Event In Colorectal Carcinogenesis Research Articles

Frequent PIK3CA gene amplification and its clinical significance in colorectal cancer

Using a DNA microarray approach to screen for gene copy number changes in 20 colorectal (CR) carcinoma samples and filtering for high-level DNA copy number changes, we detected an amplicon at 3q26 containing the PIK3CA gene. Fluorescence in situ hybridization was employed for evaluation of PIK3CA amplification on a progression CR tissue microarray containing 448 CR carcinomas, normal mucosa, and adenomas with follow-up information. PIK3CA amplification (ratio PIK3CA/centromere 3 > or = 2.0) was found in 38% of cancers, while another 19% of tumours had PIK3CA gains (ratio >1.0 but <2.0). Both PIK3CA amplification and gains were associated with high levels of PIK3CA protein expression and no association was seen between PIK3CA amplification and PIK3CA mutation. In a subset of 220 patients who received adjuvant chemotherapy and/or radiotherapy, survival in patients with PIK3CA-amplified cancers was significantly longer compared with patients with cancers without amplification. This association was independent of stage, grade, histology subtype, gender, and age categories. Interestingly, PIK3CA amplification was also seen in CR adenomas, indicating an early genetic alteration, and was also a frequent event in colorectal carcinogenesis. Furthermore, PIK3CA amplification is an independent prognostic marker for better survival and may be one of the promising markers to define CRC subsets that may maximally benefit from adjuvant therapy.

Epigenetic modulation of the retinoid X receptor α by green tea in the azoxymethane‐ApcMin/+ mouse model of intestinal cancer

We investigated the possible mechanisms of inhibition of colorectal carcinogenesis by green tea (GT) in azoxymethane-treated (AOM) Apc(Min/+) mice. Mice received water or a 0.6% (w/v) solution of GT as the only source of beverage. GT treatment commenced at the 8th week of age and lasted for 8 wk. The treatment caused a statistically significant reduction in the number of newly formed tumors (28%, P < 0.05). Immunohistochemical analysis showed that GT decreased the levels of beta-catenin and its downstream target cyclin D1. To probe a mechanism, we further investigated the expression of retinoic X receptor alpha (RXR alpha) in AOM/Apc(Min/+) tumors. Our results show that RXR alpha is selectively downregulated in AOM/Apc(Min/+) mouse intestinal tumors. In contrast, other retinoic receptors including retinoic acid receptor alpha (RAR alpha), RAR beta, RXR beta, and RXR gamma were all expressed in Apc(Min/+) adenomas. Furthermore, our results show that RXR alpha downregulation is an early event in colorectal carcinogenesis and is independent of beta-catenin expression. GT significantly increased the protein levels of RXR alpha. In addition, RT-PCR analysis showed that GT induced a similar increase in the levels of RXR alpha mRNA. Genomic bisulfite treatment of colonic DNA followed by pyrosequencing of 24 CpG sites in the promoter region of RXR alpha gene showed a significant decrease in CpG methylation with GT treatment. The results suggest that a low concentration of GT is sufficient to desilence RXR alpha and inhibit intestinal tumorigenesis in the Apc(Min/+) mouse.

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

BackgroundBRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied.MethodsMutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies.ResultsKRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191).ConclusionOur results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC.

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC Min/+ mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H 2 (PGH 2) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH 2 into PGE 2, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE 2 related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1–4) was obvious among the mPGES-1 deficient mice. PGE 2 levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH 2 derived prostanoids were generally enhanced, being most prominent for TxA 2 and PGD 2. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE 2 during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.

Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer

MicroRNAs are small, non-coding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA targets. MicroRNA expression is dysregulated in human malignancies, frequently leading to loss of expression of certain microRNAs. We report that expression of hsa-miR-342, a microRNA encoded in an intron of the gene EVL, is commonly suppressed in human colorectal cancer. The expression of hsa-miR-342 is coordinated with that of EVL and our results indicate that the mechanism of silencing is CpG island methylation upstream of EVL. We found methylation at the EVL/hsa-miR-342 locus in 86% of colorectal adenocarcinomas and in 67% of adenomas, indicating that it is an early event in colorectal carcinogenesis. In addition, we observed a higher frequency of methylation (56%) in histologically normal colorectal mucosa from individuals with concurrent cancer compared to mucosa from individuals without colorectal cancer (12%), suggesting the existence of a 'field defect' involving methylated EVL/hsa-miR-342. Furthermore, reconstitution of hsa-miR-342 in the colorectal cancer cell line HT-29 induced apoptosis, suggesting that this microRNA could function as a proapoptotic tumor suppressor. In aggregate, these results support a novel mechanism for silencing intronic microRNAs in cancer by epigenetic alterations of cognate host genes.

