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1.  Differences in DNA Methylation Signatures Reveal Multiple Pathways of Progression from Adenoma to Colorectal Cancer 
Gastroenterology  2014;147(2):418-429.e8.
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.
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.
We found genome-wide alterations in DNA methylation in the non-tumor colon mucosa adjacent to tubular adenomas 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 (adenoma-H), and low-frequency methylation (adenoma-L). Within the adenoma-H class a subset of adenomas had mutant KRAS. Additionally, the adenoma-H class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, whereas the adenoma-L class had methylation signatures similar to that of non-tumor colon tissue. The CpGs sites that were differentially methylated in these signatures are located in intragenic and intergenic regions.
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.
PMCID: PMC4107146  PMID: 24793120
epigenetic modifications; colon cancer; progression; gene regulation
2.  Inactivation of TGF-β signaling and loss of PTEN cooperate to induce colon cancer in vivo 
Oncogene  2013;33(12):1538-1547.
The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-β) signaling pathway, which is often inactivated by mutations of TGF-β type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in approximately 30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperate to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2IEKO) alone is not sufficient for intestinal tumor formation and lack of Pten (PtenIEKO) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (PtenIEKO;Tgfbr2IEKO) led to malignant tumors in both the small intestine and colon in 86.0% of the mice and to metastases in 8.1% of the tumor-bearing mice. Moreover, these tumors arose via a β-catenin independent mechanism. Inactivation of Tgf-β signaling and loss of Pten led to increased cell proliferation, decreased apoptosis, and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-β signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.
PMCID: PMC3883899  PMID: 23604118
colorectal cancer; transforming growth factor beta; PTEN; p21Cip1; colon cancer
3.  Epigenetic Biomarkers in Esophageal Cancer 
Cancer letters  2012;342(2):10.1016/j.canlet.2012.02.036.
The aberrant DNA methylation of tumor suppressor genes is well documented in esophageal cancer, including adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) as well as in Barrett's esophagus (BE), a pre-malignant condition that is associated with chronic acid reflux. BE is a well-recognized risk factor for the development of EAC, and consequently the standard of care is for individuals with BE to be placed in endoscopic surveillance programs aimed at detecting early histologic changes that associate with an increased risk of developing EAC. Yet because the absolute risk of EAC in individuals with BE is minimal, a clinical need in the management of BE is the identification of additional risk markers that will indicate individuals who are at a significant absolute risk of EAC so that they may be subjected to more intensive surveillance. The best currently available risk marker is the degree of dysplasia in endoscopic biopsies from the esophagus; however, this marker is suboptimal for a variety of reasons. To date, there are no molecular biomarkers that have been translated to widespread clinical practice. The search for biomarkers, including hypermethylated genes, for either the diagnosis of BE, EAC, or ESCC or for risk stratification for the development of EAC in those with BE is currently an area of active research. In this review, we summarize the status of identified candidate epigenetic biomarkers for BE, EAC, and ESCC. Most of these aberrantly methylated genes have been described in the context of early detection or diagnostic markers; others might prove useful for estimating prognosis or predicting response to treatment. Finally, special attention will be paid to some of the challenges that must be overcome in order to develop clinically useful esophageal cancer biomarkers.
PMCID: PMC3395756  PMID: 22406828
DNA methylation; biomarker; Barrett's esophagus; esophageal adenocarcinoma; esophageal squamous cell carcinoma
4.  Molecular alterations and biomarkers in colorectal cancer 
Toxicologic pathology  2013;42(1):124-139.
The promise of precision medicine is now a clinical reality. Advances in our understanding of the molecular genetics of colorectal cancer genetics is leading to the development of a variety of biomarkers that are being used as early detection markers, prognostic markers, and markers for predicting treatment responses. This is no more evident than in the recent advances in testing colorectal cancers for specific molecular alterations in order to guide treatment with the monoclonal antibody therapies cetuximab and panitumumab, which target the epidermal growth factor receptor (EGFR). In this review, we update a prior review published in 2010 and describe our current understanding of the molecular pathogenesis of colorectal cancer and how these alterations relate to emerging biomarkers for early detection and risk stratification (diagnostic markers), prognosis (prognostic markers), and the prediction of treatment responses (predictive markers).
PMCID: PMC3947043  PMID: 24178577
Colon Cancer; Biomarkers; EGFR; KRAS; BRAF; Microsatellite Instability; MSI; Chromosome Instability; Cetuximab; Panitumumab
5.  TGF-β signaling promotes hepatocarcinogenesis induced by p53 loss 
Hepatology (Baltimore, Md.)  2011;55(1):121-131.
