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).
Colon Cancer; Biomarkers; EGFR; KRAS; BRAF; Microsatellite Instability; MSI; Chromosome Instability; Cetuximab; Panitumumab
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.
Comparative effectiveness research; Genomics; Next generation sequencing; Randomized clinical trial; Outcomes research; Whole exome sequencing
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.
HCC; CC; AFP; Smad3; ERK1/2
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.
colon neoplasia; methylation; RET
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.
BRAF; CIMP; MLH1; risk factors; colorectal polyps
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.
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.
Colorectal cancer; Inflammatory bowel disease; Outcomes research; Cancer survival; Inflammation
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.
colorectal cancer; field cancerization; epigenetic alterations; DNA methylation
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.
Barrett's esophagus; esophageal adenocarcinoma; DNA methylation; methylation microarray
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.
Colon cancer; DNA methylation; epigenetics; biomarkers
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.
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).
Colon Cancer; Biomarkers; EGFR; KRAS; K-Ras; BRAF; Microsatellite Instability; MSI; Chromosome Instability; Cetuximab; Panitumumab; Personalized Medicine
NTRK3 is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated NTRK3 in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. NTRK3 is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. NTRK3 methylation suppresses NTRK3 expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of NTRK3 expression associates with neoplastic transformation in vitro and in vivo. We also found that a naturally occurring mutant NTRK3 found in human colorectal cancer inhibits the tumor suppressor activity of NTRK3. In summary, our findings suggest NTRK3 is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.
NTRK3 is a neurotrophin receptor and appears to be a dependence receptor in certain tissues. NTRK3 has been previously shown to be an oncogene in breast cancer and possibly hepatocellular carcinoma. Through a genome-wide methylation screen, we unexpectedly found that NTRK3 is commonly methylated in colorectal cancers but not in normal colon samples, which led us to assess whether NTRK3 could be a tumor suppressor gene in the colon. We now demonstrate that NTRK3 is frequently methylated in colorectal adenomas and cancers. Induced NTRK3 expression in the absence of its ligand, NT-3, causes apoptosis and suppresses in vitro anchorage-independent colony formation and in vivo tumor growth. Reintroduction of NT-3 releases colon cancer cells from NTRK3-mediated apoptosis, which is consistent with NTRK3 being a dependence receptor in the colon. Finally, somatic mutations of NTRK3 have been observed in primary human colorectal cancer. We provide evidence that a subset of these mutations inactivate tumor suppressor activities of NTRK3. These findings suggest that NTRK3 is a conditional tumor suppressor gene in the colon that is inactivated by both genetic and epigenetic mechanisms and whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.
It is postulated that high serum levels of insulin and insulin growth factor 1 (IGF-1) mediate obesity-associated carcinogenesis. The relationship of insulin, IGF-1 and IGF binding proteins (IGFBP) with Barrett’s oesophagus (BO) has not been well examined.
Serum levels of insulin and IGFBPs in patients with BO were compared with two separate control groups: subjects with gastro-oesophageal reflux disease (GORD) and screening colonoscopy controls. Fasting insulin, IGF-1 and IGFBPs were assayed in the serum of BO cases (n = 135), GORD (n = 135) and screening colonoscopy (n = 932) controls recruited prospectively at two academic hospitals. Logistic regression was used to estimate the risk of BO.
Patients in the highest tertile of serum insulin levels had an increased risk of BO compared with colonoscopy controls (adjusted OR 2.02, 95% CI 1.15 to 3.54) but not compared with GORD controls (adjusted OR 1.55, 95% CI 0.76 to 3.15). Serum IGF-1 levels in the highest tertile were associated with an increased risk of BO (adjusted OR 4.05, 95% CI 2.01 to 8.17) compared with the screening colonoscopy control group but were not significantly different from the GORD control group (adjusted OR 0.57, 95% CI 0.27 to 1.17). IGFBP-1 levels in the highest tertile were inversely associated with a risk of BO in comparison with the screening colonoscopy controls (adjusted OR 0.11, 95% CI 0.05 to 0.24) but were not significantly different from the GORD control group (adjusted OR 1.04, 95% CI 0.49 to 2.16). IGFBP-3 levels in the highest tertile were inversely associated with the risk of BO compared with the GORD controls (OR 0.36, 95% CI 0.16 to 0.81) and also when compared with the colonoscopy controls (OR 0.40, 95% CI 0.20 to 0.79).
