDH (compared to IgG control). The enrichment was impeded by H3k9 hypomethylation as a result of G9a knockdown. We had performed additional ChIP assays and observed that pSTAT4 bound to the promoter region of 15‐PGDH, and this binding was facilitated by G9a inhibition. Additionally, we found that inhibition of G9a by shRNA or by UNC0642 led to hypomethylation of H3K9. Our results demonstrate that 15‐PGDH expression is epigenetically silenced by G9a through methylation of H3K9 (which prevents the association of the transcription factor pSTAT4 to 15‐PGDH promoter). Furthermore, we performed bioinformatics, ChIP and immunoprecipitation assays, and demonstrated that G9a was recruited specifically by Myc/Max heterodimer through binding to the E‐box elements located in the promoter region of 15‐PGDH gene. Taken together, our findings disclosed a novel mechanism of epigenetically regulation of 15‐PGDH by G9a in CCA. Therefore, 15‐PGDH may serve as new potential therapeutic target for CCA.Support or Funding InformationThis work was supported by the National Institutes of Health grants CA102325 and CA219541This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.