Hepatocellular carcinoma (HCC) results from the accumulation of deregulated tumor suppressor genes and/or oncogenes in hepatocytes. Inactivation of TP53 and inhibition of transforming growth factor-beta (TGF-β) signaling are among the most common molecular events in human liver cancers. Thus, we assessed whether inactivation of TGF-β signaling, by deletion of the TGF-β receptor, type II (Tgfbr2), cooperates with Trp53 loss to drive HCC formation. Albumincre transgenic mice were crossed with floxed Trp53 and/or floxed Tgfbr2 mice to generate mice lacking p53 and/or Tgfbr2 in the liver. Deletion of Trp53 alone (Trp53KO) resulted in liver tumors in approximately 41% of mice by 10 months of age, while inactivation of Tgfbr2 alone (Tgfbr2KO) did not induce liver tumors. Surprisingly, deletion of Tgfbr2 in the setting of p53 loss (Trp53KO;Tgfbr2KO) decreased the frequency of mice with liver tumors to around 17% and delayed the age of tumor onset. Interestingly, Trp53KO and Trp53KO;Tgfbr2KO mice develop both HCC and cholangiocarcinomas, suggesting that loss of p53, independent of TGF-β, may affect liver tumor formation through effects on a common liver stem cell population. Assessment of potential mechanisms through which TGF-β signaling may promote liver tumor formation in the setting of p53 loss revealed a subset of Trp53KO tumors that express increased levels of alpha-fetoprotein. Furthermore, tumors from Trp53KO mice express increased TGF-β1 levels compared to tumors from Trp53KO;Tgfbr2KO mice. Increased phosphorylated Smad3 and ERK1/2 expression was also detected in the tumors from Trp53KO mice and correlated with increased expression of the TGF-β responsive genes, Pai1 and Ctgf.
TGF-β signaling paradoxically promotes the formation of liver tumors that arise in the setting of p53 inactivation.
PMCID: PMC3237853  PMID: 21898503
HCC; CC; AFP; Smad3; ERK1/2
6.  Patterns of DNA methylation in the normal colon vary by anatomical location, gender, and age 
Epigenetics  2014;9(4):492-502.
Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.
PMCID: PMC4121360  PMID: 24413027
DNA methylation; methylation microarray; normal colon methylation; colorectal cancer; field effect
7.  Complex MSH2 and MSH6 Mutations in Hypermutated Microsatellite Unstable Advanced Prostate Cancer 
Nature communications  2014;5:4988.
A hypermutated subtype of advanced prostate cancer was recently described, but prevalence and mechanisms have not been well-characterized. Here we find that 12% (7 of 60) of advanced prostate cancers are hypermutated, and that all hypermutated cancers have mismatch repair gene mutations and microsatellite instability (MSI). Mutations are frequently complex MSH2 or MSH6 structural rearrangements rather than MLH1 epigenetic silencing. Our findings identify parallels and differences in the mechanisms of hypermutation in prostate cancer compared with other MSI-associated cancers.
PMCID: PMC4176888  PMID: 25255306
8.  RET is a potential tumor suppressor gene in colorectal cancer 
Oncogene  2012;32(16):2037-2047.
Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the GDNF-family ligands, was one of the first oncogenes to be identified and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared to adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.
PMCID: PMC3465636  PMID: 22751117
colon neoplasia; methylation; RET
9.  Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers12 
Translational Oncology  2013;6(4):458-469.
Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.
PMCID: PMC3730021  PMID: 23908689
10.  Survival after inflammatory bowel disease-associated colorectal cancer in the Colon Cancer Family Registry 
AIM: To investigate the survival of individuals with colorectal cancer (CRC) with inflammatory bowel disease (IBD-associated CRC) compared to that of individuals without IBD diagnosed with CRC.
METHODS: Epidemiologic, clinical, and follow-up data were obtained from the Colon Cancer Family Registry (Colon CFR). IBD-associated cases were identified from self-report of physician diagnosis. For a subset of participants, medical records were examined to confirm self-report of IBD. Cox proportional hazards regression was applied to estimate adjusted hazard ratios (aHR) and 95%CI of mortality, comparing IBD-associated to non-IBD-associated CRC, adjusted for age at CRC diagnosis, sex, Colon CFR phase, and number of prior endoscopies. Following imputation to complete CRC stage information, adjustment for CRC stage was examined.
