Event In Colorectal Tumorigenesis Research Articles

Implication of exon 4 TP53 Gene mutations in colorectal cancers in Senegalese patients

Background: Colorectal cancer (CRC) is a major cause of death in men and women, comprising about 10% of cancer deaths after breast and lung cancer. The most significant risk factors currently implicated in the etiology of this cancer are genetic, likely due to several mutations. Methods: The study explored the involvement of genetic factors in these pathologies; specifically, exon 4 of the TP53 gene was studied in 15 CRC patients and compared with 10 healthy individuals as controls. The position of the marker mutations was determined with the Mutation Surveyor software version 5.1.2. DnaSP version 5.10, MEGA version 7.014, and the program Arlequin version 3.1 were used to highlight the parameters of variability, differentiation, and the demo-genetic evolution of our study populations. The pathogenicity of the mutations was determined through Polyphen2, TAMISER, and ClinVar. Results: Our results showed the presence of a recurrent mutation of the TP53 gene in both tumor and healthy tissue where proline was replaced with arginine at codon 72. This mutation was predicted to be benign. The presence of this mutation in healthy tissue can be considered a relatively late event in colorectal tumorigenesis. In addition, the P47L, D49A, W53S, and D48G mutations appeared to be suspicious because they were predicted to be potentially damaging. This finding suggests the genes’ involvement in the pathology of CRCs in our study population. The cancer tissue sequences contained an average of 2.59 nucleotide differences that resulted in amino acid changes. The Nei genetic distance confirms this variability between tumor tissues. Conclusion: These results suggest that variants in exon 4 of the TP53 gene may contribute to the development of CRCs. These mutations could constitute molecular markers in CRC and possibly help in the development of early diagnosis

New Insights into the Mechanism of Action of Aspirin in the Prevention of Colorectal Neoplasia.

The results of clinical studies have shown that the chronic administration of aspirin, even at the lowdoses (75-100 mg daily) recommended for the prevention of cardiovascular disease, is associated with a reduction of cancer incidence and mortality, in particular colorectal cancer (CRC). The mechanism of action of aspirin as an antineoplastic agent remains controversial. However, data of clinical pharmacology and several features of the chemopreventive effect of aspirin, emerged from clinical trials, suggest that the antiplatelet effect of aspirin plays a central role in its anticancer effects. In addition to their contribution to tumor metastasis, platelets may play a role in the early phases of tumorigenesis. In response to lifestyle and environment factors, intestinal epithelial damage/ dysfunction may be associated with platelet activation, initially as a mechanism to repair the damage. However, if the platelet response is unconstrained, it may contribute to the development of chronic inflammation. Altogether these events lead to alter the normal functions of intestinal epithelial cells and may translate into cellular transformation through several mechanisms, including the overexpression of cyclooxygenase(COX)-2 and epidermal growth factor receptor (EGFR), which are considered early events in colorectal tumorigenesis. Thus, antiplatelet agents may play a role in the prevention of CRC by modifying epigenetic events involved in early phases of colorectal tumorigenesis. Finally, we carried out a critical review of the literature on off-target mechanisms of aspirin action as anticancer drug.

Aspirin Prevents Colorectal Cancer by Normalizing EGFR Expression

BackgroundAspirin intake reduces the risk of colorectal cancer (CRC), but the molecular underpinnings remain elusive. Epidermal growth factor receptor (EGFR), which is overexpressed in about 80% of CRC cases, is implicated in the etiology of CRC. Here, we investigated whether aspirin can prevent CRC by normalizing EGFR expression. MethodsImmunohistochemistry staining was performed on paraffin-embedded tissue sections from normal colon mucosa, adenomatous polyps from FAP patients who were classified as regular aspirin users or nonusers. The interplay between cyclooxygenase-2 (COX-2) and EGFR was studied in primary intestinal epithelial cells isolated from ApcMin mice, immortalized normal human colon epithelial cells (HCECs) as well as murine embryonic fibroblasts (MEFs). ResultsImmunohistochemistry staining results established that EGFR overexpression is an early event in colorectal tumorigenesis, which can be greatly attenuated by regular use of aspirin. Importantly, EGFR and COX-2 were co-overexpressed and co-localized with each other in FAP patients. Further mechanistic studies revealed that COX-2 overexpression triggers the activation of the c-Jun-dependent transcription factor, activator protein-1 (AP-1), which binds to the Egfr promoter. Binding facilitates the cellular accumulation of EGFR and lowers the threshold required for pre-neoplastic cells to undergo transformation. ConclusionAspirin might exert its chemopreventive activity against CRC, at least partially, by normalizing EGFR expression in gastrointestinal precancerous lesions.

Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia

BackgroundLoss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3′ terminal third of Apc that does not affect Wnt signaling.ResultsA series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia.ConclusionsOur results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

Association study of APC polymorphisms with colorectal cancer in Han Chinese

ObjectivesColorectal cancer (CRC) is a common malignancy with worldwide prevalence. Familial adenomatous polyposis (FAP), a predisposition syndrome of CRC, is caused by germ line mutations in the APC gene. Mutations in APC are thought to be an early event in colorectal tumorigenesis. We hypothesized that common variants in APC might be associated with CRC. Design and methodsA case-control study genotyping ten SNPs was conducted in 312 CRC patients and 270 normal controls in the Chinese Han population. ResultsThe genotype frequency of rs2019720 showed a significant difference between cases and controls (p=0.046, after Bonferroni correction). For the three pairs of SNPs in strong LD, we carried out haplotype analyses but no significant association was detected. ConclusionOur results suggest that APC polymorphisms might be associated with CRC in the Chinese Han population.

Haploinsufficiency of SGO1 results in deregulated centrosome dynamics, enhanced chromosomal instability and colon tumorigenesis

Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1+/- mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. Enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AOM), a colon carcinogen. Together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation.

The Prognostic Value of the Immunohistochemical Expression and Mutational Pattern of the Key Mediator of Wnt Signaling

Beta-catenin plays a critical role with E-cadherin in cell-cell adhesion and is also a key molecule of the highly conserved Wnt signaling pathway that regulates cell proliferation and differentiation. Abrogation of this pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer. The objective of this study was to determine the prognostic value of β-catenin/E-cadherin complex in Tunisian patients with colorectal cancer. Matched primary tumors from 150 patients with sporadic colorectal adenocarcinomas were stained for β-catenin and E-cadherin by using immunohistochemistry. Deletion of exon 3 of CTNNB1 gene was performed by polymerase chain reaction. Our results showed that β-catenin and E-cadherin expressions were related inversely to tumor differentiation. Furthermore, the nuclear expression of β-catenin was considerably increased in advanced colorectal adenocarcinomas and was highly associated with shorter survival of patients. Deletion of exon 3 of CTNNB1 was identified in 2 cases by using polymerase chain reaction and was significantly related to tumor invasion and aberrant expression of E-cadherin. The major finding of this study is that activation of β-catenin gene by deletions involving exon 3 may be considered as an advanced event in colorectal tumorigenesis in Tunisian patients, in contrast to some worldwide studies. Moreover, disruption of β-catenin/E-cadherin complex may be considered as a dependent predictor of disease outcome.

Global histone modification of histone H3 in colorectal cancer and its precursor lesions

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.

Abstract 91: Analysis of global and repeat sequence DNA methylation in the blood and normal colonic mucosa of colorectal cancer patients, healthy individuals and patients with folate deficiency

