Search

Although somatic mutations in colorectal cancer are well characterized, little is known about the accumulation of cancer mutations in the normal colon before cancer. Here, we have developed and applied an ultrasensitive, single-molecule mutational test based on CRISPR-DS technology, which enables mutation detection at extremely low frequency (<0.001) in normal colon from patients with and without colorectal cancer. This testing platform revealed that normal colon from patients with and without colorectal cancer carries mutations in common colorectal cancer genes, but these mutations are more abundant in patients with cancer. Oncogenic KRAS mutations were observed in the normal colon of about one third of patients with colorectal cancer but in none of the patients without colorectal cancer. Patients with colorectal cancer also carried more TP53 mutations than patients without cancer and these mutations were more pathogenic and formed larger clones, especially in patients with early-onset colorectal cancer. Most mutations in the normal colon were different from the driver mutations in tumors, suggesting that the occurrence of independent clones with pathogenic KRAS and TP53 mutations is a common event in the colon of individuals who develop colorectal cancer. These results indicate that somatic evolution contributes to clonal expansions in the normal colon and that this process is enhanced in individuals with cancer, particularly in those with early-onset colorectal cancer., Significance: This work suggests prevalent somatic evolution in the normal colon of patients with colorectal cancer, highlighting the potential of using ultrasensitive gene sequencing to predict disease risk., (©2022 American Association for Cancer Research.)

Unlabelled: Peptidyl arginine deiminases (PADI) are a family of enzymes that catalyze the poorly understood posttranslational modification converting arginine residues into citrullines. In this study, the role of PADIs in the pathogenesis of colorectal cancer was investigated. Specifically, RNA expression was analyzed and its association with survival in a cohort of 98 colorectal cancer patient specimens with matched adjacent mucosa and 50 controls from donors without cancer. Key results were validated in an independent collection of tumors with matched adjacent mucosa and by mining of a publicly available expression data set. Protein expression was analyzed by immunoblotting for cell lines or IHC for patient specimens that further included 24 cases of adenocarcinoma with adjacent dysplasia and 11 cases of active ulcerative colitis. The data indicate that PADI2 is the dominantly expressed PADI enzyme in colon mucosa and is upregulated during differentiation. PADI2 expression is low or absent in colorectal cancer. Frequently, this occurs already at the stage of low-grade dysplasia. Mucosal PADI2 expression is also low in ulcerative colitis. The expression level of PADI2 in tumor and adjacent mucosa correlates with differential survival: low levels associate with poor prognosis., Implications: Downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis and points toward a possible role of citrullination in modulating tumor cells and their microenvironment. Mol Cancer Res; 14(9); 841-8. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers., Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls., Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated., Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%., Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential., (©2011 AACR.)

DNA hypomethylation is a common trait of colorectal cancer. Studies in tumor cell lines and animal models indicate that genome-wide demethylation may cause genetic instability and hence facilitate or accelerate tumor progression. Recent studies have shown that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer, but the nature of this damage has not been clearly established. Here, we show a thorough analysis of DNA methylation and genetic alterations in two series of colorectal carcinomas. The extent of DNA demethylation but not of hypermethylation (both analyzed by amplification of intermethylated sites in near 200 independent sequences arbitrarily selected) correlated with the cumulated genomic damage assessed by two different techniques (arbitrarily primed PCR and comparative genomic hybridization). DNA hypomethylation-related instability was mainly of chromosomal nature and could be explained by a genome-wide effect rather than by the concurrence of the most prevalent genetic and epigenetic alterations. Moreover, the association of p53 mutations with genomic instability was secondary to DNA hypomethylation and the correlation between DNA hypomethylation and genomic instability was observed in tumors with and without mutation in the p53 gene. Our data support a direct link between genome-wide demethylation and chromosomal instability in human colorectal carcinogenesis and are consistent with the studies in model systems demonstrating a role of DNA demethylation in inducing chromosomal instability.

Various studies have suggested the existence of different pathways of tumor progression in colorectal cancer that associate with specific molecular, chromosomal, and clinicopathological features. We hypothesize that a comprehensive analysis of cumulated genomic damage in colorectal cancers would aid the characterization of different tumor progression pathways and identify the factors determining clinical outcome of tumors of each type. Genome-wide disruption was studied by DNA fingerprinting in a series of 129 sporadic colorectal carcinomas. These results, taken together with data for DNA ploidy, microsatellite instability, p53, and K-ras mutations and clinicopathological characteristics of the patients, have been used to classify colorectal carcinomas. The following five groups can be defined based on the type and level of cumulated genomic damage: (a) tumors with microsatellite instability, right location, and good prognosis; (b) diploid tumors lacking p53 mutations, left and right location, low subchromosomal damage, and bad prognosis; (c) diploid tumors with p53 mutations, left location, high levels of subchromosomal damage, and good prognosis; (d) high aneuploid tumors, p53 mutations, left location, high levels of numerical and structural chromosomal alterations, and bad prognosis; and finally (e) low aneuploid tumors, no p53 mutations, left and right location, low levels of structural chromosomal alterations, and good prognosis. We postulate that these groups represent alternative pathways of tumor progression, each with determinants of aggressiveness. This indicates a need for different prognostic assessments depending on which group the tumor belongs to.

