MDR1 gene polymorphisms: possible association with its expression and clinicopathology characteristics in colorectal cancer patients

Aim: Over-expression of some genes, such as MDR1, can increase the level of chemotherapy drug afflux and limit the effectiveness of treatment. We aimed to investigate the effect of MDR1 polymorphisms on its expression level and related characteristics in Iranian colorectal cancer patients. Methods: Tumor, normal mucosal tissue and blood samples from CRC patients and blood samples from healthy controls (n=60) were obtained for genotyping and measuring the expression level of MDR1. Results: The expression of the MDR1 gene showed a significant increase in cancerous regions compared to adjacent normal tissue. Furthermore, the GG2677 genotype was correlated with highest while the AT 2677genotype was associated with the lowest levels of expression. In addition only the G2677T/A polymorphism showed association with histological grade of colorectal tumors. Conclusion: Our study once more emphasizes effects of MDR1 SNPs which may indirectly impact on response to drugs.     

The GSTP1 Ile105Val polymorphism is not associated with susceptibility to colorectal cancer

The glutathione S transferase (GST) family is a major part of cellular defense mechanisms against endogenous and exogenous substances, many of which have carcinogenic potential. Alteration in the expression level or structure of the glutathione-S-transferase (GST) enzymes may lead to inadequate detoxification of potential carcinogens and consequently contribute to cancer development. A member of the glutathione-S-transferase (GST) family, GSTP1, is an attractive candidate for involvement in susceptibility to carcinogen-associated colorectal cancer. An A>G transition in exon 5 resulting in an Ile105Val amino acid substitution has been identified which alters catalytic efficiency. The present study investigated the possible impact of Ile105Val GSTP1 polymorphism on susceptibility to colorectal cancer. in Jordan We examined 90 tissue samples previously diagnosed with colorectal carcinoma, and 56 non-cancerous colon tissues. DNA was extracted from paraffin embedded tissues and the status of the GSTP1 polymorphism was determined using a polymerase chain reaction restriction fragment length polymorphism (RFLP) method. No statistically significant differences were found between colorectal cancer cases and controls for the GSTP1 Ile/Ile, Ile/Val and Val/Val genotypes. The glutathione S-transferase polymorphism was not associated with risk in colorectal cancer cases in Jordan stratified by age, sex, site, grade or tumor stage. In conclusion, the GSTP1 Ile105Val polymorphism is unlikely to affect the risk of colorectal cancer.     

Polymorphisms in the genes of the urokinase plasminogen activation system in relation to colorectal cancer.

BACKGROUND: Extracellular matrix degradation, mediated by the urokinase plasminogen activation (uPA) system, is a critical step in tumor invasion and metastasis. High tumor levels of uPA and its inhibitor PAI-1 have been correlated with poor cancer prognosis. We examined four single nucleotide polymorphisms (SNPs) with a potential effect on expression of genes in the uPA system for their role in colorectal cancer susceptibility and prognosis. PATIENTS AND METHODS: We genotyped the SNPs in 308 Swedish incident colorectal cancer patients with up to 16 years of follow-up and in 585 age- and sex-matched controls. We evaluated the associations between genotypes and colorectal cancer and Dukes' stage. Survival probabilities were compared between different subgroups. RESULTS: Patients with PAI-1 -675 5G/5G genotype had better survival than patients with 4G/4G or 4G/5G genotypes when they had Dukes' stage A or B tumors (P = 0.023 and P = 0.015, respectively). No statistically significant association was observed between the SNPs and the risk of colorectal cancer or Dukes' stage. CONCLUSIONS: Our results suggest a role for the PAI-1 genotype in colorectal cancer prognosis, but further studies are needed to evaluate the impact of our finding in the clinic.     

