STAT3 polymorphisms in North Africa and its implication in breast cancer.

BACKGROUND: Only a few studies have investigated the association of single nucleotide polymorphisms in STAT3 gene with the susceptibility to cancer and response to chemotherapy. Our aim was to determine the allele frequencies of rs3869550, rs957971, and rs7211777 at the STAT3 gene in North African populations and compare them to 1000 genomes populations, and to investigate their relation with cancer. METHODS: The targeted SNPs have been analyzed in six Tunisian populations and a sample of Libyans using TaqMan® Assay. The results were compared to 1000 Genomes Project population samples. Targeting of the regions encompassing the three SNPs by micro-ARN was assessed using miR databases. RESULTS: The analysis of the 3 SNPs showed that North African populations were close to South Asians. As expected, African populations presented a significant frequency of the ancestral CCG haplotype in contrast to other populations where the fully derived TGA haplotype was more frequent. The presence and diversity of rare haplotypes at STAT3 in North African populations could have been generated by recombination between the two major haplotypes. A screening of the micro-RNA databases showed that the STAT3 region with the mutated allele of rs7211777 (G>A) could be targeted by miR hsa-miR-3606-5p, which also targets genes involved in breast cancer.

Involvement of IL17A, IL17F and IL23R Polymorphisms in Colorectal Cancer Therapy.

IL23/IL17 pathway plays an important role in the development of inflammatory bowel diseases (IBD). In general, the genes encoding the cytokines are genetically polymorphic and polymorphisms in genes IL23R and IL17 have been proved to be associated with its susceptibility to inflammatory diseases as well as cancer including colorectal cancer. Moreover, it has been shown that these interleukins are involved in anti-tumor or pro-tumor effects of various cancers. Previously, we showed that there is a significant association between IL17A, IL17F and IL23R polymorphisms as well as the occurrence of colorectal cancer and the clinical features of the disease. The purpose of the present work is to investigate an association between IL17A, IL17F and IL23R polymorphisms in 102 Tunisian patients with colorectal cancer treatment. The association was analyzed by statistical tools. We found that patients with mutated genotypes of IL17A G197A SNP could be a risk factor for the inefficiency of chemotherapy and radiotherapy. Unlike IL17F variant, patients with wild type genotypes require surgery and adjuvant chemotherapy. On the one hand, we found no evidence that supports a significant association between IL23R polymorphism and the combined genotypes of these three genes and the colorectal cancer treatment. On the other hand, we showed that there is an important interaction between IL17A/IL17F polymorphisms and the stage of the disease as well as its treatment. Finally, patients with IL17F wild type genotype highlighted that there is a valid longer OS without all treatments and with radiotherapy and a neoadjuvant chemotherapy. In contrast, we observed that there are no relationships between IL17A, IL23R and the survival of these patients neither with nor without the treatment. Our results suggest that polymorphisms in IL17A and IL17F genes may be a predictive source of colorectal cancer therapy type. Therefore, IL17F may serve as an independent prognostic factor for overall survival in patients with colorectal cancer.

Consanguinity Protecting Effect Against Breast Cancer among Tunisian Women: Analysis of BRCA1 Haplotypes.

The purpose of this study is to assess the effect of consanguinity on breast cancer incidence in Tunisia. We conducted a case-control study to evaluate the involvement of heterozygote and homozygote haplotypes of BRCA1 gene SNPs according to consanguinity among 40 cases of familial breast cancer, 46 cases with sporadic breast cancer and 34 healthy controls. We showed significant difference in consanguinity rate between breast cancer patients versus healthy controls P = 0.001. Distribution of homozygous BRCA1 haplotypes among healthy women versus breast cancer patients was significantly different; p=0.02. Parental consanguinity seems to protect against breast cancer in the Tunisian population.

MicroRNAs expression in triple negative vs non triple negative breast cancer in Tunisia: interaction with clinical outcome.