Mitochondrial D310 mutations in colorectal adenomas: An early but not causative genetic event during colorectal carcinogenesis

Somatic mutations of the D310 sequence of the mitochondrial DNA are reported in human cancers, including colorectal cancers (CRC). The presence of these mutations at early or late steps of colorectal carcinogenesis is unknown. Their prevalence increased significantly with the number of cytosines in the D310 sequence of the matched normal tissue (D310 polymorphism), suggesting that this polymorphism could be a risk factor for CRC. The aim of this study was (i) to investigate the prevalence of D310 mutations in 64 colorectal adenomas and 36 liver metastases from 15 CRC patients, (ii) to assess the relation between D310 polymorphism and the risk of colorectal adenoma in a case-control study including 613 cases with colorectal adenoma and 572 polyp-free (PF) controls. D310 mutations were found in colorectal adenomas and liver metastases from CRC patients in 27 and 33%, respectively and so are an early genetic event in colorectal carcinogenesis. The frequency of the mutations increased significantly with the number of cytosines in the matched normal tissue D310 sequence (p < 0.001) but the distribution of D310 polymorphisms was not significantly different between adenoma cases (large (>9 mm) and small (<5 mm) adenomas) and PF controls (C(4)-C(7)TC(6): 47, 52 and 49%; C(8)TC(6): 44, 39 and 41%; C(9)-C(10)TC(6): 9, 9 and 10%, respectively; p > 0.05), suggesting that germline D310 polymorphism is not a risk factor for colorectal adenomas. Considering their high frequency in colorectal adenomas, mitochondrial D310 mutations could represent a biomarker for early detection of CRC although their causative role in colorectal carcinogenesis remains uncertain.

Methylation-induced silencing of ASC/TMS1, a pro-apoptotic gene, is a late-stage event in colorectal cancer

The hypermethylation of tumor-suppressor gene promoter regions has been shown to result in the epigenetic inactivation of many genes. ASC/TMS1 is a pro-apoptotic gene that has been shown to be methylated in many different human neoplasms. The methylation status of ASC/TMS1 was analyzed in a series of colorectal cancer (CRC) cell lines, primary colorectal adenomas and cancers, and normal colorectal tissue samples using methylation-specific PCR (MSP). The gene expression of ASC/TMS1 in the CRC cell lines was analyzed using reverse-transcriptase PCR (RT-PCR). Methylation analysis showed complete methylation of ASC/TMS1 in 5 of 7 (71%) CRC cell lines. RT-PCR showed absence of mRNA expression in these same cell lines, and expression was restored after treatment with the demethylating drug 5-aza-2’-deoxyazacytidine. The two unmethylated cell lines showed ASC/TMS1 mRNA expression both before and after treatment with 5-aza-2’-deoxyazacytidine. Methylation was seen in 20 of 115 (17%) of primary colorectal cancer specimens, but no methylation was seen in 30 colorectal adenomas and 11 normal colorectal tissue samples. Methylation status of ASC/TMS1 was correlated with a series of clinicopathological variables using multivariate analysis. Methylation of ASC/TMS1 was more common in right-sided tumors (p = 0.02), concordant with hMLH1 methylation (p=0.03) and is a late stage event, occurring in 0 of 18 tubular adenomas, 0 of 12 villous adenomas, 2 of 44 (5%) Stage 1 cancers, 8 of 31 (26%) Stage 2 cancers, 8 of 21 (38%) Stage 3 cancers, and 2 of 19 (11%) Stage 4 cancers. The ASC/TMS1 gene is frequently silenced in CRC due to promoter hypermethylation. Methylation of ASC/TMS1 appears to be a late-stage event in colorectal carcinogenesis associated with invasive carcinomas but not with normal colorectal tissue or colorectal adenomas. Methylation of ASC/TMS1 may have implications for cancer prognosis.

