Cyclooxygenase-2 expression in colorectal carcinoma, adenomatous polyps and non-tumour bearing margins of resection tissues in a cohort of black Africans.

BACKGROUND: Emerging data suggest a negative role of cyclooxygenase-2 (COX-2) in colorectal carcinomas (CRC). Investigating this in developing communities such as ours helps to contribute to existing understanding of these lesions. METHODS AND FINDINGS: Formalin-fixed paraffin-embedded CRC colectomy tissues and their corresponding non-tumour margins of resected tissues were sectioned and stained with COX-2 antibody. Adenomatous polyp tissues from non-cancer bearing individuals were similarly processed for comparison. COX-2 expression was scored for percentage (< 5% = 0; 6%-25% = 1; 26%-50% = 2; 51%-75% = 3; 76%-100% = 4) and intensity (no staining = 0; yellow = 2; yellowish-brown = 3, brown = 4). Total immunoscore (percentage + intensity score) ≥ 2 was regarded as positive COX-2 expression. Outcome was statistically evaluated with clinicopathological data to determine COX-2 expression-associated and predictor variables. Ninety-five CRC cases and 27 matched non-tumour tissues as well as 31 adenomatous polyps met the inclusion criteria. Individuals with CRC had a mean age of 56.1 ± 12.6 years while those with adenomatous polyps had a median age of 65 years (range 43-88). COX-2 was differentially overexpressed in CRCs (69/95; 72.6%) and in adenomatous polyps (17/31; 54.8%) than in non-tumour tissues 5/27 (18.5%); p < 0.001). The difference in COX-2 expression between CRC and polyps was non-significant (p > 0.065). Tumour grade, advanced pT-stage, tumour-infiltrating lymphocytes, and dirty necrosis were also significantly associated with COX-2 expression (p < 0.035; 0.043, 0.035 and 0.004, respectively). Only dirty necrosis and Crohns-like lymphocytic aggregates predicted COX-2 expression (p < 0.05). CONCLUSION: This study showed a progressive increase in COX-2 expression from normal to adenomatous polyp and CRC tissues, this being associated with poorer prognostic indicators. Although COX-2 appears early in CRC, it may play a secondary role in promoting tumour growth and invasiveness.

Differential Expression of Cytochrome C Oxidase Subunit I Along the Colorectal Adenoma: Carcinoma Progression.

Loss in apoptosis competence often results in augmented genomic instability contributing to carcinogenesis. Cytochrome c oxidase subunit I (CcOI) can help assess apoptosis resistance in paraffin-embedded biopsies. In total, 50 colorectal cases including 10 control cases of colectomy for non-neoplastic condition, 15 cases of adenomatous colorectal polyps, and 25 cases of colorectal carcinoma were investigated in this retrospective study for immunohistochemical expression of CcOI. The staining pattern of CcOI was assessed and indices of aberrant expression were calculated as crypt-restricted loss and overall decreased immunostaining (ODI). ODI calculated in the adenocarcinoma tumor tissue was designated as Tr ODI. The crypt-restricted loss and ODI indices of the aberrant CcOI expression are significantly higher in the adenomatous polyps group (2.5% and 47.54%) and in the non-neoplastic mucosa among adenocarcinoma group (2.78% and 49.1%) when they are compared with the control group (0.55% and 7.32%) (P<0.001). A highly significant correlation was noted between Tr ODI and the tumor grade, the nodal status, and the stage among adenocarcinomas. In conclusion, colonic tumors arise in a field of crypts with aberrations in CcOI expression. This aberration is linked to biologically aggressive tumors. CcOI immunostaining may be applied on mucosal samples from patients with colonic adenomatous polyps and patients with previous cancer colon resection to determine individuals who are in need for frequent colonoscopies and/or chemopreventive strategies. Future follow-up studies are warranted to determine the level of expression predictive of recurrence or progression.

BMI1 and MEL18 Promote Colitis-Associated Cancer in Mice via REG3B and STAT3.