Abstract Aberrant DNA methylation is an early event in colorectal tumorigenesis and in colorectal cancer (CRC) patients these changes are detected in their normal colonic mucosa. DNA methylation changes may arise from aging or environmental exposures e.g. folate deficiency. We aimed to compare global and repeat-sequence DNA methylation in the colonic mucosa and blood of three groups of individuals – CRC patients, healthy people and patients with folate deficiency. We conducted a multisite cohort study collecting mucosa and blood from 176 patients who had undergone CRC resection and 194 healthy individuals at screening colonoscopy. Blood was also collected from 30 individuals with megaloblastic anaemia secondary to folate deficiency (red cell folate <539nM). Global DNA methylation was measured using liquid chromatography tandem mass spectrometry adapted from Quinlivan & Gregory (2008)1. Alu repeat methylation was determined using a novel, ultra-sensitive real-time PCR assay based upon methylation-sensitive restriction enzyme digestion coupled with a fluorescence-based readout2. Genotyping for a common polymorphism in the folate metabolism enzyme MTHFR (C677T) was performed using pyrosequencing. The mean age of CRC patients (68 ± 14 years) was significantly higher than healthy subjects (55 ± 14 years; p<0.01) but not significantly different from low-folate subjects (72 ± 22 years). The gender distribution was similar in all 3 groups. The prevalence of the variant T allele of MTHFR in CRC subjects (58%) and healthy controls (64%) were comparable to population-based estimates. However, the T allele was over-represented in low folate patients (79%). In the analysis of the first subset of CRC (n=38) and healthy controls (n=38), mean global DNA methylation was significantly lower in the colonic mucosa (CRC n=20: 4.1% ± 0.3; controls n=36: 4.0% ± 0.3) compared to peripheral blood (CRC n=17: 4.4% ± 0.4; controls n=36: 4.4% ± 0.3, p<0.01). No differences in DNA methylation were found in CRC patients compared to healthy subjects. Consistent with global methylation findings, Alu repeats were 2-fold less methylated in colon compared to peripheral blood in healthy (n=38) and CRC cohorts(n=36). Low-folate subjects had a trend towards lower blood global (4.26% ± 0.2) and Alu methylation (1.2-fold hypomethylated) compared to healthy subjects. To date, our results suggest that Alu repeat methylation reflect global DNA methylation and that this holds true across tissue types irrespective of cancer status. Global and Alu repeat demethylation in individuals with folate deficiency and overrepresentation of the T MTHFR allele suggests a possible link between diet and the epigenome that warrants further study. 1. Quinlivan, E.P. & Gregory, J.F., 3rd. (2008) Nucleic acids research 36, e119. 2. Rand, K. & Molloy, P. (2010) BioTechniques 49, xiii-xvii. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2011-91

Epigenetic inactivation of the NORE1gene correlates with malignant progression of colorectal tumors

BackgroundNORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.MethodsExpression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays.ResultsExpression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress.ConclusionOur data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.

Overexpression of leucine‐rich repeat‐containing G protein‐coupled receptor 5 in colorectal cancer

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a 7-transmembrane receptor reportedly expressed in stem cells of the intestinal crypts and hair follicles of mice. Overexpression of LGR5 is observed in some types of cancer; however, there has been no specific assessment in colorectal tumorigenesis. We performed quantitative RT-PCR for LGR5 expression in 37 representative cancer cell lines, and showed that LGR5 mRNA was frequently overexpressed in colon cancer cell lines. Moreover, LGR5 expression was higher in colon cancer cell lines derived from metastatic tumors compared with those from primary tumors. In clinical specimens, there was significant overexpression of LGR5 in 35 of 50 colorectal cancers (CRCs), and in seven of seven sporadic colonic adenomas, compared with matched normal mucosa. This suggests up-regulation of LGR5 from the early stage of colorectal tumorigenesis. LGR5 expression showed marked variation among CRC cases and correlated significantly with lymphatic invasion, vascular invasion, tumor depth, lymph node metastasis, and tumor stage (IIIC vs. IIIB). In addition to cancer cells, crypt base columnar cells of the small intestine and colon were shown by in situ hybridization to express LGR5. This is the first report suggesting the involvement of LGR5, not only in early events but also in late events in colorectal tumorigenesis.

Abstract 2695: Sensitive detection of K-ras mutations reveals intratumoral heterogeneity in primary colon cancers

Abstract BACKGROUND: Mutations in the K-ras oncogene occur in 30-50% of colorectal cancers and have been described as an early event in colorectal tumorigenesis. K-ras mutations have recently gained renewed interest due to their well-documented prediction of resistance against EGFR-directed therapy. In the diagnostic setting a single biopsy from each tumor is usually examined, assuming a homogeneous distribution of K-ras mutations in the tumor. Our aim was to investigate the distribution of K-ras mutations within primary colon tumors to determine whether intratumoral heterogeneity occurs. PATIENTS AND METHODS: Tumor biopsies from 158 prospectively recruited patients undergoing curative surgery for primary colon cancer were analysed for mutations in codon 12 and 13 of the K-ras gene by a sensitive and quantitative PNA clamp PCR assay. RESULTS: When analyzing single fresh-frozen biopsies from each tumor, K-ras mutations were detected in 67 (42%) of the 158 patients. Our K-ras mutation measure ddCt indicated low (ddCt<10) concentrations of mutated DNA in 13 (19 %) of the 67 patients, an observation that encouraged us to consider a heterogeneous distribution of K-ras mutations in these tumors. To verify this hypothesis, we analysed tissue sections from all available formalin-fixed tissue blocks (median three per patient) from the tumors of these 13 patients by the same K-ras PNA clamp PCR assay. Seven of the 13 patients with potential tumor heterogeneity had both tissue blocks positive and tissue blocks negative for K-ras mutations. Five of the patients had no mutations detected in any of their formalin-fixed tissue blocks, despite the detection of mutations in the fresh-frozen biopsy. This may partially be explained by the lower sensitivity of the assay in formalin-fixed samples due to poor DNA quality. The remaining patient had mutations detected in all analyzed formalin-fixed tissue sections. Thus, evidence supporting a heterogenous distribution of K-ras mutations within the tumor was obtained in 12 of the 13 investigated cases. The tumor cell fraction in all examined tissue sections was estimated by HES staining and microscopy of neighbouring tissue sections. It was above 10% in all cases and the median value was 40%. There was no correlation between the level of K-ras mutations and estimated tumor cell fraction in tissue sections with detected mutations (Pearson R=-0.11). CONCLUSION: Intratumoral heterogeneity of K-ras codon 12 and 13 mutations was confirmed in 12 (18%) of 67 primary colon tumors with K-ras mutations, with possible implications both for sampling routines in diagnostics and for our understanding of K-ras mutations in tumor biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2695.

