Regulation of SETD2, a histone methyltransferase, in advanced clear cell renal cell carcinoma (ccRCC).

Abstract

368 Background: Although the von Hippel-Lindau (VHL) tumor suppressor gene is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified loss of function mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. However it is unclear whether histone modifications are misregulated in RCC. We hypothesize that loss of SETD2 methyltransferase activity decreases H3K36 trimethylation (H3K36Me3) in ccRCC, and contributes to the cancer phenotype. Methods: To determine if loss of SETD2 protein expression alters H3K36Me3, SETD2 siRNA oligonucleotides were transfected into HEK293 (human embryonic kidney) cells and H3K36Me3 was analyzed by Western blot. To establish frequency of SETD2 loss of heterozygosity (LOH), genomic DNA was isolated from 51 VHL deficient ccRCC specimens and analyzed with Affymetrix GeneChip Mapping 250K Nsp single-nucleotide polymorphism (SNP) arrays. To evaluate alterations of histone modifications in metastatic advanced ccRCC, H3K36Me3 immunohistochemical (IHC) staining was quantitated on tissue microarrays (TMAs) representing 28 paired ccRCC specimens with unaffected kidney parenchyma controls and 40 metastases. Results: SETD2 siRNA decreases H3K36Me3 (>50%) compared to scrambled controls. Using SNP arrays, LOH at chromosome 3p (location of VHL and SETD2 genes) was detected in >90% of the tested ccRCC tumors. IHC reveals a 27.4% and 52.0% decrease in H3K36Me3 positive nuclei in primary ccRCC and metastases, respectively, when compared to matched adjacent unaffected kidney tissue (P value < 0.0001). Conclusions: SETD2 siRNA decreases H3K36Me3 indicating that SETD2 is a non-redundant H3K36 methyltransferase. LOH of VHL and SETD2 occurs in >90% of tested ccRCC tumors. H3K36Me3 is decreased in primary ccRCC and even more in metastases, suggesting that SETD2 methyltransferase activity is progressively misregulated in RCC. Loss of SETD2 methyltransferase activity may cooperate with VHL silencing to promote tumorigenesis. Future studies will focus on how SETD2 contributes to tumorigenesis and on determining the prognostic and predictive impact of SETD2 phenotypic changes in patients with ccRCC.