RESULTS: A total of 7202 CRC cases, including 250 cases of IBD-associated CRC, were analyzed. Over a twelve year follow-up period following CRC diagnosis, 2013 and 74 deaths occurred among non-IBD associated CRC and IBD-associated CRC patients, respectively. The difference in survival between IBD-associated and non-IBD CRC cases was not statistically significant (aHR = 1.08; 95%CI: 0.85-1.36). However, the assumption of proportional hazards necessary for valid inference from Cox regression was not met over the entire follow-up period, and we therefore limited analyses to within five years after CRC diagnosis when the assumption of proportional hazards was met. Over this period, there was evidence of worse prognosis for IBD-associated CRC (aHR = 1.36; 95%CI: 1.05-1.76). Results were similar when adjusted for CRC stage, or restricted to IBD confirmed in medical records.
CONCLUSION: These results support the hypothesis that IBD-associated CRC has a worse prognosis than non-IBD-associated CRC.
PMCID: PMC3671075  PMID: 23745025
Colorectal cancer; Inflammatory bowel disease; Outcomes research; Cancer survival; Inflammation
12.  Complex MSH2 and MSH6 mutations in hypermutated microsatellite unstable advanced prostate cancer 
Nature Communications  2014;5:4988.
A hypermutated subtype of advanced prostate cancer was recently described, but prevalence and mechanisms have not been well-characterized. Here we find that 12% (7 of 60) of advanced prostate cancers are hypermutated, and that all hypermutated cancers have mismatch repair gene mutations and microsatellite instability (MSI). Mutations are frequently complex MSH2 or MSH6 structural rearrangements rather than MLH1 epigenetic silencing. Our findings identify parallels and differences in the mechanisms of hypermutation in prostate cancer compared with other MSI-associated cancers.
Several patients with metastatic prostate cancer have been shown to harbour tumours with markedly high mutation rates. Here, the authors characterise hypermutation in advanced prostate cancer samples and show that these samples have somatic mismatch repair gene mutations and microsatellite instability.
PMCID: PMC4176888  PMID: 25255306
13.  Comparative effectiveness of next generation genomic sequencing for disease diagnosis: Design of a randomized controlled trial in patients with colorectal cancer/polyposis syndromes✩ 
Whole exome and whole genome sequencing are applications of next generation sequencing transforming clinical care, but there is little evidence whether these tests improve patient outcomes or if they are cost effective compared to current standard of care. These gaps in knowledge can be addressed by comparative effectiveness and patient-centered outcomes research. We designed a randomized controlled trial that incorporates these research methods to evaluate whole exome sequencing compared to usual care in patients being evaluated for hereditary colorectal cancer and polyposis syndromes. Approximately 220 patients will be randomized and followed for 12 months after return of genomic findings. Patients will receive findings associated with colorectal cancer in a first return of result visit, and findings not associated with colorectal cancer (incidental findings) during a second return of result visit. The primary outcome is efficacy to detect mutations associated with these syndromes; secondary outcomes include psychosocial impact, cost-effectiveness and comparative costs. The secondary outcomes will be obtained via surveys before and after each return visit. The expected challenges in conducting this randomized controlled trial include the relatively low prevalence of genetic disease, difficult interpretation of some genetic variants, and uncertainty about which incidental findings should be returned to patients. The approaches utilized in this study may help guide other investigators in clinical genomics to identify useful outcome measures and strategies to address comparative effectiveness questions about the clinical implementation of genomic sequencing in clinical care.
PMCID: PMC4175052  PMID: 24997220
Comparative effectiveness research; Genomics; Next generation sequencing; Randomized clinical trial; Outcomes research; Whole exome sequencing
14.  DNA methylation profiling in Barrett's esophagus and esophageal adenocarcinoma reveals unique methylation signatures and molecular subclasses 
Epigenetics  2011;6(12):1403-1412.
