DNA methylation of phosphatase and actin regulator 3 detects colorectal cancer in stool and complements FIT

Using a bioinformatics-based strategy, we set out to identify hypermethylated genes that could serve as biomarkers for early detection of colorectal cancer (CRC) in stool. In addition, the complementary value to a Fecal Immunochemical Test (FIT) was evaluated. Candidate genes were selected by applying cluster alignment and computational analysis of promoter regions to microarray-expression data of colorectal adenomas and carcinomas. DNA methylation was measured by quantitative methylation-specific PCR on 34 normal colon mucosa, 71 advanced adenoma, and 64 CRC tissues. The performance as biomarker was tested in whole stool samples from in total 193 subjects, including 19 with advanced adenoma and 66 with CRC. For a large proportion of these series, methylation data for GATA4 and OSMR were available for comparison. The complementary value to FIT was measured in stool subsamples from 92 subjects including 44 with advanced adenoma or CRC. Phosphatase and Actin Regulator 3 (PHACTR3) was identified as a novel hypermethylated gene showing more than 70-fold increased DNA methylation levels in advanced neoplasia compared with normal colon mucosa. In a stool training set, PHACTR3 methylation showed a sensitivity of 55% (95% CI: 33-75) for CRC and a specificity of 95% (95% CI: 87-98). In a stool validation set, sensitivity reached 66% (95% CI: 50-79) for CRC and 32% (95% CI: 14-57) for advanced adenomas at a specificity of 100% (95% CI: 86-100). Adding PHACTR3 methylation to FIT increased sensitivity for CRC up to 15%. PHACTR3 is a new hypermethylated gene in CRC with a good performance in stool DNA testing and has complementary value to FIT.     

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment,tracing a biomarker signature specific for this tumor

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non‐invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell‐free circulating DNA from CRC patients.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

Methylation and expression of p16INK4 tumor suppressor gene in primary colorectal cancer tissues

It is known that p16(INK4) tumor suppressor gene expression in colon cancer cells is repressed by methylation at the CpG island of promoter, but in vivo silencing of p16 gene is not fully understood. Some studies showed that primary colorectal cancer (CRC) tissues often overexpress the p16 protein, while others showed the high incidence of p16 methylation. The aim of this study was to clarify p16 gene regulation in vivo. We used real-time methylation-specific PCR (MSP) to examine density of p16 methylation, and immunohistochemistry, Western blot analysis to determine p16 protein expression. Methylation was detected in 5 CRC cell lines tested and 9 of 21 (42.9%) CRCs. Four of 5 CRC cell lines did not express p16 mRNA, but 6 of 9 CRCs did express p16 mRNA even with methylation. Real-time MSP showed that CRC tissues had a wide variety in methylation density (methylation index: 0.28-0.91) and that highly methylated CRC tissues displayed significantly lower p16 mRNA expression than those with no-methylation or low-methylation. Immunohistochemistry showed that the majority of CRCs (53 of 55: 96.4%) overexpressed the p16 protein. Low p16 expression was associated with lymph node metastasis (p=0.003) and large tumor size (p=0.048). Western blot in a subset of non-tumor and tumor samples showed a consistent overexpression of the p16 protein. These results showed that CRC tissues displayed variable methylation density, which may be characteristics of p16 gene methylation in vivo. Our data suggest that a low p16 expression due to methylation may contribute to tumor enlargement and expansion of CRC.     

CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5' CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.     

Identification of transgelin‐2 as a biomarker of colorectal cancer by laser capture microdissection and quantitative proteome analysis

To search for potential protein markers of colorectal cancer (CRC), the changes in protein expression levels between microdissected tumor cells and normal mucosa epithelia were analyzed by an acetylation stable isotopic labeling method coupled with linear quadrupole ion trap fourier transform mass spectrometry (LTQ‐FTMS). In total, 137 proteins were up‐regulated or down‐regulated significantly in cancer by at least two‐fold. Based on gene ontology analysis, the largest part of differential proteins were unknown for both subcellular localization and biological process. In particular, the significant up‐regulation of transgelin‐2 (TAGLN2) in CRC was validated by Western blot analysis and further evaluated by immunohistochemistry in paired tumor and normal mucosa samples from 120 consecutive CRC patients, 20 adenomas, and eight synchronous hepatic metastases of CRC. TAGLN2 expression was frequently observed in cancer cells, precancerous lesions, and hepatic metastases, whereas in normal epithelia expression was rarely observed. The overexpression of TAGLN2 was associated with lymph node and distant metastasis, advanced clinical stage (P < 0.001), and shorter overall survival in CRCs. Cox regression analysis indicated that high tumor‐TAGLN2 expression represents an independent prognostic factor. Consequently, over‐expression of TAGLN2 may serve as a new biomarker for predicting progression and prognosis of CRC. (Cancer Sci 2009)     