PAI-1 promoter polymorphism modulates uPA-PAI complex accumulation by breast cancer cells

OBJECTIVES: The uPA-PAI system has been shown to play a role in the development of a more aggressive tumor phenotype. The PAI-1 promoter 4G/5G polymorphism, furthermore, regulates free plasma PAI-1 levels in patients with myocardial infarction. Our aim was to verify if the different polymorphisms in the PAI-1 promoter are also associated with alterations in the intracellular accumulation of uPA-PAI complexes in human breast cancer. METHODS: Accumulation of uPA-PAI complexes inside the tumor cells was determined by means of immunohistochemistry, as previously described by our own group, and two extremely different sets of tumors were chosen, one of them with strong uPA-PAI complex reactivity inside more than 50% of tumor cells, the other with no demonstrable reactivity at all. Finally, the 4G/5G polymorphism of the PAI-1 promoter was studied in all of them by means of DNA extraction, PCR amplification of the PAI promoter sequence, and restriction polymorphism typing. RESULTS: Absence of intracellular uPA-PAI complex accumulation was significantly associated with the prevalence of the 4G allele and, conversely, the presence of uPA-PAI complexes inside the tumor cells was significantly associated with 5G/5G homozygosity (logistic regression, p = 0.0128). Furthermore, none of the 7 5G/5G homozygous tumors showed histological grade 3, as did 6/21 tumors in the group where the 4G allele was present. In spite of the low case number, this association of the 5G/5G polymorphism with a less aggressive phenotype almost reached statistical significance (Spearman's correlation test, p = 0.118). CONCLUSIONS: The 4G/5G polymorphism of the PAI-1 promoter seems indeed to be associated with different rates of uPA-PAI complex internalization by breast cancer cells. Complex accumulation inside the tumor cells is significantly related to 5G/5G homozygosity, and this shows a trend towards an association with a less aggressive, better-differentiated tumor phenotype.     

Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer

INTRODUCTION: EGF/EGFR interactions are important mechanisms behind colorectal tumour development and growth. Recently a single nucleotide polymorphism in the EGF gene has been identified (EGF A61G). It may be a potential predictor for survival of patients receiving EGFR-inhibitor cetuximab treatment, but the clinical importance and the functional influence on EGF gene expression levels in colorectal cancer (CRC) patients have not yet been further assessed. The aim of the present study was to investigate the relationship between EGF A61G genotype and EGF gene expression levels in colorectal adenocarcinomas and normal colon tissue. MATERIAL AND METHODS: Eighty-one CRC patients were included in the study. Tissue samples from normal colon, adenocacinomas and corresponding blood samples were analysed by real-time PCR for EGF gene expression and EGF A61G genotype, respectively. RESULTS: Thirty-three percent were AA, 48% and 19% A/G and G/G respectively. We found a significantly lower median age in the A/A group compared to the G/G group, suggesting a later time of diagnosis in the G/G patients. There was a significant difference between the median EGF gene expression among the three genotypes in normal colon (p < 0.001) but not in adenocarcinomas. Furthermore, the median EGF gene expression was lower in CRC tissue than in normal colon samples, (0.13 (range 0.01-6.4) vs. 0.76, (range 0.013-5.55)). CONCLUSION: We suggest that EGF A61G genotype has a functional influence on EGF gene expression in normal colon in CRC patients. The clinical implications warrant further investigations in prospective trials.     

Significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) expression in human colorectal carcinomas.