Transforming growth factor beta 1 and metalloproteinase-9 overexpression in colorectal cancer (CC) and adenoma

Although the role of transforming growth factor beta (TGFbeta) 1 and metalloproteinase-9 (MMP-9) is well documented in colorectal cancer (CC), there is a little evidence supporting its role in early carcinogenesis. The aim of the study was to determine the pattern of immunohistochemical expression of TGFbeta1, MMP-9, and Ki-67 in CC and adenomatous polyps. The study group comprised 50 patients with colorectal polyps and 33 patients with CC. Endoscopically removed polyps and CC biopsies had been evaluated with histopatologic examination and immunohistochemistry. The biopsies from 30 healthy objects served as a control group. For all antibodies labeling indices (LI) had been calculated. Among 62 adenomas, 33 high-grade dysplasia (HGD) and 29 low-grade dysplasia (LGD) had been detected. Mean TGFbeta1, MMP-9, and Ki-67 LI in CC were significantly higher (p < 0.01, 0.01, and 0.01, respectively) than in HGD polyps. Mean TGFbeta1, MMP-9, and Ki-67 LI in HGD polyps were significantly higher than in LGD polyps (p < 0.01, 0.01, and 0.01, respectively). There had been no statistical difference in TGFbeta1, MMP-9, and Ki-67 LI between LGD and the control group (p > 0.05, 0.05, and 0.05, respectively). There was a positive correlation between TGFbeta1 and MMP-9 (r = 0.898), Ki-67 and MMP-9 (r = 0.938), and TGFbeta1 and Ki-67 (r = 0.913). We did not observe any correlation between TGFbeta1, MMP-9, Ki-67 LI and the clinical parameters evaluated. The increased expression of TGFbeta1, MMP-9 observed in colorectal adenomas seems to be related to the grade of dysplasia. We assume that overexpression of TGFbeta1, MMP-9 represent an early event in colorectal carcinogenesis and may possibly have the prognostic value.

Systemic analysis of the differential gene expression profile in a colonic adenoma−normal SSH library

Background The discovery of differentially expressed genes of colonic adenoma minus normal mucosa enables the understanding of early molecular events in colorectal carcinogenesis. In our previous study, we have developed an adenoma minus normal mucosa suppression subtractive hybridization (SSH) library and identified 109 differentially expressed clones. Methods An in-house EST pipeline and the Gene Ontology web-based tool ( http://genereg.ornl.gov/gotm) were used to analyze these clones. Realtime quantitative RT-PCR (Q-PCR) was applied to detect the expression of 14-3-3 zeta, REG4 and 6 ribosomal protein genes ( RPS2, RPS12, RPS27A, RPL5, RPL7a and RPL10a) in 14 adenomas (8 with concurrent cancers) and 44 colorectal adenocarcinomas with paired normal mucosa. Results Sixty-two candidate genes were obtained from this library. Bioinformatics analysis indicated that both ribosomal protein genes and immune-related genes were enriched. REG4 was significantly upregulated in colorectal adenomas (medium fold: 1.676, p < 0.05, Wilcoxon test) and 14-3-3 zeta in cancers (medium fold: 1.202, p < 0.01, Wilcoxon test), as compared with those of paired normal mucosa. However, all ribosomal protein genes were not significantly overexpressed in colorectal adenomas or cancers. Conclusions A differential gene expression profile in A−N SSH library may be helpful in understanding the molecular mechanism of colorectal cancer initiation and progression. REG4 and 14-3-3 zeta may be potential biomarkers for early colorectal cancer detection.

Suppression of human selenium‐binding protein 1 is a late event in colorectal carcinogenesis and is associated with poor survival

The purpose of this study was to analyze altered protein expression in cancer tissues and determine its relationship to prognosis in colorectal carcinomas. We performed proteomic expression analysis on 14 colorectal carcinomas and matched nontumorous colonic mucosa by 2-DE and MALDI-TOF-MS. Comparative analysis of the respective spot patterns on 2-DE showed 14 spots that were markedly changed in the colorectal carcinomas. Among them, selenium-binding protein 1 (SELENBP1) was markedly decreased in 12 (85%) carcinomas. The reduced expression of SELENBP1 was further supported by Western blot analysis and immunohistochemistry. Suppression of SELENBP1 was further analyzed in another eight-paired adenomas and carcinomas from the same patients using Western blot analysis and immunohistochemistry, and revealed that one adenoma and seven carcinomas exhibited markedly reduced SELENBP1 expression. Patients with low levels of SELENBP1 expression had significantly lower overall survival rates (72 vs. 85%, p = 0.021) among the 240 stages II and III colorectal carcinomas by using tissue microarray analysis. Our findings indicate that suppression of SELENBP1 is a frequent and late event in colorectal carcinogenesis, and may contribute to the rapid progression of colorectal carcinoma.