Cancer cells progress through the accumulation of genetic alterations. Familial adenomatous polyposis (FAP) tumors provide an excellent model to unravel the molecular steps underlying malignant transformation. Global genomic damage was assessed in 56 adenomas and 3 carcinomas from six FAP patients and compared with that of sporadic adenomas and carcinomas. Evolutive trees were traced after application of maximum likelihood clustering and split decomposition methods to the analysis of comprehensive genetic profiles generated by diverse molecular approaches: arbitrarily primed PCR, comparative genomic hybridization, and flow cytometry. Overall, genomic damage as assessed by arbitrarily primed PCR was lower in familial adenomas than in sporadic adenomas and carcinomas. Comparative genomic hybridization data also show a low number of alterations in the majority of FAP adenomas. Tumors of the same patient were likely to share specific genetic alterations and may be grouped into one or two clusters. Putative common pathways were also identified, which included tumors of up to three different patients. According to our data, FAP tumors accumulate specific genetic alterations and in a preferred order that is characteristic of each individual. Moreover, the particular genetic background and environmental conditions of a FAP patient restrain the molecular evolution portrait of synchronous tumors.

The effects of retinol (vitamin A) depend on its transport and binding to nuclear receptors. The cellular retinol-binding protein 1 (CRBP1) and the retinoic acid receptor beta2 (RARbeta2) are key components of this process. Loss of CRBP1 expression occurs in breast tumors, but the mechanism is not known. We examined whether CpG island hypermethylation of CRBP1 was responsible for its inactivation in cancer cell lines (n = 36) and primary tumors (n = 553) and its relation to RARbeta2 methylation. Hypermethylation of CRBP1 was common in tumors and cancer cell lines, with the highest frequency in lymphoma and gastrointestinal carcinomas. Hypermethylation correlated with loss of CRBP1 mRNA, and in vitro treatment with the demethylating agent 5-aza-2'-deoxycytidine reactivated CRBP1 expression. CRBP1 methylation appeared in premalignant lesions and frequently occurred with RARbeta2 hypermethylation in the same tumors. Finally, we observed that a higher dietary retinol intake was associated with reduced frequencies of methylation of both genes. Epigenetic disruption of CRBP1 is a common event in human cancer that may have important implications for cancer prevention and treatment using retinoids.

Aberrant DNA methylation is recognized as being a common feature of human neoplasia.CpG island hypermethylation and global genomic hypomethylation occur simultaneously in the cancer cell. However, very little is known about the interindividual inherited susceptibility to these epigenetic processes. To address this matter, we have genotyped in 233 cancer patients (with colorectal, breast, or lung tumors), four germ-line variants in three key genes involved in the metabolism of the methyl group, methylene-tetrahydrofolate reductase, methionine synthase, and cystathionine beta-synthase, and analyzed their association with DNA methylation parameters. The epigenetic features analyzed were the 5-methylcytosine content in the genome of the tumors and their normal counterparts, and the presence of CpG island hypermethylation of tumor suppressor genes (p16(INK4a), p14(ARF), hMLH1, MGMT, APC, LKB1, DAPK, GSTP1, BRCA1, RAR beta 2, CDH1, and RASSF1). Two positive associations were found. First, carriers of genotypes containing the methylene-tetrahydrofolate reductase 677T allele show constitutive low levels of 5-methylcytosine in their genomes (P = 0.002), and tumors in these patients do not achieve severe degrees of global hypomethylation (P = 0.047). Second, tumors occurring in homozygous carriers of the methionine synthase 2756G allele show a lower number of hypermethylated CpG islands of tumor suppressor genes (P = 0.029). The existence of these associations may provide another example of the interplay between genetic and epigenetic factors in the cancer cell.

Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

The INK4a/ARF locus encodes two distinct tumor suppressors, p16INK4a and p14ARF. Although the contribution of p16INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14ARF lesions and their consequences. Recent data indicate that p14ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14ARF methylation. Taken together, these data support that epigenetic silencing of p14ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.

Germ-line mutations in the tumor suppressor gene APC are associated with hereditary familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal tumors. We now report that methylation in the promoter region of this gene constitutes an alternative mechanism for gene inactivation in colon and other tumors of the gastrointestinal tract. The APC promoter is hypermethylated in 18% of primary sporadic colorectal carcinomas (n = 108) and adenoma (n = 48), and neoplasia with APC methylation fails to express the APC transcript. Methylation affects only wild-type APC in 95% of cases and is not observed in tumors from FAP patients who have germ-line APC mutations. As with APC mutation, aberrant APC methylation occurs early in colorectal carcinogenesis. When other tumor types are analyzed (n = 208), methylation of the APC promoter is not restricted to the colon but is present in tumors originating elsewhere in the gastrointestinal tract but rarely in other tumors. Our data suggest that hypermethylation of APC provides an important mechanism for impairing APC function and further underscores the importance of the APC pathway in gastrointestinal tumorigenesis.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.