BACKGROUND & AIMS: Polycomb group proteins are epigenetic factors that silence gene expression; they are dysregulated in cancer cells and contribute to carcinogenesis by unclear mechanisms. We investigated whether BMI1 proto-oncogene, polycomb ring finger (BMI1), and polycomb group ring finger 2 (PCGF2, also called MEL18) are involved in the initiation and progression of colitis-associated cancer (CAC) in mice. METHODS: We generated mice containing floxed alleles of Bmi1 and/or Mel18 and/or Reg3b using the villin-Cre promoter (called Bmi1ΔIEC, Mel18ΔIEC, DKO, and TKO mice). We also disrupted Bmi1 and/or Mel18 specifically in intestinal epithelial cells (IECs) using the villin-CreERT2-inducible promoter. CAC was induced in cre-negative littermate mice (control) and mice with conditional disruption of Bmi1 and/or Mel18 by intraperitoneal injection of azoxymethane (AOM) followed by addition of dextran sulfate sodium (DSS) to drinking water. Colon tissues were collected from mice and analyzed by histology and immunoblots; IECs were isolated and used in cDNA microarray analyses. RESULTS: Following administration of AOM and DSS, DKO mice developed significantly fewer polyps than control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice. Adenomas in the colons of DKO mice were low-grade dysplasias, whereas adenomas in control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice were high-grade dysplasias with aggressive invasion of the muscularis mucosa. Disruption of Bmi1 and Mel18 (DKO mice) during late stages of carcinogenesis significantly reduced the numbers of large adenomas and the load of total adenomas, reduced proliferation, and increased apoptosis in colon tissues. IECs isolated from DKO mice after AOM and DSS administration had increased expression of Reg3b compared with control, Bmi1ΔIEC, or Mel18ΔIEC mice. Expression of REG3B was sufficient to inhibit cytokine-induced activation of STAT3 in IECs. The human REG3ß protein, the functional counterpart of mouse REG3B, inhibited STAT3 activity in human 293T cells, and its expression level in colorectal tumors correlated inversely with pSTAT3 level and survival times of patients. CONCLUSIONS: BMI1 and MEL18 contribute to the development of CAC in mice by promoting proliferation and reducing apoptosis via suppressing expression of Reg3b. REG3B negatively regulates cytokine-induced activation of STAT3 in colon epithelial cells. This pathway might be targeted in patients with colitis to reduce carcinogenesis.

Correlation between Faecal Tumour M2 Pyruvate Kinase and Colonoscopy for the Detection of Adenomatous Neoplasia in a Secondary Care Cohort.

BACKGROUND AND AIMS: Colorectal cancer screening programmes that target detection and excision of adenomatous colonic polyps have been shown to reduce colorectal cancer related mortality. Many screening programmes include an initial faecal occult blood test (FOBt) prior to colonoscopy. To refine the selection of patients for colonoscopy other faecal-based diagnostic tools have been proposed, including tumour M2-pyruvate kinase (tM2-PK). To determine whether tM2-PK quantification may have a role in diverse settings we have assessed the assay in a cohort of patients derived from both the England bowel cancer screening programme (BCSP) and symptomatic individuals presenting to secondary care. METHOD: Patients undergoing colonoscopy provided faecal samples prior to bowel preparation. Faecal tM2-PK concentrations were measured by ELISA. Sensitivity, specificity, positive predictive value, negative predictive value and ROC analyses were calculated. RESULTS: Ninety-six patients returned faecal samples: 50 of these with adenomas and 7 with cancer. Median age was 68. Median faecal tM2-PK concentration was 3.8 U/mL for individuals without neoplastic findings at colonoscopy, 7.7 U/mL in those with adenomas and 24.4 U/mL in subjects with colorectal cancer (both, p=0.01). ROC analysis demonstrated an AUROC of 0.66 (sensitivity 72.4%, specificity 48.7%, positive predictive value 67.7%, negative predictive value 36.7%). Amongst BCSP patients with a prior positive FOBt faecal tM2-PK was more abundant (median 6.4 U/mL, p=0.03) and its diagnostic accuracy was greater (AUROC 0.82). CONCLUSION: Our findings confirm that faecal tM2-PK ELISA may have utility as an adjunct to FOBt in a screening context, but do not support its use in symptomatic patients.

[Prevalence of altered mismatch repair protein nuclear expression detected by immunohistochemistry on adenomas with high-grade dysplasia and features associated with this risk in a population-based study]. / Prevalencia de alteración de expresión nuclear de proteínas reparadoras con inmunohistoquímica sobre adenomas con displasia de alto grado y características asociadas a dicho riesgo en un estudio de base poblacional.