Barrett's esophagus (BE) is a metaplastic process whereby the normal stratified, squamous esophageal epithelium is replaced by specialized intestinal epithelium. Barrett's is the only accepted precursor lesion for esophageal adenocarcinoma (EAC), a solid tumor that is rapidly increasing in incidence in western countries. BE evolves into EAC through intermediate steps that involve increasing degrees of dysplasia. Current histologic criteria are quite subjective and the clinical behavior of BE is highly variable and difficult to predict using these standards. It is widely believed that molecular alterations present in BE and EAC will provide more precise prognostic and predictive markers for these conditions than the current clinical and histologic features in use. In order to further define molecular alterations that can classify unique groups of BE and EAC, we utilized methylation microarrays to compare the global gene methylation status of a collection of normal squamous, BE, BE + high-grade dysplasia (HGD), and EAC cases. We found distinct global methylation signatures, as well as differential methylation of specific genes, that discriminated these histological groups. We also noted high and low methylation epigenotypes among the BE and EAC cases. Additional validation of those CpG sites that distinguished BE from BE + HGD and EAC may lead to the discovery of useful biomarkers with potential clinical applications in the diagnosis and prognosis of BE and EAC.
PMCID: PMC3256330  PMID: 22139570
Barrett's esophagus; esophageal adenocarcinoma; DNA methylation; methylation microarray
15.  Epigenetics and Colorectal Cancer 
Colorectal cancer is a leading cause of cancer deaths in the world. It results from an accumulation of genetic and epigenetic changes in colon epithelial cells that transforms them into adenocarcinomas. There have been major advances in our understanding of cancer epigenetics over the last decade, particularly regarding aberrant DNA methylation. Assessment of the colon cancer epigenome has revealed that virtually all colorectal cancers have aberrantly methylated genes and the average colorectal cancer methylome has hundreds to thousands of abnormally methylated genes. As with gene mutations in the cancer genome, a subset of these methylated genes, called driver genes, is presumed to play a functional role in colorectal cancer. The assessment of methylated genes in colorectal cancers has also revealed a unique molecular subgroup of colorectal cancers called CpG Island Methylator Phenotype (CIMP) cancers; these tumors have a particularly high frequency of methylated genes. The advances in our understanding of aberrant methylation in colorectal cancer has led to epigenetic alterations being developed as clinical biomarkers for diagnostic, prognostic, and therapeutic applications. Progress in the assessment of epigenetic alterations in colorectal cancer and their clinical applications has shown that these alterations will be commonly used in the near future as molecular markers to direct the prevention and treatment of colorectal cancer.
PMCID: PMC3391545  PMID: 22009203
Colon cancer; DNA methylation; epigenetics; biomarkers
16.  Genomic Aberrations Occurring in Subsets of Serrated Colorectal Lesions but not Conventional Adenomas 
Cancer research  2013;73(9):10.1158/0008-5472.CAN-12-3462.
A subset of aggressive colorectal cancers exhibit BRAF mutation, MLH1 methylation, and a CpG island methylator phenotype (CIMP), but precursors are poorly established. In this study, we determined the status of these markers in colorectal polyps and evaluated associated risk factors. The study included 771 polyp cases and 1,027 controls who were ages 24-80, part of a group health program, received a colonoscopy from 1998-2007, and completed a structured questionnaire assessing risk factors. Following standard pathology review, polyps were assayed for BRAF mutation (V600E) and tested for MLH1 and CIMP methylation, the latter including the genes: CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1. Polytomous logistic regression was used to estimate odds ratios and 95% confidence intervals for the association between molecularly-defined subsets of polyps and potential risk factors. There were 580 conventional adenomas and 419 serrated lesions successfully assayed. For adenomas, the prevalence of each marker was ≤1%. In contrast, 55% of serrated lesions harbored mutant BRAF, 26% were CIMP-high, and 5% had methylated MLH1. In these lesions, the highest prevalence of markers was in sessile serrated polyps (SSPs) of ≥10 mm that were in the right-side/cecal regions of the colon. Risk factors for CIMP-high serrated lesions included Caucasian race, current smoking status, and a history of polyps, whereas for serrated lesions with mutant BRAF the significant risk factors were male sex, current smoking status, obesity, and a history of polyps. Our results suggest that SSPs and other large, right-sided serrated lesions have a unique molecular profile that is similar to CIMP-high, BRAF-mutated colorectal cancers.
PMCID: PMC3815695  PMID: 23539450
BRAF; CIMP; MLH1; risk factors; colorectal polyps
17.  Colorectal Cancer Molecular Biology Moves Into Clinical Practice 
Gut  2010;60(1):116-129.
The promise of personalized medicine is now a clinical reality, with colorectal cancer genetics at the forefront of this next major advance in clinical medicine. This is no more evident than in the recent advances in testing of colorectal cancers for specific molecular alterations in order to guide treatment with the monoclonal antibody therapies cetuximab and panitumumab, which target the epidermal growth factor receptor (EGFR). In this review, we examine genetic mechanisms of colorectal cancer and how these alterations relate to emerging biomarkers for early detection and risk stratification (diagnostic markers), prognosis (prognostic markers), and the prediction of treatment responses (predictive markers).