Coexistence of K-ras mutations and HPV infection in colon cancer.

Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer.Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact testsResults HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis.Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

Aberrant expression of krÜppel-like factor 6 protein in colorectal cancers

To investigate whether krUppel-like factor 6 (KLF6) plays an important role in the development and/or progression of colorectal cancer. A total of 123 formalin-fixed and paraffin-embedded colorectal cancer specimens were analyzed by immunohistochemistry using tissue microarray for the expression of KLF6 protein. The specimens were collected over a 3-year period in the laboratories at our large teaching hospital in Seoul, Republic of Korea. The correlation of KLF6 expression with clinicopathologic parameters was analyzed by chi2 test and Bartholomew test. Normal colonic epithelium showed weak to moderate expression of KLF6, whereas reduced KLF 6 expression or loss of KLF6 expression was seen in 45 (36.6%) of the 123 colorectal carcinoma specimens. Interestingly, aberrant expression of KLF6 was detected in 25 (43.1%) of 58 cases with metastasis to regional lymph node and in 31 (47.0%) of 66 tumors more than 5 cm in size. Statistically, loss of KLF6 expression was significantly associated with tumor size (P < 0.05). However, there was no significant correlation between KLF6 expression and Dukes'stage (Bartholomew test, P > 0.05), tumor location and lymph node metastasis (chi2 test, P > 0.05). Loss of KLF6 expression may be a common and early event in colorectal carcinogenesis.

DAPK promotor methylation is an early event in colorectal carcinogenesis

Death-associated protein kinase (DAPK) is frequently inactivated by promotor hypermethylation in various human cancers. At present, little is known about the significance of DAPK inactivation in colorectal carcinogenesis. We therefore, investigated macrodissected samples of 22 formalin-fixed and paraffin-embedded T1-carcinomas showing normal colon mucosa, intraepithelial neoplasia and carcinoma tissue on the same slice. Dissected carcinoma areas showed a higher frequency of DAPK promotor methylation (81.2%) compared to intraepithelial neoplasia (68.2%). Colon mucosa adjacent to intraepithelial neoplasia or carcinoma showed DAPK promotor methylation in only two of eight cases (25%). We suggest that DAPK promotor hypermethylation may play an important role in the early steps of tumor progression in colorectal carcinoma.

Ionomycin downregulates β-catenin/Tcf signaling in colon cancer cell line

Functional activation of beta-catenin/Tcf signaling plays an important role in the early events in colorectal carcinogenesis. We examined the effect of ionomycin against beta-catenin/Tcf signaling in colon cancer cells. Reporter gene assay showed that ionomycin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by ionomycin in HEK293 cells transiently transfected with a constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism is related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunoprecipitation analysis, western blot and electrophoretic mobility shift assay. As a result, our data reveal that the association of beta-catenin and Tcf-4 is disrupted and the amount of beta-catenin product in the nucleus is decreased by ionomycin in a concentration-dependent manner. Moreover, ionomycin strongly suppressed the binding of the Tcf complexes to its specific DNA-binding sites. The significance of the current work is that ionomycin is a negative regulator of beta-catenin/Tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear beta-catenin products and to the suppressed binding of Tcf complexes to consensus DNA.

Regulation of AKT1 expression by beta-catenin/Tcf/Lef signaling in colorectal cancer cells