INTRODUCTION: MicroRNAs are small, non coding regulatory molecules containing approximately 21 to 25 nucleotides. They function as controllers of expression at post transcriptional levels of most human protein-coding genes and play an essential role in cell signaling pathways. The objective of the present study is to evaluate the expression profile of the following micro-RNAs: miR-10b, miR-17, miR-21, miR-34a, miR-146a, miR-148a and miR-182, and to determine their possible interaction in triple-negative and non triple-negative primary breast cancers based on clinical outcome. METHODS: 60 triple-negative and non triple-negative breast cancer cases, along with their corresponding normal samples were investigated in relation to the expression of the seven studied miRNAs using qPCR Syber Green. RESULTS: We observed that miR-21, miR-146a and miR-182 were significantly over expressed in triple negative breast cancer. Moreover, miR-10b, miR-21 and miR-182 were significantly associated to lymph node metastases occurrence in triple negative breast carcinoma while only miR-10b was associated with grade III in non triple negative breast cancer cases. Almost all the analyzed microRNAs were strongly associated with patients' genico-obstetric history in non triple negative breast cancer cases except for miR-34a. All the studied microRNAs were strongly correlated with the use of the contraceptive pills in non triple negative breast cancer groups. The additive effect of hormonal factors in triple negative breast cancer cases showed an association with all the studied miRs except for miR-34 and miR-146a. CONCLUSION: The studied microRNAs are strongly influenced by environmental factors especially with hormonal patients' history. Moreover, miR-10b, miR-21 and miR-182 could be defined as biomarkers in breast cancer to predict both lymph node metastases and grade III occurrence.

Wild-type genotypes of BRCA1 gene SNPs combined with micro-RNA over-expression in mammary tissue leading to familial breast cancer with an increased risk of distant metastases' occurrence.

Germ line deleterious mutations of BRCA1 gene are not the unique factor that could inactivate BRCA1 protein which leads to familial breast cancer onset with distant metastases' occurrence. The present research explores the role that could be assigned to BRCA1 SNPs to inactivate BRCA1 protein and therefore to the occurrence of familial breast cancer with an increased risk of distant metastases' occurrence. The presence or the absence of BRCA1 protein was first analyzed by applying the immunohistochemistry technique to the tumors with sporadic and familial breast cancer. Then, a case-control study was conducted including 40 patients with familial breast cancer, 46 ones with sporadic breast cancer and 34 healthy controls based on the genotyping of nine BRCA1 SNPs (c.442.58delT, c.2082C>T, c.2311T>C, c.2612C>T, c.3113A>G, c.3119G>A, c.3548A>G, c.4308T>C and 4837A>G) via direct sequencing. Finally, the functional role that could be assigned to these SNPs was focused upon. miRbase site was used as a bioinformatics tool to predict potential micro-RNAs (miRs) targeting SNPs that are associated with familial breast cancer according to the results of this research. These predicted miRs were confirmed by Q-PCR analysis and correlated with BRCA1 protein expression among patients along with potential distant metastases. Clinical outcome showed that distant metastasis concerned 45 % of familial breast cancer patients and 19.5 % with sporadic breast cancer. Analysis of BRCA1 protein expression revealed a negative staining among 46.6 % of familial breast cancer patients and only 16.6 % within sporadic breast cancer ones. The association of four variants was identified within BRCA1 gene (c.442.58 delT, c.2311T>C, c.2612C>T and c.4308T>C) to familial breast cancer across their wild genotypes. miR-1179 was selected as potential miR that targets the region of BRCA1 mRNA containing the c.2311T>C variant within the TT genotype. The expression of miR-1179 was significantly associated with familial breast cancer patients without BRCA1 deleterious mutations compared to those with sporadic breast cancer according to TT genotype along with BRCA1 negative staining and according to the occurrence of distant metastases. Combination between TT genotype of c.2311T>C and miR-1179 over-expression could generate a lack of BRCA1 protein leading to a high risk of familial breast cancer with distant metastases.

MicroRNAs 146a and 147b biomarkers for colorectal tumor's localization.