These results provide support for the hypothesis that the insulin/IGF signalling pathways have a role in the development of BO.
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.
DNA methylation; azoxymethane; colorectal cancer; epigenetics
Genetic influences may be discerned in families that have multiple affected members and may manifest as an earlier age of cancer diagnosis. In this study we determine whether cancers develop at an earlier age in multiplex Familial Barrett’s Esophagus (FBE) kindreds, defined by 3 or more members affected by Barrett’s esophagus (BE) or esophageal adenocarcinoma (EAC).
Information on BE/EAC risk factors and family history was collected from probands at eight tertiary care academic hospitals. Age of cancer diagnosis and other risk factors were compared between non-familial (no affected relatives), duplex (two affected relatives), and multiplex (three or more affected relatives) FBE kindreds.
The study included 830 non-familial, 274 duplex and 41 multiplex FBE kindreds with 274, 133 and 43 EAC and 566, 288 and 103 BE cases, respectively. Multivariable mixed models adjusting for familial correlations showed that multiplex kindreds were associated with a younger age of cancer diagnosis (p = 0.0186). Median age of cancer diagnosis was significantly younger in multiplex compared to duplex and non-familial kindreds (57 vs. 62 vs. 63 yrs, respectively, p = 0.0448). Mean body mass index (BMI) was significantly lower in multiplex kindreds (p = 0.0033) as was smoking (p < 0.0001), and reported regurgitation (p = 0.0014).
Members of multiplex FBE kindreds develop EAC at an earlier age compared to non-familial EAC cases. Multiplex kindreds do not have a higher proportion of common risk factors for EAC, suggesting that this aggregation might be related to a genetic factor.
These findings indicate that efforts to identify susceptibility genes for BE and EAC will need to focus on multiplex kindreds.
Esophageal adenocarcinoma; Barrett’s esophagus; genetics; family history
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
DNA methylation; CpG island; MGMT; epigenetics
Supplement use among cancer patients is high, and folic acid intake in particular may adversely affect the progression of colorectal cancer. Few studies have evaluated the use of folic acid-containing supplements (FAS) and its predictors in colorectal cancer patients.
To assess the use of FAS, change in use, and its predictors after colorectal cancer diagnosis.
We used logistic regression models to investigate predictors of FAS use and its initiation after colorectal cancer diagnosis in 1,092 patients recruited through the Colon Cancer Family Registry (C-CFR).
The prevalence of FAS use was 35.4% before and 55.1% after colorectal cancer diagnosis (p=0.004). Women were more likely than men to use FAS after diagnosis (OR 1.47, 95% CI 1.14-1.89), as were those consuming more fruit (ptrend<0.0001) or vegetables (ptrend=0.001), and US residents (p<0.0001). Less likely to use FAS after diagnosis were non-white patients (OR 0.66, 95% CI 0.45-0.97), current smokers (OR 0.67, 95% CI 0.46-0.96), and those with higher meat intake (ptrend=0.03). Predictors of FAS initiation after diagnosis were generally similar to those of FAS use after diagnosis, though associations with race and vegetable intake were weaker and those with exercise stronger.
Our analysis showed substantial increases in the use of folic acid-containing supplements after diagnosis with colorectal cancer, with use or initiation more likely among women, Caucasians, U.S. residents, and those with a health-promoting lifestyle.
Studies of cancer prognosis that rely on pre-diagnostic exposure information may result in substantial misclassification.
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.
TGF-β; Smad; nonSmad; p21; apoptosis; signaling
Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7−/−; p53−/− carcinomas exhibit a CIN+ phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.
Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia.
Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26–81) were included in the study. Using the SFP10 LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p<0.01). The majority of differentially methylated gene targets occurred at or close to specific chromosomal locations such as previously described HPV methylation “hotspots” and viral integration sites.
We have identified a panel of differentially methlylated CpG loci across the spectrum of HPV-associated squamous neoplasia of the anus. To our knowledge, this is the first reported application of large-scale high throughput methylation analysis for the study of anal neoplasia. Our findings support further investigations into the role of host-genome methylation in HPV-associated anal carcinogenesis with implications towards enhanced diagnosis and screening strategies.
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.
colon cancer; transforming growth factor beta; KRAS; transformation; epidermal growth factor
Defining our understanding of the association between DNA alkylation and colon carcinogenesis
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.
Thrombospondin; transforming growth factor β; methylation; colorectal cancer; epigenetics