Abstract A1-05: Elucidation of epigenetic driver genes in clear cell renal cell carcinoma using a newly developed assay, AcceSssIble
Authors: Becket Elinne Coral , Duymich Christopher , Chang Yin-Wei , Pandiyan Kurinji , Nichols Peter , Jones Peter , Gill Inderbir , Liang Gangning 
Published: November 2015

Abstract
Background: While many studies have uncovered genetic mutations that drive tumorigenesis, far fewer have described epigenetic changes, such as nucleosome positioning and DNA methylation, which lead to the development of cancer. Therefore, accurately mapping these changes between normal and tumor tissue will provide novel information to identify genes that undergo epigenetic changes that drive tumorigenesis (“epigenetic driver genes”). In this study, we used an assay developed in our laboratory to investigate the epigenetic changes between clear cell renal cell carcinoma (ccRCC, the most common subtype of renal carcinoma) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis.
Methods: Current methods to investigate epigenomic changes in clinical samples are expensive and require abundant biological sample material for analysis. We have developed a novel assay (“AcceSssIble”) to simultaneously determine DNA methylation and chromatin accessibility in clinical samples. It is rapid and cost-effective, only requiring 20 mg of tissue, the Infinium HumanMethylation450 BeadChip platform, and the CpG methyltransferase M.SssI. We used this method to measure the changes in DNA methylation and chromatin accessibility in 9 matched pairs of ccRCC tumors and adjacent normal tissue from different patients, and intersected this data with RNA-seq data of 72 matched ccRCC samples and DNA methylation data of 160 matched ccRCC samples from The Cancer Genome Atlas (TCGA). Genes that were revealed to have the most changes in chromatin structure and expression were then targeted by siRNA knockdown for functional validation in ccRCC.
Results: From the AcceSssIble assay on 9 pairs of ccRCC patient tumor/normal samples, we uncovered 438 genes whose promoters change in chromatin accessibility in at least 2 ccRCC samples, both dependent and independent of DNA methylation changes, and have an accompanying change in gene expression in TCGA RNA-seq data. The results produce a striking figure in which chromatin accessibility changes are inversely correlated with DNA methylation but directly correlated with gene expression changes. Interestingly, loss of (DNA methylation change-dependent) accessibility preferentially occurred within CpG islands, while gain of (DNA methylation change-dependent) accessibility was strongly biased towards non-CpG islands. Meanwhile, chromatin accessibility changes independent of DNA methylation changes do not show preference in CpG content. Furthermore, pathway analyses reveal involvement of HIF1α signaling, cAMP-mediated signaling, and G-protein Coupled Receptor Signaling in the development of ccRCC. Lastly, we performed siRNA knockdown experiments on several top genes most changing in expression and accessibility, which revealed two genes, encoding type IV collagen and an RNA-binding protein, whose knockdown resulted in a significant increase in proliferation in normal kidney epithelial cells.
Conclusions: Our study revealed a vast number of chromatin accessibility and accompanying gene expression changes that occur in gene promoters in the development of ccRCC, both dependent and independent of DNA methylation changes. Each individual tumor has a unique profile of epigenetic alterations. Moreover, almost none of the genes that were found to undergo epigenetic and resulting gene expression changes overlap with TCGA's findings of commonly mutated genes in ccRCC. Overall, these studies represent novel approaches that can help identify new therapeutic target genes and treatment strategies for ccRCC, including personalized approaches.
Citation Format: Elinne Coral Becket, Christopher Duymich, Yin-Wei Chang, Kurinji Pandiyan, Peter Nichols, Peter Jones, Inderbir Gill, Gangning Liang. Elucidation of epigenetic driver genes in clear cell renal cell carcinoma using a newly developed assay, AcceSssIble. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-05.