PMCID: PMC3006043  PMID: 20921207
Colon Cancer; Biomarkers; EGFR; KRAS; K-Ras; BRAF; Microsatellite Instability; MSI; Chromosome Instability; Cetuximab; Panitumumab; Personalized Medicine
18.  Field cancerization in the colon: a role for aberrant DNA methylation? 
Gastroenterology Report  2014;2(1):16-20.
Colorectal cancer is the third most common cancer worldwide and arises secondary to the progressive accumulation of genetic and epigenetic alterations in normal colon cells, which results in a polyp-to-cancer progression sequence. It is known that individuals with a personal history of colon adenomas or cancer are at increased risk for metachronous colon neoplasms. One explanation for this increased risk could be field cancerization, which is a phenomenon in which the histologically normal tissue in an organ is primed to undergo transformation. Epigenetic alterations appear to be promising markers for field cancerization. In this review, we discuss field cancerization in the colon and the data supporting the use of methylated DNA as a biomarker for this phenomenon.
PMCID: PMC3920999  PMID: 24760232
colorectal cancer; field cancerization; epigenetic alterations; DNA methylation
19.  Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model 
Molecular carcinogenesis  2010;49(1):94-103.
Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer-specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19Arf, Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.
PMCID: PMC2875385  PMID: 19777566
DNA methylation; azoxymethane; colorectal cancer; epigenetics
20.  CIMP and colon cancer gets more complicated 
Gut  2007;56(11):1498-1500.
Evidence for a subset of colon cancers with low‐level CIMP that has unique molecular and clinical features compared with cancers with no CIMP and high‐level CIMP
PMCID: PMC2095640  PMID: 17938428
DNA methylation; CpG island; MGMT; epigenetics
21.  TGF-β receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-β 
Biochimica et biophysica acta  2009;1793(7):1165-1173.
TGF-β is a pluripotent cytokine that mediates its effects through a receptor composed of TGF-β receptor type II (TGFBR2) and type I (TGFBR1). The TGF-β receptor can regulate Smad and nonSmad signaling pathways, which then ultimately dictate TGF-β's biological effects. We postulated that control of the level of TGFBR2 is a mechanism for regulating the specificity of TGF-β signaling pathway activation and TGF-β's biological effects. We used a precisely regulatable TGFBR2 expression system to assess the effects of TGFBR2 expression levels on signaling and TGF-β mediated apoptosis. We found Smad signaling and MAPK-ERK signaling activation levels correlate directly with TGFBR2 expression levels. Furthermore, p21 levels and TGF-β induced apoptosis appear to depend on relatively high TGFBR2 expression and on the activation of the MAPK-ERK and SMAD pathways. Thus, control of TGFBR2 expression and the differential activation of TGF-β signaling pathways appears to be a mechanism for regulating the specificity of the biological effects of TGF-β.
PMCID: PMC2700179  PMID: 19339207
TGF-β; Smad; nonSmad; p21; apoptosis; signaling
22.  TGF-β receptor inactivation and mutant Kras induce intestinal neoplasms in mice via a β-catenin independent pathway 
Gastroenterology  2009;136(5):1680-8.e7.
Background & Aims
During colorectal cancer pathogenesis, mutations and epigenetic events cause neoplastic behavior in epithelial cells by deregulating the Wnt, Ras–Raf–ERK, and transforming growth factor (TGF)-β signaling pathways, among others. The TGF-β signaling pathway is often inactivated in colon cancer cells by mutations in the gene encoding the TGF-β receptor TGFBR2. The Ras–Raf–ERK pathway is frequently upregulated in colon cancer via mutational activation of KRAS or BRAF. We assessed how these pathways interact in vivo and affect formation of colorectal tumors.
We analyzed intestinal tumors that arose in mice that express an oncogenic (active) form of Kras and that have Tgfbr2 mutations––2 common genetic events observed in human colorectal tumors. LSL-KrasG12D mice were crossed with mice with Villin-Cre;Tgfbr2E2flx/E2flx mice, which do not express Tgfbr2 in the intestinal epithelium.