INTRODUCTION: Alteration of mismatch repair system protein expression detected by immunohistochemistry (IHQ) in tumoural tissue is a useful technique for Lynch Syndrome (LS) screening. A recent review proposes LS screening through immunohistochemical study not only in all diagnosed cases of colorectal cancer (CRC) but also in advanced adenomas, especially in young patients. OBJECTIVE: To assess the prevalence of altered IHQ carried out in all adenomas with high-grade dysplasia (HGD) diagnosed in our community in 2011, as well as the variables associated with this alteration. METHODS: We included all the cases of adenomatous polyps with HGD diagnosed in the three public pathology laboratories of Navarre during 2011 and performed a statistical study to assess the association between different patient and lesion characteristics and altered IHQ results. RESULTS: A total of 213 colonic adenomas with HGD were diagnosed, and 26 (12.2%) cases were excluded from the final analysis (2 known LS, 22 without IHQ study and 2 with inconclusive IHQ studies). The final number of adenomas included was 187. Pathologic results were found in 10 cases (5.35%)-6 cases in MLH1 and PMS2, 2 cases in PMS2, 1 case in MSH6 and 1 case in MSH2 and MSH6. The factors showing a statistically significant association with the presence of abnormal proteins were the synchronous presence of CRC, the presence of only one advanced adenoma, proximal location of HGD and age <50 years. CONCLUSIONS: The percentage of pathologic nuclear expression found in IHQ is high. Consequently, screening of all diagnosed HGD could be indicated, especially in young patients, with a single AA and proximal HGD.

Upregulation of nemo-like kinase is an independent prognostic factor in colorectal cancer.

AIM: To investigate the expression and oncogenic role of nemo-like kinase (NLK) in colorectal cancer. METHODS: Expression of NLK protein was assessed by immunohistochemistry in tissue specimens from 56 cases of normal colorectal mucosa, 51 cases of colorectal adenoma, and 712 cases of colorectal cancer. In addition, NLK expression was knocked down using a lentivirus carrying NLK small hairpin RNA in colorectal cancer cells. Cell viability methylthiazoletetrazolium assays, colony formation assays, flow cytometry cell cycle assays, Transwell migration assays, and gene expression assays were performed to explore its role on proliferation and migration of colorectal cancer. RESULTS: Expression of NLK protein progressively increased in tissues from the normal mucosa through adenoma to various stages of colorectal cancer. Overexpression of NLK protein was associated with advanced tumor-lymph node-metastasis stages, poor differentiation, lymph node and distant metastases, and a higher recurrence rate of colorectal cancer (P < 0.05). Multivariate analyses showed that NLK expression was an independent prognostic factor to predict overall survival (hazard ratio 2.57, 95% confidence interval: 1.66-3.98; P < 0.001) and disease-free survival (hazard ratio 1.96, 95% confidence interval: 1.40-2.74: P < 0.001) of colorectal cancer patients. Furthermore, knockdown of NLK expression in colorectal cancer cell lines reduced cell viability, colony formation, and migration, and arrested tumor cells at the G0/G1 phase of the cell cycle. At the gene level, knockdown of NLK expression inhibited matrix metalloproteinase-2 expression in colorectal cancer cells. CONCLUSION: NLK overexpression is an independent prognostic factor in colorectal cancer and knockdown of NLK expression inhibits colorectal cancer progression and metastasis.

Expression of 11ß-hydroxysteroid dehydrogenase type 2 is deregulated in colon carcinoma.

Although the effects of glucocorticoids on proliferation, differentiation and apoptosis are well known, and steroid hormones have been identified to play a role in pathogenesis and the development of various cancers, limited data are available regarding the relationship between the local metabolism of glucocorticoids and colorectal adenocarcinoma (CRC) formation. Glucocorticoid metabolism is determined by 11ß-hydroxysteroid dehydrogenases type 1 and 2 (11HSD1, 11HSD2), which increase the local concentration of cortisol due to the reduction of cortisone, or decrease this concentration due to the oxidation of cortisol. The objective of this study was to evaluate the extent of 11HSD1 and 11HSD2 mRNA in pre-malignant colorectal polyps and in CRC. The specimens were retrieved from patients by endoscopic or surgical resection and the expression of 11HSD1 and 11HSD2 was measured by real-time PCR. The polyps were of the following histological types: hyperplastic polyps and adenomas with low- or high-grade dysplasia. The neoplastic tissue of CRC obtained during tumor surgery was also studied. It was found that 11HSD2 was not only downregulated in CRC but already in the early stages of neoplastic transformation (adenoma with low-grade dysplasia). In contrast, the level of 11HSD1 was significantly increased in CRC but not in pre-malignant polyps. The results demonstrate that the downregulation of 11HSD2 gene expression is a typical feature of the development of colorectal polypous lesions and their transformation into CRC.