Despite the advances in the medical care of colorectal carcinoma patients, the prognosis has improved only marginally over recent decades. Thus, additional prognostic indicators would be of great clinical value to select patients for adjuvant therapy. In the present study, the antigen levels of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and their immunohistochemical staining were compared in paired colorectal tumor (n = 64) and background colon tissue of the same patients with clinical and pathological staging. The antigen levels, measured with an ELISA method, were found to be significantly higher in cancer tissue (mean 1.92 ng/mg protein for uPA and 7.08 for PAI-1) than in corresponding normal mucosa (0.29 ng/mg protein for uPA and 1.11 ng/mg protein for PAI-1). There was a positive correlation between uPA and PAI-1 antigen levels and clinicopathological parameters such as grade (p < 0.001 and p = 0.01, respectively), while for Dukes' stage, only PAI-1 correlated positively (p = 0.018). Nodal status correlated positively with uPA but not with PAI-1 antigen levels. Immunohistochemical localization of both antigens was observed mainly in cancer cells and much less in stromal cells. Staining intensity increased from adenoma to adenocarcinoma. The degree of staining was associated with grade, Dukes' stage and nodal status for uPA (p < 0.001, p = 0.002, p < 0.001, respectively) and only with grade for PAI-1 (p = 0.007).     

急性白血病患者的纤溶酶原激活剂抑制物-1及其基因启动子区4G/5G多态性的调查

目的探讨纤溶酶原激活剂抑制物-1（plasminogen activatorinhibitor-1,PAI-1）基因启动子区4G/5G多态性与急性白血病的关系.方法应用等位基因特异性引物PCR分析30例急性白血病患者和30名健康人的PAI-1基因4G/5G多态性.发色底物法测PAI-1活性.结果 PAI-14G/5G基因型多态性与PAI-1活性水平相关,4G/4G活性最高;急性白血病组的pAI-1活性明显低于对照组（P＜0.01）;PAI-14G/5G基因型、基因分布频率在急性白血病组和对照组之间无差异（P＞0.05）.结论PAI-1基因4G/5G多态性可能不是急性白血病的发病危险因素.急性白血病患者PAI-1活性明显降低,在其出血中起到一定的作用.     

CCND1 polymorphism and age of onset of hepatoblastoma

Cyclin D1, encoded by the gene CCND1, is a major regulator of the cell cycle transition from G1 phase to S phase. A CCND1 polymorphism (G to A) at codon 242, the boundary of exon 4 and intron 4, affects splicing such that exon 5 is not expressed in the A allele. Since exon 5 is involved in rapid turnover, the variant cyclin D1 corresponding to the A allele may have a longer half-life. A previous study demonstrated that in families with hereditary nonpolyposis colorectal cancer, the age of onset of colorectal cancer varied according to variation at this polymorphic site. We examined this CCND1polymorphism in a series hepatoblastoma, a childhood liver cancer that shares other molecular features with colon cancer. We determined in an analysis of 84 children with hepatoblastoma that the G/A exon 4 polymorphism in CCND1 is correlated with the age of onset of hepatoblastomas. The A/A genotype is associated with an earlier age of onset compared to the G/A or G/G genotype. The median age of patients with the G/G genotype was 22 months, compared to 17 months in patients with the G/A genotype and 11 months for the A/A genotype. These findings suggest that the CCND1 A polymorphism may contribute to tumor development in children with hepatoblastoma.     

Cytochrome P450 17A1 and catechol O-methyltransferase polymorphisms and age at Lynch syndrome colon cancer onset in Newfoundland.

PURPOSE: Lynch syndrome is a cancer predisposition syndrome which includes colon cancer. It is caused by inherited defects in DNA mismatch repair genes. Sporadic colon cancers are influenced by exogenous hormones (e.g., postmenopausal hormones); we hypothesized that polymorphisms which influence endogenous hormones would therefore modify age at colon cancer onset among Lynch syndrome mutation carriers. EXPERIMENTAL DESIGN: We genotyped 146 Caucasian Lynch syndrome mutation carriers for a 5'-untranslated region polymorphism in cytochrome P450 17A1 (CYP17; c.-34T-->C) and an exon 4 polymorphism in catechol O-methyltransferase (COMT; c.472G-->A); 50 mutation carriers had developed colon or rectal cancer at last contact. We used chi(2) tests to assess differences in counts. Kaplan-Meier survival curves and Cox proportional hazard models assessed age at onset of colorectal cancer stratified by CYP17 and COMT genotypes. RESULTS: Homozygous carriers of the CYP17 C allele were diagnosed with colorectal cancer 18 years earlier than homozygous carriers of the T allele. Hazard ratios identified that, relative to homozygous carriers of the T allele (T/T), carriers of one copy (T/C) and two copies (C/C) of the rare allele were, respectively, at 1.9-fold and 2.9-fold increased the risk of colon cancer at any age. The COMT rare allele suggested a nonstatistically significant trend of decreased colon cancer risk. CONCLUSIONS: This study showed that a polymorphism in CYP17 (c.-34T-->C) modifies age at onset of Lynch syndrome. Because of the high risk of colorectal cancer among this group, knowledge of the CYP17 genotype is warranted for genetic counseling and risk assessment. Future work should assess polymorphisms associated with steroid hormones in Lynch syndrome mutation carriers.     