Neither inactivation of Tgfbr2 nor expression of oncogenic Kras alone was sufficient to induce formation of intestinal neoplasms. Histologic abnormalities arose in mice that expressed Kras, but only the combination of Tgfbr2 inactivation and Kras activation led to intestinal neoplasms and metastases. The cancers arose via a β-catenin–independent mechanism; the epidermal growth factor signaling pathway was also activated. Cells in the resulting tumors proliferated at higher rates, expressed decreased levels of p15, and expressed increased levels of cyclin D1 and cdk4, compared to control cells.
A combination of inactivation of the TGF-β signaling pathway and expression of oncogenic Kras leads to formation of invasive intestinal neoplasms through a β-catenin–independent pathway; these adenocarcinomas have the capacity to metastasize.
PMCID: PMC2782436  PMID: 19208363
colon cancer; transforming growth factor beta; KRAS; transformation; epidermal growth factor
23.  DNA alkylation and DNA methylation: cooperating mechanisms driving the formation of colorectal adenomas and adenocarcinomas? 
Gut  2007;56(3):318-320.
Defining our understanding of the association between DNA alkylation and colon carcinogenesis
PMCID: PMC1856827  PMID: 17339242
24.  The aberrant methylation of TSP1 suppresses TGF-β1 activation in colorectal cancer 
Colorectal cancer arises from the progressive accumulation of mutations and epigenetic alterations in colon epithelial cells. Such alterations often deregulate signaling pathways that affect the formation of colon cancer, such as the Wnt, RAS-MAPK and TGF-β pathways. The tumor promoting effects of mutations in genes, such as APC, have been demonstrated in cancer cell lines and in mouse models of intestinal cancer; however, the biological effects of most epigenetic events identified in colorectal cancer remain unknown. Consequently, we assessed whether the aberrant methylation of TSP1, the gene for thrombospondin 1, a regulator of TGF-β ligand activation, is an epigenetic mechanism for inhibiting the TGF-β signaling pathway. We found methylated TSP1 occurs in colon cancer cell lines (33%), colon adenomas (14%) and colon adenocarcinomas (21%). In primary colorectal cancers, loss of TSP1 expression correlated with impaired TGF-β signaling as indicated by decreased Smad2 phosphorylation and nuclear localization. Furthermore, methylation-induced silencing of TSP1 expression reduced the concentration of secreted active TGF-β1 and attenuated TGF-β signaling. Reversal of TSP1 methylation resulted in increased TSP1 mediated activation of the latent LAP:TGF-β complex and subsequent TGF-β receptor activation. Our results demonstrate that the aberrant methylation of TSP1 has biological consequences and provide evidence that the aberrant methylation of TSP1 is a novel epigenetic mechanism for suppressing TGF-β signaling in colorectal cancer.
PMCID: PMC2777657  PMID: 18425817
Thrombospondin; transforming growth factor β; methylation; colorectal cancer; epigenetics
25.  Colorectal endoscopy, advanced adenomas, and sessile serrated polyps: implications for proximal colon cancer 
Colonoscopy is associated with a decreased risk of colorectal cancer but may be more effective in reducing the risk of distal than proximal malignancies. To gain insight into the differences between proximal and distal colon endoscopic performance, we conducted a case-control study of advanced adenomas, the primary targets of colorectal endoscopy screening, and sessile serrated polyps (SSPs), newly recognized precursor lesions for a colorectal cancer subset that occurs most often in the proximal colon.
The Group Health-based study population included: 213 advanced adenoma cases, 172 SSP cases, and 1,704 controls ages 50–79, who received an index colonoscopy from 1998–2007. All participants completed a structured questionnaire covering endoscopy history. Participants with polyps underwent a standard pathology review to confirm the diagnosis and reclassify a subset as advanced adenomas or SSPs. Logistic regression analyses were conducted to estimate adjusted odds ratios (OR) and 95% confidence intervals (CI) for the association between endoscopy and advanced adenomas and SSPs separately; site-specific analyses were completed.
Previous endoscopy was associated with decreased risk of advanced adenomas in both the rectum/distal colon (OR=0.38; 95% CI: 0.26–0.56) and proximal colon (OR=0.31; 95% CI: 0.19–0.52), but there was no statistically significant association between prior endoscopy and SSPs (OR=0.80; 95%CI: 0.56–1.13).
Our results support the hypothesis that the effect of endoscopy differs between advanced adenomas and SSPs. This may have implications for proximal colon cancer prevention and be due to the failure of endoscopy to detect/remove SSPs, or the hypothesized rapid development of SSPs.
PMCID: PMC3418887  PMID: 22688851

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