The serine/threonine kinase AKT plays a critical role in controlling the balance between cell survival and apoptosis. Several reports implicated AKT in the molecular pathogenesis of different human malignancies and overexpression of AKT was recently demonstrated to be an early event in colorectal carcinogenesis. We report here the identification of nine putative Tcf/Lef-binding elements (TBEs) upstream to the ATG initiation site of the AKT1 gene. Four of these TBEs are located upstream of the transcriptional start, whereas five TBEs are situated in Exon 1 of the AKT1 gene. Accordingly, we hypothesized that AKT1 expression might be regulated by Wnt/beta-catenin signaling. To elucidate the regulation of AKT expression in colon cancer cells, we generated reporter constructs containing the luciferase gene under the control of different regions derived from the AKT1 promoter/enhancer. Transient expression of the constructs in colorectal cancer (CRC) cell lines resulted in significant activation of the reporter gene. Luciferase was stimulated 20- to 50-fold in SW480, SW948 and HCT116 CRC cells. In contrast, the AKT1 promoter/enhancer constructs showed only a weak response in 293 embryonic kidney cells. Coexpression of a constitutively active beta-catenin mutant in colon cancer cells further enhanced reporter gene activation from the AKT1 promoter/enhancer, whereas it was downregulated by introduction of either wild-type APC or dnTcf-4. In addition, immunohistochemical staining of tumor sections derived from CRC patients showed elevated expression levels of AKT1, correlating with enhanced cytoplasmic/nuclear expression of beta-catenin. In summary our data suggest that beta-catenin/Tcf contributes to the transcriptional regulation of the AKT1 gene.

Quercetin, a potent inhibitor against β-catenin/Tcf signaling in SW480 colon cancer cells

Dysregulation of Wnt/β-catenin pathway plays a central role in early events in colorectal carcinogenesis. We examined the effect of quercetin, a famous anti-tumor agent, against β-catenin/Tcf signaling in SW480 cells. Quercetin inhibited the transcriptional activity of β-catenin/Tcf in SW480 and also in HEK293 cells transiently transfected with constitutively active mutant β-catenin gene, whose product is not induced to be degraded by APC–Axin–GSK3β complex, so we concluded that its inhibitory mechanism was related to β-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed EMSA showing that binding of the Tcf complexes to its specific DNA-binding sites was strongly suppressed by quercetin. Immunoprecipitation analysis also showed that the binding of β-catenin to Tcf-4 was also disrupted by quercetin. Western blot analysis proved these decreased bindings resulted from decreased level of β-catenin and Tcf-4 product in nucleus caused by quercetin. Together, we suggest that quercetin is an excellent inhibitor of β-catenin/Tcf signaling in SW480 cell lines, and the reduced β-catenin/Tcf transcriptional activity is due to the decreased nuclear β-catenin and Tcf-4 proteins.

Dietary factors and truncating APC mutations in sporadic colorectal adenomas.

Inactivating mutations in APC are thought to be early, initiating events in colorectal carcinogenesis. To gain insight into the relationship between diet and inactivating APC mutations, we evaluated associations between dietary factors and the occurrence of these mutations in a Dutch case-control study of sporadic colorectal adenomas (278 cases; 414 polyp-free controls). Direct-sequencing was used to screen adenomas for mutations in the mutation cluster region of APC; truncating mutations were detected in 161 (58%) of the adenomas. Red meat consumption was significantly differently related to polyps with truncating APC mutation (APC(+) polyps) compared to polyps without truncating APC mutation (APC(-) polyps) (highest vs. lowest tertile, odds ratio [OR] = 0.5, 95% confidence interval [CI] = 0.3-1.0). High intake of red meat and fat seemed to increase the risk of APC(-) polyps only (APC(+) vs. controls: red meat, OR = 1.0, 95% CI = 0.6-1.6; fat, OR = 1.1, 95% CI = 0.6-1.9; APC(-) vs. controls: red meat, OR = 1.8, 95% CI = 1.0-3.1; fat, OR = 1.9, 95% CI = 1.0-3.7). Intake of carbohydrates was inversely associated with both polyp groups, most noticeably with APC(-) polyps. Most other evaluated dietary factors were not distinctively associated with a specific APC status. None of the dietary factors was specifically associated with a particular type of truncating APC mutation. Our data suggest that red meat and fat may increase the risk of APC(-) polyps in particular, whereas carbohydrates may especially decrease the risk of APC(-) polyps. However, most examined dietary factors do not appear to be specifically associated with the occurrence of truncating APC mutations in colorectal adenomas but seem to affect both pathways equally.

Expression of the Low Molecular Weight Cyclin E is Early Event in Colorectal Carcinogenesis.