Associations between Single Nucleotide Polymorphisms of COX-2 and MMP-2 Genes and Colorectal Cancer Susceptibility in the Saudi Population

Background: It has been reported that COX-2 expression is associated with MMP-2 expression in thyroid and breast cancers, suggesting that MMPs are linked to COX-2-mediated carcinogenesis. Several polymorphisms within the MMP2 promoter region have been reported in cases with oncogenesis and tumor progression, especially in colorectal carcinogenesis. Materials and Methods: This research evaluated risk of association of the SNPs, including genes for COX-2 (A/G transition at +202) and MMP-2 (C/T transition at-1306), with colorectal cancer in 125 patients and 125 healthy controls. Results and Conclusions: Our data confirmed that MMP2 C-1306 T mutations were significantly more common in colon cancer patients than in our control Saudi population; p=0.0121. On the other hand in our study, there was no significant association between genotype distribution of the COX2 polymorphism and colorectal cancer; p=0.847. An elevated frequency of the mutated genotype inthe control group as compared to the patients subjects indeed suggested that this polymorphism could decrease risk in the Saudi population. Our study confirmed that the polymorphisms that could affect the expressions of MMP-2 and COX-2 the colon cancer patients were significantly higher than that in the COX-2 negative group. The frequency of individuals with MMP2 polymorphisms in colon cancer patients was higher than individuals with combination of COX2 and MMP2 polymorphisms. Our study confirmed that individuals who carried the polymorphisms that could affect the expressions of COX2 are more susceptible to colon cancer. MMP2 regulatory polymorphisms could be considered as protective; further studies need to confirm the results with more samples and healthy subjects.     

Identification of mitochondrial FoF1-ATP synthase involved in liver metastasis of colorectal cancer

Liver metastasis is a major cause of poor survival of colorectal cancer patients. In order to identify the proteins associated with liver metastasis in colorectal cancer, we carried out two-dimensional gel electrophoresis-based comparative proteomic analysis of normal colon mucosa, primary colon cancer tissue and corresponding metastatic tumor tissue in liver. The proteins identified were further validated by immunohistochemical analysis of 67 quadruplet samples of normal colon primary colorectal cancer and normal liver-synchronous liver metastasis, and 251 colorectal cancers as well as in vitro invasion assay of the human colon cancer cell line, SNU-81. From proteomic assessment, the mitochondrial FoF1-ATP synthase (ATP synthase) alpha-subunit was identified as a protein that is upregulated in liver metastasis compared with the primary tumor. Immunohistochemical analyses confirmed a significant increase in the expression of ATP synthase alpha- and d-subunits in synchronous liver metastasis compared with primary tumor and normal mucosa, respectively. ATP synthase alpha- and d-subunits were overexpressed in 197 (78.5%) and 190 (75.7%), respectively, of the 251 colorectal cancers. The alpha- and d-subunits were significantly associated with liver metastasis (P < 0.05) as well as low histological grade (P < 0.0001). The d-subunit also correlated with venous invasion (P = 0.026) and distant metastasis (P = 0.032). In stage III cancers, d-subunit expression was independently associated with poor survival (P = 0.017). Furthermore, transfection of small interfering RNA targeted to ATP synthase alpha- and d-subunits resulted in decreased in vitro invasiveness of the human colon cancer cell line. Our overall findings demonstrate that increased ATP synthase is associated with liver metastasis of colorectal cancer.     

