Abstract 1035: SETD2, a histone methyltransferase, is misregulated in advanced clear cell renal cell carcinoma (ccRCC)
Authors: Ho Thai H. , Monzon Federico A. , Hoang Anh , Tamboli Pheroze , Furge Kyle , Dykema Karl , Petillo David , Teh Bin Tean , Tannir Nizar , Jonasch Eric , Hung Mien-Chie 
Published: August 2012

Background: Although the von Hippel-Lindau (VHL) tumor suppressor is inactivated in more than 91% of clear cell renal cell carcinoma (ccRCC), VHL deletion in mice is insufficient for tumorigenesis, suggesting that additional mutations are required. Exome sequencing of early stage (I-III) ccRCC tumors identified missense and truncating mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. However it is unclear whether these mutations alter histone modifications. We hypothesize that loss of SETD2 histone methyltransferase activity decreases H3K36 trimethylation (H3K36Me3) in ccRCC. 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. In parallel, H3K36Me3 immunohistochemical (IHC) staining was quantitated in nephrectomy tissue with a mutant SETD2 genotype. To establish the frequency of SETD2 loss of heterozygosity (LOH), genomic DNA was isolated from 51 formalin-fixed paraffin-embedded ccRCC specimens and analyzed with Affymetrix GeneChip Mapping 250K Nsp single-nucleotide polymorphism (SNP) arrays. To determine if there are alterations of histone modifications in metastatic ccRCC, H3K36Me3 IHC staining was performed on tissue microarrays (TMAs) representing 28 paired ccRCC specimens with unaffected kidney parenchyma controls and 40 metastases (bone, brain and lungs). Results: SETD2 siRNA decreases H3K36Me3 (&gt;50%) compared to scrambled controls suggesting that SETD2 is a non-redundant H3K36 methyltransferase. Nephrectomy specimens with SETD2 truncating mutations also have decreased H3K36Me3 (&gt;50%) compared to matched uninvolved kidney tissue. Using SNP arrays, LOH at chromosome 3p (location of VHL and SETD2 genes) was detected in &gt;90% of the tested ccRCC tumors. H3K36Me3 IHC staining was quantitated in TMAs representing advanced ccRCC specimens. 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 &lt;0.0001). Discussion: SETD2 siRNA transfected cell lines and patient-derived tissue with truncating SETD2 mutations have decreased H3K36Me3. In advanced ccRCC, LOH occurs in &gt;90% of tested tumors. H3K36Me3 is decreased in primary ccRCC and even more in metastases, suggesting that SETD2 methyltransferase activity is progressively misregulated in RCC. VHL loss may cooperate with loss of SETD2 histone methyltransferase activity to promote tumorigenesis. Future studies will focus on the mechanism of how SETD2 contributes to tumorigenesis and on determining the prognostic and predictive impact of SETD2 phenotypic changes in patient-derived tissue.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1035. doi:1538-7445.AM2012-1035