Ornithine decarboxylase and glutamate decarboxylase 65 as prognostic markers of gallbladder malignancy: a clinicopathological study in benign and malignant lesions of the gallbladder.

Ornithine decarboxylase (ODC) plays a critical role in cell proliferation and is overexpressed in a variety of cancers. Furthermore, γ-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity are increased in neoplastic tissues in colon and breast cancer. However, few studies have examined these molecules in gallbladder cancer specimens. We observed the expression levels of ODC and GAD65 in benign and malignant lesions of the gallbladder and investigated their clinicopathological significance for the first time. The expression levels of ODC and GAD65 in specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15) and chronic cholecystitis (n=35) were detected using immunohistochemical methods. Kaplan-Meier survival and Cox regression analyses were carried out to explore the clinical and pathological correlations. The levels of positive staining of ODC and GAD65 were significantly higher in gallbladder adenocarcinoma than in peritumoral tissues, adenomatous polyps and chronic cholecystitis. The Kaplan­Meier survival analysis and Cox regression analysis showed that the expression of ODC and GAD65 correlated significantly with the one-year survival rate and the mean survival time of the patients postoperatively. We conclude that the overexpression of ODC and GAD65 are significant in the carcinogenesis and progression of gallbladder adenocarcinoma. They may be important biological markers for the evaluation of biological behaviors and the prognosis of gallbladder adenocarcinoma.

Loss of SIRT1 histone deacetylase expression associates with tumour progression in colorectal adenocarcinoma.

BACKGROUND: The class III histone deacetylase SIRT1 is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, and has been reported to serve diverse roles in various biological processes, such as caloric restriction, apoptosis, neuronal protection, cell growth, differentiation and tumourigenesis. With respect to tumourigenesis, there have been conflicting data supporting whether SIRT1 act as a tumour promoter or as a tumour suppressor. METHODS: SIRT1 protein expression, determined by immunohistochemistry, was investigated in human normal colonic mucosa, adenoma, adenocarcinoma and metastatic tissue samples. RESULTS: All normal colonic mucosa showed SIRT1 expression with no exception, and 42 (80.8%) of 52 adenomatous polyps were positive for SIRT1. However, only 208 (41.9%) of 497 colorectal adenocarcinomas were positive. Moreover, 45 (35.7%) of 126 metastatic tissues were positive. Collectively, the SIRT1 expression was gradually decreased during carcinogenesis and tumour progression. The associations between SIRT1 expression and clinicopathological parameters revealed that loss of SIRT1 expression was associated with proximal tumour location, mucinous histology and defective mismatch repair protein expression. This suggests that loss of SIRT1 expression is associated with the microsatellite instability phenotype of colorectal adenocarcinoma. In survival analyses, the loss of SIRT1 expression was significantly associated with overall survival (p=0.027, log-rank test) in univariable analysis, but multivariable analysis failed to achieve significance. CONCLUSIONS: SIRT1 expression was gradually decreased during the normal-adenoma-adenocarcinoma-metastasis sequence, suggesting a possible role of SIRT1 in tumour suppression in the colorectum, and a probable link to the microsatellite instability pathway.

Linkage disequilibrium and haplotype analysis of COX-2 and risk of colorectal adenoma development.