The recently identified class of microRNAs (miRs) provided a new insight into cancer research, since abnormalities of members of microRNAs family have been found in various types of cancer. However, the relationship between five miRNAs (miR146a, miR155, miR21, miR135a, and miR147b) and colorectal cancer remains unclear. In the present study, we examined expression of these miRNAs in 25 pair-matched colon cancer tissues and normal colon mucosa. The expression levels of miR146a, miR155, miR21, miR135a, and miR147b were quantified by real-time PCR. We found that miR21, miR146a, and miR135a were all expressed at higher levels in colon tumors. On the other hand, miR146a and miR147b expressions are significantly higher in left colon compared to right colon. These two miRs, especially miR146a, seemed to be markers for the left colon tumors. Moreover, significant proportional and inverse correlations were found between miR expressions in tumor and healthy tissue, and the correlations profiles were different depending on cancer localization. Taken together, these results lead us to suggest the presence of different mechanisms regulating miRs expression and consequently their target genes in left and right colon. So the pathway of colorectal carcinogenesis would be different according to the site of the tumor.

3020insC NOD2/CARD15 polymorphism associated with treatment of colorectal cancer.

Chronic inflammation is closely linked to cancer. The risk of damage by colorectal cancer (CRC) may increase due to autoimmune disease and cryptogenic inflammation. Therefore, genetic factors implicated in the chronic irritation in inflammatory bowel disease such as NOD2/CARD15 may predispose to CRC. In this report, we shed the light on the possible contribution of the NOD2 3020insC variant to CRC risk in a series of 246 Tunisian subjects including 101 patients with CRC and 145 healthy controls. NOD2/CARD15 polymorphism was genotyped by sizing fluorescently labeled PCR products and automated sequencers. We analyzed the association between the molecular features at this gene in relation to tumor and patient characteristics and treatments. Through this qualitative analysis, we found that CRC patients with mutant allele of NOD2/CARD15 were suffering from Crohn's disease (CD) with canonic presentation. We also observed a positive association between 3020insC polymorphism and surgery and chemotherapy. We suppose that around 3% of colorectal cases which happen at an age older than 50 years are associated with the canonic form of CD along with the 3020insC mutation and that these patients are in need for chemotherapy.

Significant association between interleukin-17A polymorphism and colorectal cancer.

Interleukin (IL) 17A is an inflammatory cytokine expressed by Th 17 cells and plays a role in tissue inflammation by inducing release of proinflammatory and neutrophil-mobilizing cytokines. We have investigated the association between colorectal cancer and polymorphisms of IL17A (rs2275913. G197A). The study was performed in 241 subjects (102 with colorectal cancer and 139 healthy controls). Genotypes were determined by fluorescent-based restriction fragment length polymorphism method. The association between the molecular features at the gene in relation to tumor and patient clinical characteristics was analyzed. There was a significant difference between the genotype frequencies of IL17A G197A of control subjects (GG 68.34 % and GA + AA 31.65 %) and patients with colorectal cancer (GG 47.05 % and GA + AA 52.94 %) (p = 0.001 with odds ratio (OR) 2.45 (1.43-4.11)). IL17A G197A polymorphism is particularly associated with colon cancer. Indeed, the IL17A GG genotype could be considered as a protective factor against colon cancer (p = 0.00001) with OR 3.77 (2.04-6.99). We have noted a significant association of IL17A G197A polymorphism not only with tumor localization (p = 0.003) but also with tumor differentiation (p = 0.0005) in CRC patients. We have also showed a significant association of G197A variant with an increased risk of advanced stage (p = 0.005). Our result suggests that the A allele of IL17A gene is involved in susceptibility to colorectal cancer and is associated with clinical features as tumor location, tumor differentiation, and TNM stage. IL17A polymorphism may serve as biomarker of disease location and progression.

Significant association between IL23R and IL17F polymorphisms and clinical features of colorectal cancer.