UGT1A1 polymorphism can predict hematologic toxicity in patients treated with irinotecan.

PURPOSE: Irinotecan (CPT-11) is approved in metastatic colorectal cancer treatment and can cause severe toxicity. The main purpose of our study was to assess the role of different polymorphisms on the occurrence of hematologic toxicities and disease-free survival in high-risk stage III colon cancer patients receiving 5-fluorouracil (5FU) and CPT-11 adjuvant chemotherapy regimen in a prospective randomized trial. EXPERIMENTAL DESIGN: Four hundred patients were randomized in a phase III trial comparing LV5FU2 to LV5FU2 + CPT-11. DNA from 184 patients was extracted and genotyped to detect nucleotide polymorphism: 3435C>T for ABCB1, 6986A>G for CYP3A5, UGT1A1*28 and -3156G>A for UGT1A1. RESULTS: Genotype frequencies were similar in both treatment arms. In the test arm, no significant difference was observed in toxicity or disease-free survival for ABCB1 and CYP3A5 polymorphisms. UGT1A1*28 homozygous patients showed more frequent severe hematologic toxicity (50%) than UGT1A1*1 homozygous patients (16.2%), P = 0.06. Moreover, patients homozygous for the mutant allele of -3156G>A UGT1A1 polymorphism showed more frequent severe hematologic toxicity (50%) than patients homozygous for wild-type allele (12.5%), P = 0.01. This toxicity occurred significantly earlier in homozygous mutant than wild-type homozygous patients (P = 0.043). In a Cox model, the hazard ratio for severe hematologic toxicity is significantly higher for patients with the A/A compared with the G/G genotype [hazard ratio, 8.4; 95% confidence interval, 1.9-37.2; P = 0.005]. CONCLUSIONS: This study supports the clinical utility of identification of UGT1A1 promoter polymorphisms before LV5FU2 + CPT-11 treatment to predict early hematologic toxicity. The -3156G>A polymorphism seems to be a better predictor than the UGT1A1 (TA)(6)TAA>(TA)(7)TAA polymorphism.     

Energy balance, insulin-related genes and risk of colon and rectal cancer

Energy balance, or the ability to maintain body weight by balancing energy intake with energy expenditure, appears to be important in the etiology of colon cancer. One possible mechanism whereby energy balance may be associated with colorectal cancer is through its association with insulin. In our study, we evaluate the interaction between polymorphisms in 4 genes thought to be involved in insulin-related functions and components of energy balance with risk of colorectal cancer. Data from 2 population-based case-control studies of colon and rectal cancer conducted in Utah and Northern California were used to evaluate associations between body mass index (BMI), physical activity, energy intake and sucrose-to-fiber ratio and a CA repeat polymorphism of the IGF1 gene, the A/C polymorphism at nucleotide -202 of the IGFBP3, the G972R polymorphism of the IRS1 gene and the G1057D polymorphism of the IRS2 gene. A total of 1,346 incident colon cancer cases and 1,544 population-based controls and 952 incident rectal cancer cases and 1,205 controls were available for analysis. Inconsistent associations were identified between BMI, physical activity, energy intake and insulin-related genes. The 192/192 IGF1 genotype was associated with significant reduction in colon cancer risk among those with high physical activity (odds ratio [OR] 0.57; 95% confidence interval [CI] 0.39-0.83; p interaction 0.01). Although there was no significant pattern of interaction between either BMI or energy intake and polymorphisms assessed, specific sources of energy did appear to be more related to colon cancer risk in the presence of specific IRS2 and IGF1 genotypes. A high sucrose-to-fiber ratio increased risk of colon cancer in men who had the IRS2 DD genotype and among men who did not have the 192/192 IGF1 genotype. In summary, these data support the importance of components of energy balance in risk of colorectal cancer. Obesity, physical activity and energy intake appear to alter risk of colorectal cancer; however, the risk appears to be minimally influenced by genetic variants evaluated.     