Single nucleotide polymorphisms (SNPs) in the promoter and untranslated region of cyclooxygenase (COX)-2, an inducible enzyme responsible for the synthesis of prostaglandins, have been reported to modulate the risk for many human cancers. We performed comprehensive linkage disequilibrium (LD) and haplotype analyses of 13 single nucleotide polymorphisms of the COX-2 gene and examined its susceptibility to adenoma development in 72 African American cases and 142 controls. Results revealed significant variation in LD patterns with consequence for adenoma development. Two distinct haplotype blocks were identified; one block covered the coding regions of exon 1, introns and a section of the 3'-unstranslated region (3'-UTR), whereas the second block resided solely in the 3'-UTR region. A haplotype in block 1 increased the risk of adenoma development by threefold (odds ratio [OR]= 2.9, confidence interval [CI]= 1.8-3.7, P= 0.002). Regression analysis showed, increase in copies of minor alleles of 6,064(T>C) polymorphism associated with increased odds of adenoma development by 80% (OR = 1.80, CI = 1.09-3.21, P= 0.034), 10,848(G>A) by 84% (OR = 1.84, CI = 1.05-3.23, P= 0.034) and 10,935(A>G) by 32% (OR = 1.32, CI = 1.12-3.69, P= 0.036). These results support the hypothesis that COX-2 gene might play a role in the etiology of colon cancer and warrant further investigation in other cancers. Besides, these variations should be taken into account for disease-based association studies in which the COX-2 polymorphism is considered as a candidate gene. Clin Trans Sci 2012; Volume 5: 60-64.

Association of the DNMT3B polymorphism with colorectal adenomatous polyps and adenocarcinoma.

DNMT3B is an important enzyme to modulate the methylation status in mammalian cells. The aim of this study is to investigate the correlation of the DNMT3B G39179T polymorphism with the susceptibilities of colorectal adenomatous polyps and adenocarcinoma. This case-control study included 146 colorectal adenomatous polyps, 170 colorectal adenocarcinoma patients, and 157 normal controls. DNMT3B polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. Family history of colorectal cancer significantly increases the risk of developing colorectal adenomatous polyps and adenocarcinoma. The genotype frequency of DNMT3B polymorphism (T/T and G/T + G/G) in adenocarcinoma patients was significantly different from that in controls (P value = 0.01). Compared with DNMT3B T/T genotype, the G allelotype (G/T + G/G genotype) had lower risk to develop colorectal adenocarcinoma (OR = 0.50, 95% CI = 0.29-0.87); while there was no significant difference between the colorectal adenomatous polyps patients and controls (OR = 0.63, 95% CI = 0.37-1.09), although descending tendency could be found in this polyps group. In the stratification analysis, a significant association was confined to subgroups of age < 55 (OR = 0.31, 95% CI = 0.12-0.84) and males (OR = 0.35, 95% CI = 0.17-0.71). Meanwhile, combined G/T + G/G genotypes were found to have a lower risk in non-drinkers to develop both colorectal adenomatous polyps and adenocarcinoma (OR = 0.54, 95% CI = 0.31-0.96 and OR = 0.48, 95% CI = 0.27-0.84, respectively). This study also showed a distinct difference in the distribution of DNMT3B G39179T SNP in different ethnics. DNMT3B G39179T SNP may be a potential genetic susceptibility factor for adenocarcinoma of the colon, especially in younger Chinese Han non-drinker men.

COX-2 expression in sporadic colorectal adenomatous polyps is linked to adenoma characteristics.

AIMS: To assess cyclooxygenase-2 (COX-2) expression in sporadic colonic adenomas and to explore the association of COX-2 positivity with adenoma characteristics linked to increased risk of malignant transformation. METHODS AND RESULTS: COX-2 expression and localization were assessed in 64 colorectal adenomas and 35 paired adjacent normal colonic mucosal biopsy specimens. The number of adenoma specimens was then extended to include polyps exhibiting an increasing degree of epithelial dysplasia. Forty colonic hyperplastic polyps were also identified from the pathology diagnostic database and included in the analysis. Immunohistochemistry was performed with the Envision+ peroxidase-linked biotin-free system incorporating a signal amplification step. There was a statistically significant increase in COX-2 expression in colonic polyps compared with paired adjacent normal mucosa, chi(2) = 40.1, P = 0.001. The probability of COX-2 expression increased along with increasing adenoma size and increasing degree of epithelial dysplasia. Fifty-five per cent of the hyperplastic polyp specimens expressed COX-2. CONCLUSIONS: This study associates COX-2 epithelial expression with a number of adenoma characteristics that convey an increased risk of malignant transformation. This is in keeping with a positive role for COX-2 in early colorectal carcinogenesis.

DNA methyltransferase and alcohol dehydrogenase: gene-nutrient interactions in relation to risk of colorectal polyps.

