Arginine methylation by PRMT6 as a novel stemness and metabolic regulator in hepatocellular carcinoma

Abstract

Objective: Arginine methylation is a common posttranslationalmodification that plays pivotal roles in signaltransduction. However, its function in human diseases is poorlyunderstood. Protein methyltransferase 6 (PRMT6) belongs tothe type I PRMT enzyme family, responsible for catalyzing theasymmetric dimethylation of arginine residues in proteins. Theaim of this study was to delineate the effects of loss of PRMT6in regulating hepatocellular carcinoma (HCC) stemness andglycolysis, cancer characteristics that we now know contributeto tumor recurrence and therapy resistance.

Methods: Cancer stemness and glycolysis related functional/cell biological studies were performed on HCC/non-tumor livercell lines and organoid cultures modulated by lentiviral basedstrategies, as well as in both immunocompromised xenograftsand immunocompetent PRMT6-/- mice models. Transcriptomeprofiling coupled with protein-protein interaction studieswas utilized to delineate possible PRMT6 protein interactingpartners and altered molecular mechanisms by which lossof PRMT6 drives HCC stemness and glycolysis, followed byconfirmation with co-immunoprecipitation and in vitro/in vivokinase and methylation assays. Results: PRMT6 was frequently down-regulated in HCC andits expression was negatively correlated with aggressive cancerfeatures in HCC patients. Silencing of PRMT6 promoted thetumor-initiating, metastasis, therapy resistance, and glucosemetabolism potential of HCC cell lines and patient-derivedorganoids, whereas overexpression of PRMT6 led to an oppositeeffect. The findings of our lentiviral based functional studieswere further substantiated by PRMT6 wild-type and catalyticinactive methyltransferase mutant overexpression. Consistently,loss of PRMT6 expression aggravated liver tumorigenesis inchemical-induced HCC PRMT6 knockout mice. Contrary toits usual localization in the nucleus where it has previouslybeen identified to play a role in histone modifications in othertumor types, we did not find that PRMT6 altered the H3R2mark in HCC cells. Interestingly, we identified a previouslyunappreciated role of PRMT6 in the cytoplasm. Integratedtranscriptome and protein-protein interaction studies revealedan enrichment of genes implicated in RAS signaling and thatPRMT6 interacted with CRAF on arginine 100 and as a resulthindered its RAS binding potential and altered its downstreamMEK/ERK signaling. As a consequence, down-regulation ofPRMT6 in HCC results in activation of ERK-mediated cancer stemness via regulating CD133, SOX2, and NANOG, as well asERK-mediated glucose metabolic reprogramming via regulatingPKM2. Conclusions: Our work uncovered a critical repressivefunction of PRMT6 in maintenance of HCC cells by regulatingthe MEK/ERK pathway via arginine dimethylation of CRAF,providing a new avenue of molecular mechanism by whichERK mediated stemness and altered metabolic properties ofHCC cells are developed. DOI: 10.20892/j.issn.2095-3941.2018.S098