Kelly T. Carter

Differences in DNA Methylation Signatures Reveal Multiple Pathways of Progression From Adenoma to Colorectal Cancer

BACKGROUND & AIMS Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation. METHODS We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays. RESULTS We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions. CONCLUSIONS Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6–65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression.

Significance Rett syndrome is a neurodevelopmental disorder in girls who are heterozygous for a mutation in the X-linked gene MeCP2. Because cells in these individuals will be missing MeCP2 function only when the wild-type copy of the gene is on the inactive X, reactivation of the silenced copy of MeCP2 presents a potential therapeutic strategy. To identify genes that silence MeCP2 on the inactive X and that could therefore prove valuable as therapeutic targets, we carried out a screen for genes whose down-regulation reactivated a MeCP2 reporter on the inactive X. The 30 genes we have identified reveal a genetic circuitry required for maintenance of X-chromosome inactivation in differentiated cells and a large number of targets suitable for pharmacologic intervention. Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG–binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.

TGF-β signaling alters the pattern of liver tumorigenesis induced by Pten inactivation

Hepatocarcinogenesis results from the accumulation of genetic and epigenetic changes in liver cells. A common mechanism through which these alterations induce liver cancer is by deregulating signaling pathways. A number of signaling pathways, including the PI3K/PTEN/AKT and transforming growth factor β (TGF-β) pathways have been implicated in normal liver development as well as in cancer formation. In this study, we assessed the effect of the TGF-β signaling pathway on liver tumors induced by phosphatase and tensin homolog (Pten) loss. Inactivation of only the TGF-β receptor type II, Tgfbr2, in the mouse liver (Tgfbr2LKO) had no overt phenotype, while inactivation of Pten alone (PtenLKO), resulted in the formation of both hepatocellular carcinomas and cholangiocarcinomas (CC). Interestingly, deletion of both Pten and Tgfbr2 (PtenLKO;Tgfbr2LKO) in the mouse liver resulted in a dramatic shift in tumor type to predominantly CC. Assessment of the PI3K/PTEN/AKT pathway revealed increased phosphorylation of AKT and glycogen synthase kinase 3 beta (GSK-3β) in both the PtenLKO and PtenLKO;Tgfbr2LKO mice, suggesting that this pathway is constitutively active regardless of the status of the TGF-β signaling pathway. However, phosphorylation of p70 S6 kinase was observed in the liver of all three phenotypes (Tgfbr2LKO, PtenLKO, PtenLKO;Tgfbr2LKO) indicating that the loss of Tgfbr2 and/or Pten leads to an increase in this signaling pathway. Analysis of markers of liver progenitor/stem cells revealed that the loss of TGF-β signaling resulted in increased expression of c-Kit and CD133. Furthermore, in addition to increased c-Kit and CD133, Scf and EpCam expression were also increased in the double knock-out mice. These results suggest that the alteration in tumor types between the PtenLKO mice and PtenLKO;Tgfbr2LKO mice is secondary to the altered regulation of stem-cell features induced by the loss of TGF-β signaling.

Methylated B3GAT2 and ZNF793 Are Potential Detection Biomarkers for Barrett's Esophagus

Background: Barretts esophagus (BE) is a preneoplastic condition in which normal esophageal squamous epithelium (SQ) is replaced by specialized intestinal metaplasia. It is the presumed precursor for esophageal adenocarcinoma (EAC) as well as the strongest risk factor for this cancer. Unfortunately, many patients with BE go undiagnosed under the current BE screening guidelines. The development of noninvasive and accurate BE detection assays could potentially identify many of these undiagnosed BE patients. Methods: DNA methylation is a common epigenetic alteration in BE. Therefore, we conducted a genome-wide methylation screen to identify potential BE biomarkers. Samples from SQ (N = 12), stomach (N = 28), and BE (N = 29) were analyzed and methylation levels at over 485,000 CpG sites were compared. Pyrosequencing assays were used to validate the results and MethyLight assays were developed to detect the methylated alleles in endoscopic brushings. Results: We discovered two genes, B3GAT2 and ZNF793, that are aberrantly methylated in BE. Clinical validation studies confirmed B3GAT2 and ZNF793 methylation levels were significantly higher in BE samples (median = 32.5% and 33.1%, respectively) than in control tissues (median = 2.29% and 2.52%, respectively; P < 0.0001 for both genes). Furthermore, gene-specific MethyLight assays could accurately detect BE (P < 0.0001 for both) in endoscopic brushing samples. Conclusion: B3GAT2 and ZNF793 are hypermethylated in BE, and the methylation status of these genes can be used to detect BE in tissue samples. Impact: These findings support the development of methylated B3GAT2 and ZNF793 as biomarkers for noninvasive assays for the detection of BE. Cancer Epidemiol Biomarkers Prev; 24(12); 1890–7. ©2015 AACR.

Transposon mutagenesis identifies candidate genes that cooperate with loss of Transforming Growth Factor-beta signaling in mouse intestinal neoplasms

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF‐β) pathway. Importantly, the effects of TGF‐β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF‐β signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF‐β signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF‐β signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF‐β receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene‐centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/β‐catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

A five-CpG DNA methylation score to predict metastatic-lethal outcomes in men treated with radical prostatectomy for localized prostate cancer

Prognostic biomarkers for localized prostate cancer (PCa) could improve personalized medicine. Our group previously identified a panel of differentially methylated CpGs in primary tumor tissue that predict disease aggressiveness, and here we further validate these biomarkers.

Abstract 1157: Increased expression of RecQ helicases in sporadic primary colorectal cancers

The RecQ helicase family of enzymes regulate nucleic acid metabolism through the major activity of unwinding double stranded nucleic acids. Heritable loss of RECQ helicase expression results in human syndromes associated with an elevated risk of cancers including colorectal cancer (CRC). In vitro studies have shown that loss of function of WRN and/or BLM increases sensitivity to killing by DNA damaging chemotherapeutic agents. Thus, over-expression of these helicases may mediate tumor resistance to DNA damaging chemotherapeutic agents, whereas under-expression may identify a population of tumors that may be more susceptible. Currently, the literature with regards to the expression status of these enzymes in CRC is sparse. In this study, we assess the expression of all five members of the RECQ helicase family (WRN, BLM, RECQL, RECQL4 and RECQL5) in 32 sporadic primary colorectal cancer cases with matched normal colonic mucosa using quantitative real-time PCR. The results show a significant increase in the fold change of RECQ helicase expression in tumor as compared to the matched normal mucosa, using the Wilcoxon Signed Rank test with a theoretical median of 1. The results are as follows: WRN median=1.191, mean=1.347, SD=0.6189, p-value=0.0043; BLM median=4.146, mean=12.88, SD=24.86, p-value 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 1157. doi:1538-7445.AM2012-1157