Disturbances in DNA methylation are a characteristic of colorectal carcinogenesis. Folate-mediated one-carbon metabolism is essential for providing one-carbon groups for DNA methylation via DNA methyltransferases (DNMTs). Alcohol, a folate antagonist, could adversely affect one-carbon metabolism. In a case-control study of colorectal polyps, we evaluated three single nucleotide polymorphisms (-149C>T, -283T>C, -579G>T) in the promoter region of the DNMT3b gene, and a functional polymorphism in the coding region of the alcohol dehydrogenase ADH1C gene, ADH1C *2. Cases had a first diagnosis of colorectal adenomatous (n = 530) or hyperplastic (n = 202) polyps at the time of colonoscopy, whereas controls were polyp-free (n = 649). Multivariate logistic regression analysis was used to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI). There were no significant main associations between the DNMT3b or ADH1C polymorphisms and polyp risk. However, DNMT3b -149TT was associated with an increase in adenoma risk among individuals with low folate and methionine intake (OR, 2.00; 95% CI, 1.06-3.78, P interaction = 0.10). The ADH1C *2/*2 genotype was associated with a possibly elevated risk for adenomatous polyps among individuals who consumed >26 g of alcohol/d (OR, 1.95; 95% CI, 0.60-6.30), whereas individuals who were wild-type for ADH1C were not at increased risk of adenoma (P interaction = 0.01). These gene-diet interactions suggest that polymorphisms relevant to DNA methylation or alcohol metabolism may play a role in colorectal carcinogenesis in conjunction with a high-risk diet.

CYP27A1 and CYP24 expression as a function of malignant transformation in the colon.

Vitamin D deficiency is strongly associated with the risk of developing colorectal cancer (CRC). Because of the propensity of bioactive 1,25-dihydroxyvitamin D3 to cause toxic hypercalcemia, considerable effort has been directed to identifying safer drugs while retaining the efficacy of the parent compound. However, vitamin D precursors do not present toxicity concerns and may be sufficient for CRC chemoprevention or chemotherapy, providing the appropriate enzymes are present in colonic epithelia. We previously showed that CYP27B1 is present at equally high levels in the colon and CRC irrespective of differentiation but was not present in metastases. In this study we used quantitative immunohistochemistry to show that CYP27A1, converting D3 to 25-hydroxycholecalciferol, is present in increasing concentrations in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. Whereas total cellular CYP27A1 remains high in CRC and lymph node metastases, the amount of enzyme present in the nuclei decreases with tumor cell dedifferentiation while rising in the cytoplasm. Similarly, increasing amounts of the deactivating enzyme CYP24 are present in the nuclei of normal colonic epithelia, ACFs, and adenomatous polyps. Although the amount of total CYP24 decreases slightly in CRC as a function of tumor cell dedifferentiation and metastasis, location of this enzyme shifts almost entirely from the nuclear compartment to the cytoplasmic compartment. These data indicate that non-toxic vitamin D precursors should be sufficient for CRC chemoprevention, but that neither vitamin D nor its precursors may be sufficient for CRC chemotherapy.

Disease-associated casein kinase I delta mutation may promote adenomatous polyps formation via a Wnt/beta-catenin independent mechanism.

The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKIdelta and epsilon) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKIdelta/epsilon might upregulate CKIdelta/epsilon activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKIepsilon and CKIdelta regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKIdelta missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKIdelta(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKIepsilon. Second, CKIdelta(R324H) is more potent than wildtype CKIdelta in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKIdelta kinase activity in an in vitro kinase assay or Wnt/beta-catenin signal transduction as assessed by a beta-catenin reporter assay, it alters morphogenetic movements via a beta-catenin-independent mechanism in early Xenopus development. This novel human CKIdelta mutation may alter the physiological role and enhance the transforming ability of CKIdelta through a Wnt/beta-catenin independent mechanism and thereby influence colonic adenoma development.

Genetic polymorphisms of MMP1, MMP3 and MMP7 gene promoter and risk of colorectal adenoma.

