The effect of loss of SETD2 on mitochondrial dysfunction in clear cell renal cell carcinoma.

Abstract

567 Background: SET domain-containing protein 2 ( SETD2) is among the most commonly mutated genes in clear cell renal cell carcinoma (ccRCC). Classically, SETD2 modulates chromatin structure via the methylation of lysine 36 on histone H3. However, histone-independent functions of SETD2 are emerging. We sought to discover novel SETD2-dependent changes to the cellular lysine methylation landscape during kidney cancer tumorigenesis. Methods: In this study, we used HKC human proximal tubule kidney cell lines, the likely nephrogenic region of origin for ccRCC. The proteomes of wild type (WT) and SETD2-knock out (KO) cells were labeled using stable isotope labeling by amino acids in cell culture (SILAC). Proteins were trypsinized and lysine-methylated peptides were immunoprecipitated. Relative abundance of lysine-methylated peptides and total peptides were calculated in the WT and SETD2-KO using nano-liquid chromatography/tandem mass spectrometry (LC/MS-MS). Results: > 30,000 peptides were quantified, including > 50 lysine methylated peptides. We observed decreased lysine methylation of eukaryotic elongation factor 1A1 (eEF1A1), including K165 trimethylation (K165me3) and K318 monomethylation (K318me1). eEF1A1 is classically known for delivery of aminoacyl-tRNA to the ribosome, and its function is modulated via the lysine methyltranferases eEF1AKMT1-4. eEF1AKMT3 is responsible for trimethylation of K165, and expression of this enzyme was decreased in SETD2-KO cells. In addition, we observed significant decrease in expression in proteins of the electron transport chain (ETC) complex 1 in SETD2-KO cells yet normal gene expression, suggesting a translational defect. Functionally, SETD2-KO cells are characterized by decreased cellular oxygen consumption rate and increased mitochondrial mass, suggestive of ETC dysfunction. Conclusions: We observe decreased expression of eEF1AKMT3 in SETD2-KO cells, resulting in decreased K165me3 of eEFA1A, decreased translation of ETC proteins, and mitochondrial dysfunction. As mitochondrial dysfunction is commonly observed in RCC, we expect this represents a novel mechanism of SETD2-mediated tumorigenesis.