Comparing the DNA Hypermethylome with Gene Mutations in Human Colorectal Cancer

A previous version of this article appeared as an Early Online Release on July Comparing the DNA Hypermethylome with Gene Mutations in Human Colorectal Cancer Kornel E. Schuebel 0 Wei Chen 0 Leslie Cope 0 Sabine C. Glo ckner 0 Hiromu Suzuki 0 Joo-Mi Yi 0 Timothy A. Chan 0 Leander Van Neste 0 Wim Van Criekinge 0 Sandra van den Bosch 0 Manon van Engeland 0 Angela H. Ting 0 Kamwing Jair 0 Wayne Yu 0 Minoru Toyota 0 Kohzoh Imai 0 Nita Ahuja 0 James G. Herman 0 Stephen B. Baylin 0 0 1 Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins , Baltimore , Maryland, United States of America, 2 Predoctoral Training Program in Human Genetics, The Johns Hopkins University , Baltimore , Maryland, United States of America, 3 Biometry and Clinical Trials Division, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins , Baltimore , Maryland, United States of America, 4 Department of Surgery, The Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 5 First Department of Internal Medicine, Sapporo Medical University , Sapporo , Japan , 6 Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University , Ghent, Belgium, 7 Oncomethylome Sciences, Liege , Belgium , 8 Department of Pathology, University of Maastricht , Maastricht , The Netherlands , 9 Bionumerik Pharmaceuticals Inc. , San Antonio, Texas , United States of America We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumorspecific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken. - It is now well established that loss of proper gene function in human cancer can occur through both genetic and epigenetic mechanisms [1,2]. The number of genes mutated in human tumor samples is being clarified. Recently, Sjo blom et al. [3] sequenced 13,023 genes in colorectal cancer (CRC) and breast cancer, and estimated an average of 14 significant mutations per tumor, suggesting that a relatively small number of genetic events may be sufficient to drive tumorigenesis. In contrast, the full spectrum of epigenetic alterations is not well delineated. The best-defined epigenetic alteration of cancer genes involves DNA hypermethylation of clustered CpG dinucleotides, or CpG islands, in promoter regions associated with the transcriptional inactivation of the affected genes [2]. These promoters are located proximal to nearly half of all genes [4] and are thought to remain primarily methylation free in normal somatic tissues. The exact number of such epigenetic lesions in any given tumor is not precisely known, although a growing number of screening approaches, none covering the whole genome efficiently, are identifying an increasing number of candidate genes [513]. Given the large number of potential target promoters present in the genome, we hypothesized that many more hypermethylated genes await discovery. Herein, we describe a whole human transcriptome microarray screen to identify genes silenced by promoter hypermethylation in human CRC. The approach readily identifies candidate cancer genes in single tumors with a high efficiency of validation. By comparing the list of candidate hypermethylated genes with mutated genes recently identified in CRC [3], we establish key relationships between the altered tumor genome and the gene hypermethylome. Our studies provide a platform to understand how epigenetic and genetic alterations drive human tumorigenesis. Developing the Whole Transcriptome Approach Our first step towards a global identification of hypermethylation-dependent gene expression changes was made by comparing, in a genome-wide expression array-based approach, wild-type HCT116 CRC cells with isogenic partner cells carrying individual and combinatorial genetic deletions of two major human DNA methyltransferases (Figure 1A) [14]. Editor: Jeannie T. Lee, Massachusetts General Hospital, United States of America Loss of gene expression in association with aberrant accumulation of 5-methylcytosine in gene promoter CpG islands is a common feature of human cancer. Here, we describe a method to discover these genes that permits identification of hundreds of novel candidate cancer genes in any cancer cell line. We now estimate that as much as 5% of colon cancer genes may harbor aberrant gene hypermethylation and we term these the cancer promoter CpG island DNA hypermethylome. Multiple mutated genes recently identified via cancer resequencing efforts are shown to be within this hypermethylome and to be more likely to undergo epigenetic inactivation than genetic alteration. Our approach allows derivation of new potential tumor biomarkers and potential pathways for therapeutic intervention. Importantly, our findings illustrate that efforts aimed at complete identification of the human cancer genome should include analyses of epigenetic, as well as genetic, changes. Importantly, in the DNMT1( / )DNMT3B( / ) double knockout (DKO) HCT116 cells, which have virtually complete loss of global 5-methylcytosine, all previously individually examined hypermethylated genes lacking basal expression in wild-type cells undergo promoter demethylation with concomitant gene re-expression [10,1416]. By stratifying genes according to altered signal intensity on a 44K Agilent Technologies array platform, we observe a unique spike of gene expression increases in the DKO cells when compared to the isogenic wildtype parental cells, or isogenic cell lines in which DNMT1 or DNMT3B have been individually deleted and which harbor minimal changes in DNA methylation (Figure 1B). This minimal change in the DNMT1( / )cells may, in part, be due to recently identified alternative transcripts arising from the DNMT1 locus [17,18]. We tested our approach using a pharmacologic strategy based on our previous approach [10], but now markedly modified to provide whole-transcriptome coverage, to identify silenced hypermethylated genes in any cancer cell line. For densely hypermethylated and transcriptionally inactive genes, the DNA demethylating agent 5-aza-29-deoxycytidine (DAC) has a well established capacity to induce gene re-expression [19,20]. On the other hand, for these same genes, the class I and II histone deac (...truncated)