Genetic pathways to colorectal cancer

The colorectal cancer paradigm explains how genetic and histological changes lead normal epithelial cell to transform into pre-malignant adenomas then progress to malignant carcinomas. Using the Genetic Alterations in Cancer Knowledge System intragenic allele loss and gene mutation data from approximately 9000 colorectal tumors were compared to the model of colorectal tumor development. The distribution of mutations along the TP53 codons as a function of tumorigenesis also was analyzed. Alterations of APC, KRAS and TP53 were observed in a higher percentage of adenocarcinomas compared to adenomas ( P < 0.05) indicating that the alterations accumulated with malignancy. Alterations in BRAF, CTNNB, HRAS and NRAS were infrequent regardless of morphology. Differences were observed in the distribution of TP53 mutations with tumorigenesis. Mutations (single base substitutions) occurred most frequently at codons 175 and 273 in both tumor types; however, in adenocarcinomas the mutation incidence at codon 248 was approximately three times that reported in adenomas. It is proposed that the higher incidence of mutation at codon 248 is a later event in colorectal tumorigenesis that occurs as the tumors become malignant.

Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost beta-catenin binding sites. APC-free beta-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3' untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis.

Epigenetic-Genetic Interactions in theAPC/WNT, RAS/RAF, andP53Pathways in Colorectal Carcinoma

Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O(6)-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C-->A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist. We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas. We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non-MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002). Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.

Frequent Alteration of XAF1 in Human Colorectal Cancers: Implication for Tumor Cell Resistance to Apoptotic Stresses

X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) is a candidate tumor suppressor located at the chromosome 17p13 region, but the molecular basis underlying its inactivation in human tumors and growth-inhibiting function has not been well defined. We explored the candidacy of XAF1 as a suppressor in colorectal tumorigenesis. XAF1 expression was characterized by polymerase chain reaction-based cloning, isoform-specific polymerase chain reaction, ribonuclease protection, and immunoblot assays. Allelic loss of the gene was evaluated by loss of heterozygosity (LOH) assay, and promoter CG dinucleotide (CpG) site methylation was determined using bisulfite sequencing. The effect of XAF1 on tumor growth was examined using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, colony formation, and viability assays. Expression of 5 XAF1 variants including 2 novel transcripts was down-regulated concomitantly in 11 of 20 (55%) cell lines and 26 of 65 (40%) primary tumors. XAF1 reduction was tumor-specific and showed a correlation with advanced stage and high grade of tumor. LOH of the gene was found in 12 of 33 (36%) tumors. Promoter CpG site methylation was observed frequently in both cell lines and tumor tissues including many LOH tumors, suggesting that biallelic inactivation of XAF1 might be common in colorectal cancers. XAF1 expression suppressed tumor cell growth and enhanced cellular response to various apoptotic stimuli, such as 5-fluorouracil, etoposide, H(2)O(2), gamma-irradiation, ultraviolet, and tumor necrosis factor-alpha, whereas knockdown of its expression protected cells from the stresses. Genetic and epigenetic alteration of XAF1 is a common event in colorectal tumorigenesis and contributes to the malignant tumor progression by providing survival advantages for tumor cells under various stress conditions.

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.

