The utility of genome-wide methylation profiles in defining biologically and clinically relevant sub-classes of cancer has been established for multiple cancer types and is attributed to high correlation between epigenetic alterations across CpG loci.16
The definition of methylation-based classes has revealed associations between these distinct classes and clinicopathologic tumor characteristics17–20
as well as environmental exposures.17,20,21
The goal of the present study was to understand how methylation-based profiles are associated with expression of the genes responsible for establishing and maintaining methylation marks in the context of RCC. The use of an array for identifying methylation profiles provides a broad genomic assessment of epigenetic alterations, reducing the bias of a candidate gene approach and allowing for a robust statistical assessment for finding correlations with a range of covariates. Our method for clustering samples, RPMM, allows for data-driven clustering based on methylation, and subsequent examination of the biological or clinical meaning of these clusters.
While much is known about the genetics of RCC, only limited work has been done toward understanding the contributions of epigenetics to the disease. For example, the von Hippel Lindau (VHL) tumor suppressor, which had long been recognized as playing a central role in development of sporadic clear cell RCC due to decreased expression by mutational inactivation or loss of heterozygosity, has also been shown to be silenced by DNA methylation in a significant proportion of tumors.7
As appreciation for the role of epigenetics in renal carcinogenesis has grown, several other epigenetically altered genes have been identified in RCC including SFRP1
and Wnt-antagonist DKK2
Initially, RPMM was applied to methylation data from both tumor and normal samples, and demonstrated that the pattern of methylation in cancer-related genes varies between tumor and normal samples. This analysis resulted in six classes, two of which contained all the normal samples, suggesting that normal tissues have similar methylation profiles to one another and are distinct from the majority of tumors. The observation that normal tissues fell into classes along with some tumor samples suggests an increased presence of non-malignant cells, a less aggressive phenotype, or both for those tumors. RPMM on RCCs alone resulted in only three classes, suggesting a decrease in heterogeneity of methylation profiles upon removal of normal kidney tissues. Mean methylation extent across all loci decreased progressively from Classes 1–3.
An altered epigenetic landscape can originate from dysregulation in the expression of genes that control DNA methylation or regulate epigenetic processes by direct or indirect mechanisms. Therefore, we evaluated the expression of key epigenetic regulatory genes DNMT3B, DNMT1, VEZF1
in tumors. An association was noted between class membership and EZH2
expression, where methylation Class 3 tumors demonstrated higher expression of EZH2
than tumors in the other two classes. Class 3 was also comprised of only tumors of low stage (I and II), whereas the prevalence of higher stage tumors (III and IV) was seen to increase progressively from Class 2 to Class 1. With the lower extent of aberrant promoter hypermethylation observed in Class 3 tumors, our data suggest that tumors overexpressing EZH2
are less advanced and demonstrate fewer epigenetic abnormalities. In fact, this finding is consistent with recent work showing that while EZH2
expression is significantly elevated in primary RCC tumors compared with histologically normal kidney samples, higher EZH2
expression among tumors was characteristic of less aggressive tumors and a more favorable disease prognosis.14
Taken together, these data suggest that EZH2 may be more important in tumor initiation than in progression of RCC.
Our analysis of global methylation markers revealed that the extent of methylation at both LINE-1 and AluYb8 was significantly higher in Classes 2 and 3 as compared to Class 1. Thus, methylation of these repetitive elements was inversely related to the extent of gene-associated methylation across classes, a pattern consistent with the gene-specific regional hypermethylation and concomitant global genomic hypomethylation that is a hallmark of neoplasia.26
Our study is limited in its sample size to more precisely quantify this relationship, and further studies focused on the relationship between LINE-1 and AluYb8 methylation and gene-specific methylation in low-grade tumors is warranted.
Considering that methylation profiles of samples were associated with EZH2
expression, and EZH2 is the major catalytic subunit of the histone methyltransferase complex PRC2, the distribution of PcG target genes across loci with differential methylation was of great interest. The significant contribution of aberrant chromatin structure in RCC was highlighted in a recent finding, demonstrating that mutations of the SWI/SNF chromatin remodeling complex gene PBRM1
existed in 41% of RCC tumors in the study.27
Our classification of CpGs based on methylation state revealed that classes of CpG loci with relatively low methylation extent in tumors were significantly enriched for loci associated with PcG target genes. Expanding this examination to include a comparison with non-diseased kidney tissue led to the observation that these same classes were relatively less correlated between tumor and normal tissue, and likely include genes targeted for alteration in carcinogenesis. Interestingly, this suggests that while there is a broad range of methylation degree among cancer-related genes in RCC, the genes that are most highly methylated among tumors are not necessarily those that drive the cancer phenotype, which is characterized by gene-specific hypermethylation in tumors compared to normal tissue. In addition, PcG target genes associate with a large proportion of the loci that are differentially methylated (specifically hypermethylated) in tumors compared to normal tissue. This phenomenon may be explained by a new body of literature describing the epigenetic landscape of cancer stem cells. These studies present a model whereby PcG target genes, specifically regulatory targets of EZH2
, which in normal cells carry the distinctive mark of trimethylated lysine 27 of histone 3, are coordinately regulated with multiple genes that become aberrantly hypermethylated in cancer.28–30
Collectively, these data suggest that EZH2
pre-marks genes for ultimate “deep silencing” by DNA methylation and epigenetic silencing in the context of carcinogenesis.
The pivotal role that EZH2 plays in RCC carcinogenesis exemplifies its newly understood function as a mechanistic connector between the two major modes of epigenetic gene repression, namely polycomb group repression via histone remodeling and DNA methylation.12
The present work strongly supports the existing model that these two mechanisms, which act together to determine the accessibility of chromatin to transcriptional machinery, are inextricably linked. In addition to showing that patterns of tumor methylation are associated with the expression of a gene, EZH2
, which is indispensable for establishing these marks, our work demonstrates that these methylation patterns may be reflective of important and early gene-specific alterations which have shaped the tumor epigenome.