Aberrant promoter methylation of multiple genes during multistep pathogenesis of colorectal cancers

Aberrant methylation of 5'gene promoter regions associated with gene silencing is an epigenetic phenomenon responsible for silencing of tumor suppressor genes in many cancer types. The aims of our study were to study the role of methylation of a large panel of genes during multistage pathogenesis and to correlate our findings with patient age and other clinico-pathological features. We investigated the aberrant promoter methylation profile of 19 genes in 92 colorectal cancers (CRCs) and corresponding nonmalignant epithelia (NME) (n = 57), and selected 15 genes for studying 26 colorectal adenomas (CAs). On the Basis of our results, the genes could be divided into 3 groups. Group 1 consisted of 13 genes whose methylation was tumor-specific. For 8 of these genes, the methylation frequencies in CAs were similar to those of CRCs, but significantly different from the frequencies in NME. Group 2, consisting of 2 genes demonstrating little or no methylation, were present in any sample type. In Group 3, consisting of 4 genes, relatively frequent methylation was present in both CRCs and NME, and the differences between these specimen types were not significant. Methylation of Group 1 genes were tightly correlated with each other, and these genes demonstrated increased methylation frequencies in CRCs with increasing age. Methylation was not correlated with other clinico-pathological features. In general, methylation frequencies of CAs were intermediate between CRCs and NME. Our study constitutes the most comprehensive methylation profile of CRCs, demonstrates that methylation commences early during CRC pathogenesis and is an age-related phenomenon.     

Methyl-CpG binding column-based identification of nine genes hypermethylated in colorectal cancer

DNA methylation is an epigenetic event that plays a role in gene expression regulation. Alterations in DNA methylation contribute to cancer development and progression. The aim of this study was to identify gene promoters aberrantly methylated in colorectal tumor tissue in comparison to normal colonic mucosa. Analyses were performed on two pooled DNA samples: from normal and cancerous tissue obtained from CRC patients. DNA was fractionated according to methylation degree with the use of affinity column containing methyl-CpG binding domain. To identify novel hypermethylated gene promoters, methylated DNA from normal and from cancerous tissues were analyzed with the use of promoter microarrays. We identified nine novel genes hypermethylated in colorectal cancer. The frequency of their promoter methylation was assessed in the larger group of patients (n = 77): KCNK12 (methylated in 41% of CRC patients), GPR101 (40%), CDH2 (45%), BARX1 (56%), CNTFR (22%), SYT6 (64%), SMO (21%), EPHA5 (43%), and GSPT2 (21%). The results of gene expression level analysis suggest the role of promoter methylation in downregulation of six out of nine genes examined. We did not find correlation between gene methylation and age, gender, tumor grade or stage. Importantly, in stage IV CRC methylation of GPR101 correlated with longer time to progression (P = 0.0042; HR = 2.5468; 95% CI 1.5391-10.0708).     

Identification of novel hypermethylated genes and demethylating effect of vincristine in colorectal cancer

Background

Colorectal cancer (CRC) arises as a consequence of genetic events such as gene mutation and epigenetic alteration. The aim of this study was to identify new hypermethylated candidate genes and methylation-based therapeutic targets using vincristine in CRC.

Methods

We analyzed the methylation status of 27,578 CpG sites spanning more than 14,000 genes in CRC tissues compared with adjacent normal tissues and normal colon tissues using Illumina bead chip array. Twenty-one hypermethylated genes and 18 CpG island methylator phenotype markers were selected as candidate genes. The methylation status of 39 genes was validated by quantitative methylation-specific polymerase chain reaction in CRC tissues, adjacent normal tissues, normal colon cells, and three CRC cell lines. Of these, 29 hypermethylated candidate genes were investigated using the demethylating effects of 5-aza-2′-deoxycytidine (5-aza-dC) and vincristine in CRC cells.

Results

Thirty-two out of 39 genes were hypermethylated in CRC tissues compared with adjacent normal tissues. Vincristine induced demethylation of methylated genes in CRC cells to the same extent as 5-aza-dC. The mRNA expression of AKR1B1, CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304 was restored by treatment with 5-aza-dC and vincristine.

Conclusion

These results suggest that these novel hypermethylated genes AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 may be potential methylation biomarkers and therapeutic targets of vincristine in CRC.     

Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing

Thyroid-stimulating hormone receptor (TSHR) expression is frequently silenced in epithelial thyroid cancers associated with decreased or absent TSH-promoted iodine uptake. To study the underlying molecular mechanism of decreased TSHR expression, we examined the methylation status of the TSHR gene promoter by sequencing bisulfite-treated DNA from thyroid tumors. After identification of methylated sites by sequencing bisulfite-treated DNA, we used methylation-specific polymerase chain reaction and found frequent CpG methylation in papillary thyroid cancer (23 of 39 patients; 59%) and follicular thyroid cancers (7 of 15 patients; 47%). In contrast, we saw no methylation in normal thyroid tissues and benign adenomas (0 of 8 patients; 0%). In human thyroid tumor cell lines, we observed that TSHR was normally expressed at the protein and mRNA level in cells where the TSHR gene was unmethylated, whereas it was silenced in cell lines where the TSHR promoter was hypermethylated. Treatment of the latter cells with a demethylating agent partially restored TSHR expression. We thus demonstrate aberrant methylation of human TSHR as a likely molecular pathway responsible for the silencing of this gene in thyroid cancers. We propose that methylation of TSHR may provide a novel diagnostic marker of malignancy and a basis for potential use of demethylating agents in conjunction with TSH-promoted radioiodine therapy for epithelial thyroid cancers.     

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.     

The APC E1317Q variant in adenomatous polyps and colorectal cancers.

Genetic susceptibility may play a role in many colorectal cancers (CRCs). Known syndromes such as familial adenomatous polyposis and hereditary nonpolyposis CRC account for <5% of CRCs. The germ-line missense variant of the APC gene, E1317Q, has been proposed to confer a risk for colonic adenomatous polyps (adenomas), but not for CRCs in the general population. These findings are contradictory and controversial. In the present study, 608 cases (377 patients with CRC, 145 patients with 4-100 lifetime adenomas, and 86 with < or =3 lifetime adenomas), and 679 controls (362 spouses and 317 patients with normal colonoscopy) were screened for the APC E1317Q variant. The frequency of heterozygotes for E1317Q among patients with CRC (2.4%), patients with 4-100 adenomas (1.4%), and < or =3 adenomas (3.5%) did not differ from spouse controls (2.8%). When CRC patients were examined by DNA mismatch repair status, age at onset (< or =age 50 versus >50), or family history of CRC, no differences in the frequency of E1317Q were found. The APC variant E1317Q does not appear to be associated with increased risk for colorectal neoplasia in the general population. However, when we used normal colonoscopy controls (E1317Q carrier frequency = 0.3%), the prevalence of E1317Q was significantly increased in CRC patients, in patients with < or =3 adenomas, and in CRC patients with intact mismatch repair status, suggesting a possible role for E1317Q in colorectal tumorigenesis. These results underscore the importance of carefully defining the controls to be used in comparisons of allele frequencies.     

SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2'deoxycytidine to increase SPARC expression and improve therapy response

Poor clinical outcomes in cancer can often be attributed to inadequate response to chemotherapy. Strategies to overcome either primary or acquired chemoresistance may ultimately impact on patients' survival favourably. We previously showed that lower levels of SPARC were associated with therapy-refractory colorectal cancers (CRC), and that upregulating its expression enhances chemo-sensitivity resulting in greater tumour regression in vivo. Here, we examined aberrant hypermethylation of the SPARC promoter as a potential mechanism for repressing SPARC in CRCs and whether restoration of its expression with a demethylating agent 5-Aza-2'deoxycytidine (5-Aza) could enhance chemosensitivity. Initially, the methylation status of the SPARC promoter from primary human CRCs were assessed following isolation of genomic DNA from laser capture microdissected specimens by direct DNA sequencing. MIP101, RKO, HCT 116, and HT-29 CRC cell lines were also used to evaluate the effect of 5-Aza on: SPARC promoter methylation, SPARC expression, the interaction between DNMT1 and the SPARC promoter (ChIP assay), cell viability, apoptosis, and cell proliferation. Our results revealed global hypermethylation of the SPARC promoter in CRCs, and identified specific CpG sites that were consistently methylated in CRCs but not in normal colon. We also demonstrate that SPARC repression in CRC cell lines could be reversed following exposure to 5-Aza, which resulted in increased SPARC expression, leading to a significant reduction in cell viability (by an additional 39% in RKO cells) and greater apoptosis (an additional 18% in RKO cells), when combined with 5-FU in vitro (in comparison to 5-FU alone). Our exciting findings suggest potential diagnostic markers of CRCs based on specific methylated CpG sites. Moreover, the results reveal the therapeutic utility of employing demethylating agents to improve response through augmentation of SPARC expression.