Quantitative analysis of lymphangiogenic markers in human colorectal cancer

Lymphatic spread of colorectal cancer cells to regional lymph nodes is one of the early events in metastatic cancer, and is often associated with distant metastatic spread and a poor prognosis. This study examined lymphangiogenic factors, and in particular a panel of newly discovered lymphangiogenic markers, in colorectal cancer tissues from a cohort of patients. Paired samples (background normal mucosa and cancer) of colon tissue were obtained from patients with colorectal cancer. The expression and levels of the VEGF-C and VEGF-D cytokines, the VEGF receptors VEGFR-2 and VEGFR-3, and newly described lymphatic endothelial markers, LYVE-1, Prox-1, podoplanin and 5'-nucleotidase were assessed. RNA was extracted from the frozen colon tissues. The level of expression for each factor/marker was determined using RT-PCR and quantified using a real-time quantitative PCR (RT-QPCR) technique, with respective cloned cDNA plasmids as internal standards. VEGF-D was expressed to a significantly higher degree in the colon tumour tissues. There was no significant difference between the expression levels for both VEGF-C and its receptor, VEGFR-2, in background and cancer tissues. However, levels of the VEGFR-3 receptor were found to be significantly higher in colon cancer than the normal background tissues. LYVE-1 levels were below detection in most cases. There was a significant increase in the degree of Prox-1 and 5'-nucleotidase expression in colon cancer tissue. Podoplanin expression was also increased in the cancer samples. These markers indicate an increase in lymphangiogenesis in colon cancer, and may therefore have prognostic value for colon cancer patients.     

Methylation and regulation of expression of different retinoic acid receptor beta isoforms in human colon cancer

Tumor suppressor genes can become inactivated in cancer via hypermethylation of their promoter. The retinoic acid receptor beta (RARbeta) gene is expressed from two distinct promoters, both of which have CpG islands. RARbeta1 is expressed primarily during embryogenesis, whereas RARbeta2 is expressed in adult tissues and hypermethylated in a number of cancer cells. We used combined bisulfite restriction analysis to evaluate their methylation in colorectal mucosa and tumors. Methylation of RARbeta1 was detected, with a mean of 2% in normal colon tissues in young subjects (< 32 years), and 16% in older subjects (> 75 years) (P < 0.001). Using paired normal/tumor tissue samples, we found higher mean methylation rate in tumors than in adjacent normal tissue (mean, 46% versus 16%; P < 0.001) and hypermethylation of RARbeta1 in all eight cell lines examined. By RT-PCR, RARbeta1 was not expressed in normal adult colon tissues and its expression could not be efficiently activated in most cell lines by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR). RARbeta2 methylation was also observed in normal colon tissues and was lower in young individuals than in older ones (mean, 11% versus 23%; P < 0.05). Among paired samples, RARbeta2 methylation was higher in tumor tissue than in normal tissue in 14 cases, vice versa in 7 cases, and equal in 6 cases. All eight cell lines were hypermethylated and did not express RARbeta2, but RARbeta2 expression could be reactivated easily by 5-Aza-CdR. We suggest that the embryonic RARbeta1 isoform is readily hypermethylated in aging colon mucosa and all colorectal cancers because of its lack of expression in normal tissues. The adult RARbeta2 isoform also shows age-related methylation in normal tissues but more variable methylation in colorectal cancer, perhaps because its expression offers continued protection against methylation or its silencing does not provide a selective advantage in the early stages of the disease.