BACKGROUND: Matrix metalloproteinases (MMP) have been shown to play a role in colorectal cancer (CRC). More recently, MMP1, MMP3 and MMP7 functional gene promoter polymorphisms have been found to be associated with CRC occurrence and prognosis. To document the role of MMP polymorphisms in the early step of colorectal carcinogenesis, we investigated their association with colorectal adenoma risk in a case-control study comprising 295 patients with large adenomas (LA), 302 patients with small adenomas (SA) and 568 polyp-free (PF) controls. METHODS: Patients were genotyped using automated fragment analysis for MMP1 -1607 ins/del G and MMP3 -1612 ins/delA (MMP3.1) polymorphisms and allelic discrimination assay for MMP3 -709 A/G (MMP3.2) and MMP7 -181 A/G polymorphisms. Association between MMP genotypes and colorectal adenomas was first tested for each polymorphism separately and then for combined genotypes using the combination test. Adjustment on relevant variables and estimation of odds ratios were performed using unconditional logistic regression. RESULTS: No association was observed between the polymorphisms and LA when compared to PF or SA. When comparing SA to PF controls, analysis revealed a significant association between MMP3 -1612 ins/delA polymorphism and SA with an increased risk associated with the 6A/6A genotype (OR = 1.67, 95% CI: 1.20-2.34). Using the combination test, the best association was found for MMP3.1-MMP1 (p = 0.001) with an OR of 1.88 (95% CI: 1.08-3.28) for the combined genotype 2G/2G-6A/6A estimated by logistic regression. CONCLUSION: These data show a relation between MMP1 -1607 ins/del G and MMP3 -1612 ins/delA combined polymorphisms and risk of SA, suggesting their potential role in the early steps of colorectal carcinogenesis.

Enhanced expression of MMP-7 and MMP-13 in inflammatory bowel disease: a precancerous potential?

Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP-2, MMP-9, MMP-7, and MMP-13. The aim of this study was to determine the expression levels of MMP-2, MMP-9, MMP-7, MMP-13, and MMP-14 and their specific inhibitor TIMP-1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohn's disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohn's disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real-time polymerase chain reaction to study MMP and TIMP-1 gene expression in both pathological and normal mucosal specimens. For MMP-2, MMP-9, and TIMP-1, protein expression also was quantified with sandwich enzyme-linked immunosorbent assay. In biopsy specimens of Crohn's disease and ulcerative colitis, significantly increased levels of MMP-2, MMP-7, and MMP-13 mRNA were found. MMP-2 and MMP-9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP-7 and MMP-13 genes. MMP-14 mRNA was decreased in APs. MMPs, especially MMP-7 and MMP-13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP-7 and MMP-13 may serve as an early indicator for colorectal carcinogenesis.

Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation.

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.

Prostacyclin synthase and arachidonate 5-lipoxygenase polymorphisms and risk of colorectal polyps.

Prostacyclin synthase (PGIS) and arachidonate 5-lipoxygenase (ALOX5) are enzymes relevant to prostaglandin and leukotriene synthesis, both important pathways for colon cancer risk. We hypothesized that genetic variation altering the function of these enzymes would modify risk of colorectal polyps. In a Minnesota-based case-control study of adenomatous (n = 517) or hyperplastic (n = 192) polyps versus polyp-free controls (n = 618), we investigated the role of promoter repeat polymorphisms in PGIS and ALOX5 as well as ALOX5 -1700 G>A. Having fewer than six repeats on both PGIS alleles (<6R/<6R) was associated with an increased risk of adenomas compared with the 6R/6R (wild-type) genotype (OR, 1.90; 95% CI, 1.09-3.30). Having more repeats (>6R/> or =6R) reduced risk (OR, 0.73; 95% CI, 0.40-1.35; P(trend) = 0.03). In allele-based analyses, fewer repeats were associated with a modestly increased risk of adenomas and perhaps hyperplastic polyps. There were no risk differences for either the ALOX5 VNTR or -1700 G>A polymorphisms. Associations with regular use of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) differed by PGIS genotype. Among individuals with at least one wild-type allele, NSAID use was associated with a decreased risk; however, those with fewer PGIS repeats (<6R/<6R) did not benefit (P(interaction) = 0.06). There was also evidence of an interaction between the COX-2 -765 G>C and ALOX5 -1700 G>A genotypes (P(interaction) = 0.07). The PGIS promoter polymorphism may affect risk of colorectal polyps and modify the effects of NSAID use on polyp risk. A more comprehensive investigation of genetic variability in prostaglandin synthesis in relation to risk of colorectal neoplasia and NSAID pharmacogenetics is warranted.