Th17cells are involved in inflammatory and autoimmune diseases. These cells may be involved in pathological processes mainly producing pro-inflammatory cytokines. Recently, it was shown that the IL23/IL17 pathway plays an important role in the development of inflammatory bowel disease. In general, genes encoding cytokines are genetically polymorphic and polymorphisms in genes IL23R el IL17F were shown associated with susceptibility to Crohn's disease and ulcerative colitis which in their turn are considered as risk factors for developing colorectal cancer (CRC). Our approach is to study IL17F and IL23R polymorphisms as risk factor associated with CRC in the Tunisian population in patients and healthy controls. Interesting, we noted a significant association between IL17F and IL23R polymorphisms and tumor location (p=0.0001 and p=0.049, respectively), tumor histology (p=0.007 and p=0.049, respectively) and tumor architecture (p=0.0000000001 and p=0.07, respectively) in CRC patients. We also showed a significant association of IL17F variant with an increased risk of TNM stage III/IV (p=0.007), showing an increased risk of advanced stage. Finally, we observed a positive link between IL17F polymorphism and CRC patients with lymph nodes (p=0.0000000001) and metastasis (p=0.00000009). However, we found no evidence to support a significant association between IL17F and IL23R polymorphisms and colorectal cancer susceptibility. Our findings suggest that IL17F and IL23R polymorphisms were significantly associated with clinical features variables. The IL17F cytokine appear to be involved in the control of tumor growth and invasion of gastrointestinal tumors. IL17 and IL23 polymorphisms or those of their receptors as important determinants of susceptibility to colorectal cancer are still subject to questioning.

Positive link between variant Toll-like receptor 4 (Asp299Gly and Thr399Ile) and colorectal cancer patients with advanced stage and lymph node metastasis.

Toll-like receptors (TLRs) are considered as major endotoxin-signaling receptor and as crucial sensors of innate immunity. TLRs recognize pathogen-associated molecular patterns; induce effectors genes involving inflammatory cytokines and therefore initiation of adaptative immune responses against pathogens. Recently, it has been shown that TLRs are involved in tumor progression. In fact, increased level of TLR4 is associated with progression of colon malignancies. Even, TLR4 polymorphism has been shown associated with susceptibility to have colorectal cancer. Our study aimed to investigate an association between TLR4 Asp299Gly (D299G) and Thr399Ile (T399I) polymorphisms in Tunisian patients with colorectal cancer. Using a primer extension method (SNaPshot), we genotyped two variants of TLR4 D299G and T399I in 100 patients with colorectal cancer and 140 healthy controls in Tunisian population. Interesting, we noted a significant association between T399I polymorphism and tumor differentiation (p = 0.027) and tumor architecture (p = 0.02) in colorectal cancer (CRC) patients. We also showed a significant association of D299G with an increased risk of advanced stage (p = 0.03). Finally, we observed a positive link between D299G and T399I polymorphisms and CRC patients with lymph node (p = 0.00024; p = 0.0005, respectively) and metastasis (p = 0.001; p = 0.002, respectively). However, we found no evidence to support a significant association between TLR4 D299G and T399I polymorphisms and colorectal cancer susceptibility. Our findings suggest that TLR4 D299G and T399I polymorphisms are significantly associated with clinical features variables. TLR4 polymorphisms may serve as biomarker of disease progression. Therefore, our results need confirmation in even larger studies.

In vivo prevention of bladder urotoxicity: purified hydroxytyrosol ameliorates urotoxic effects of cyclophosphamide and buthionine sulfoximine in mice.

Urotoxicity is a troublesome complication associated with cyclophosphamide (CP) and L-buthionine-SR-sulfoximine (BSO) treatment in chemotherapy. With this concern in mind, the present study investigated the potential effects of a hydroxytyrosol extract from olive mill waste (OMW) on urotoxicity induced by acute CP and BSO doses using a Swiss albino mouse model. Toxicity modulation was evaluated by measuring lipid peroxidation (LPO) and antioxidants in urinary bladder. The findings revealed that the hydroxytyrosol extract exerted a protective effect not only on LPO but also on enzymatic antioxidants. When compared to the controls, the CP-treated animals underwent significant decreases in the glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GP), and catalase (CAT) activities. The level of glutathione (GSH) was also reduced with increased doses of LPO in the CP-treated animals. L-Buthionine-SR-sulfoximine treatment exerted an additive toxic effect on the CP-treated animals. Interestingly, pretreatment with the hydroxytyrosol extract restored the activities of all enzymes back to normal levels and exhibited an overall protective effect on the CP- and BSO-induced toxicities in urinary bladder. The restoration of GSH through the treatment with the hydroxytyrosol extract can play an important role in reversing CP-induced apoptosis and free radical-mediated LPO.