Cyclin E is essential for the transition from the G1 to S-phase of the cell cycle, and plays important roles in carcinogenesis in many cancers. Especially, low molecular weight cyclin E is overexpressed in breast cancer and its level of expression correlates well with the progression and prognosis. Although the cyclin E level is amplified, and overexpressed, in many cancers, including colorectal cancer, the role of low molecular weight cyclin E in colorectal cancer remains to be studied. Therefore, the expression of low molecular weight cyclin E in various stages of colorectal tumors was studied. The expression of low molecular weight cyclin E was analyzed in 45 tumors, and compared with paired normal mucosa from the same patients (6 adenomas, 11 stage A, 14 stage B and 14 stage C colorectal cancers) by Western blot analysis. The expres sion of low molecular weight cyclin E was also analyzed in normal colon mucosa from 12 healthy normal controls. The low molecular weight cyclin E was expressed exclusively in all stages of colon tumors, but not in the normal mucosa from the same patients or in the normal controls. However, there was no correlation between tumor progression and the degree of expression of low molecular weight cyclin E. The expression of low molecular weight cyclin E is suggested to be an early event in colorectal carcinogenesis.

A better cell cycle target for gene therapy of colorectal cancer: cyclin G.

The purpose of this study was to evaluate the overexpression of cyclin G in colorectal neoplasia, which may be a more frequent event than cyclin D1 during the cell cycle and thus may have a more enhanced therapeutic potential in treating colorectal cancer. Ninety formalin-fixed, paraffin-embedded human colon and rectal specimens were obtained from the Pathology Department of Norris Cancer Center/University of Southern California. The tissues had been obtained after surgical resection between 1995 and 2001, and had been processed by routine clinical histopathologic methods. Ninety-one percent of colorectal tumors had cyclin G overexpression. These cyclin-positive patients were evenly distributed between men and women, and between tumor locations, that is, 36% rectal tumors and 34% right-sided tumors. Thirty-two percent were well differentiated, and 66% were moderately differentiated. Thirty patients (38%) had stage I disease, 16 (20%) had stage II disease, 25 (32%) had stage III, and seven (9%) had stage IV disease. Eight patients (10%) in this group had recurrent disease during follow-up. There was no correlation between cyclin G overexpression and clinical and pathologic characteristics. Cyclin D1 overexpression was found to be present in only 42% of colorectal adenocarcinomas. There was no correlation between cyclin D1 overexpression and clinical and pathologic characteristics. The present study demonstrates that cyclin G overexpression is a frequent event in colorectal cancer. This frequent event in colorectal carcinogenesis may facilitate new therapeutic approaches acting as a target for gene therapy, possibly directed at downregulating cyclin G in colorectal cancer.

Nitric oxide-donating aspirin inhibits beta-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear beta-catenin-TCF association.

Dysregulation of the Wnt pathway and altered Beta-catenin expression are central early events in colorectal carcinogenesis. We studied the ortho, meta, and para (o-, m-, and p-) positional isomers of NO-donating aspirin (NO-ASA), a chemopreventive agent against colon cancer, for their effect on Beta-catenin/T cell factor (TCF) signaling. In human SW480 colon carcinoma cells, cell-growth inhibition by NO-ASA [IC50 values for p-, o-, and m- were 48.1 +/- 4.3 (mean +/-SEM), 60.4 +/- 2.1, and 900 +/-50 microM, respectively] was accompanied by significant inhibition of Beta-catenin signaling. We determined Beta-catenin-dependent TCF-4 transcriptional activity by measuring the activity of the luciferase gene placed under the control of TCF-4 regulatory sequences. The IC50 values for Beta-catenin/TCF-4-signaling inhibition by NO-ASA were: o-, 2.6 +/- 0.4; m-, 15 +/- 5; p-, 1.1 +/- 0.1 microM; and for ASA, >5,000 microM. Total or nuclear levels of Beta-catenin and its distribution in the cell were not altered by NO-ASA, as judged by protein expression levels and semiquantitative immunofluorescence analysis. NO-ASA disrupted the association of Beta-catenin and TCF-4 in the nucleus, whereas ASA did not affect it. NO-ASA reduced the expression of cyclin D1, a downstream target gene that plays an important role in colon carcinogenesis. In contrast, a structural analog of NO-ASA lacking the -NO2 moiety did not affect TCF-4 transcriptional activity. Thus, NO-ASA inhibits Beta-catenin-mediated TCF activity by preventing the formation of the Beta-catenin/TCF-4 complex. This effect, occurring at NO-ASA concentrations far below those required to inhibit cell growth, may be a critical early event in the chemopreventive activity of NO-ASA against colon cancer.