Subcellular localization of β-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions

Adenomatous polyposis coli (APC) is a tumor suppressor gene whose main function is the destabilization of β-catenin, a key effector of the Wnt signaling pathway. This gene is defective in familial adenomatous polyposis (FAP), a dominantly inherited disease, but inactivation of APC has been reported also in most sporadic colorectal tumors and it is considered an early event in colorectal tumorigenesis. The aim of the present study was to evaluate the intracellular ultrastructural distribution of β-catenin and APC proteins in epithelial cells of normal colorectal mucosa, aberrant crypt foci (ACF, an early premalignant lesion) and cancer. We used the immunogold electron microscopic method to identify both proteins. Normal colonic epithelial cells showed a strong membranous expression of β-catenin and lacked cytoplasmic and nuclear expression. Normal cells showed APC localization pattern characterized by diffuse nuclear expression and along the plasma membrane. In ACF and in carcinoma an absent or reduced membranous expression of β-catenin was associated with an increased nuclear and cytoplasmatic expression. In aberrant crypt foci and carcinoma, APC was evident inside the nucleus and at the level of cell-cell junctions, but it was decreased in the cytoplasm. This method allowed the accurate localization of proteins of the Wnt signaling pathway in the early steps of colorectal carcinogenesis. The similar pattern of subcellular distribution of APC and β-catenin in dysplastic ACF and colorectal cancer suggests that ACF are precursor lesions of sporadic and FAP-associated colorectal carcinoma.

The order of genetic events associated with colorectal cancer progression inferred from meta‐analysis of copy number changes

To identify chromosomal aberrations that differentiate among the Dukes' stages of colorectal cancer (CRC) as well as those that are responsible for the progression into liver metastases, we performed a meta-analysis of data obtained from 31 comparative genomic hybridization (CGH) studies comprising a total of 859 CRCs. Individual copy number profiles for 373 primary tumors and 102 liver metastases were recorded and several statistical analyses, such as frequency, multivariate logistic regression, and trend tests, were performed. In addition, time of occurrence analysis was applied for the first time to copy number changes identified by CGH, and each genomic imbalance was thereby classified as an early or late event in colorectal tumorigenesis. By combining data from the different statistical tests, we present a novel genetic pathway for CRC progression that distinguishes the Dukes' stages and identifies early and late events in both primary carcinomas and liver metastases. Results from the combined analyses suggest that losses at 17p and 18 and gains of 8q, 13q, and 20 occur early in the establishment of primary CRCs, whereas loss of 4p is associated with the transition from Dukes' A to B-D. Deletion of 8p and gains of 7p and 17q are correlated with the transition from primary tumor to liver metastasis, whereas losses of 14q and gains of 1q, 11, 12p, and 19 are late events. We supplement these findings with a list of potential target genes for the specific alterations from a publicly available microarray expression dataset of CRC.

Analysis of SMAD4/DPC4 gene alterations in multiploid colorectal carcinomas

Although recent animal studies have shown that SMAD4/DPC4 gene alterations are essential for late-stage intestinal tumorigenesis, the role of SMAD4/DPC4 gene alterations in primary human colorectal carcinomas is not fully understood. Therefore, we attempted to clarify the role of the SMAD4/DPC4 gene during tumor progression of colorectal carcinoma. Differences in allelic imbalance (AI) and mutations of the SMAD4/DPC4 gene between diploid and aneuploid populations were analyzed for 30 sporadic DNA multiploid colorectal carcinomas (used as a tumor progression model and defined as the coexistence of diploid and aneuploid cells within the same tumor). The crypt isolation technique was coupled with DNA cytometric sorting and a polymerase chain reaction assay. In addition, hypermethylation of the promoter region was examined to clarify whether inactivation of gene expression occurred. Although a SMAD4/DPC4 gene AI was detected in only 5 of 27 informative diploid populations, 25 of 27 aneuploid populations had a SMAD4/DPC4 gene AI. Mutation of the SMAD4/DPC4 gene was detected in only one aneuploid population of multiploid colorectal carcinomas, but not in the corresponding diploid population. In total, 20 available multiploid carcinomas were selected for methylation analysis, and no evidence of hypermethylation of the promoter region was found. We suggest that, although mutation of the SMAD4/DPC4 gene and hypermethylation of the promoter region are infrequent events in colorectal tumorigenesis, AI at the SMAD4/DPC4 gene locus may play a key role in the progression of colorectal carcinomas.