Zbigniew Morawiec

Polymorphisms of the promoter regions of matrix metalloproteinases genes MMP-1 and MMP-9 in breast cancer

SummaryPurposeMatrix metalloproteinases play a crucial role in the cancer invasion and metastasis, angiogenesis and tumorigenicity. A single guanine insertion – the 1G/2G polymorphism in the promoter of the matrix metalloproteinase 1 (MMP-1) gene creates a binding site for the transcription factor AP-1 and thus may affect the transcription level of MMP-1. The C→T substitution at the polymorphic site of the MMP-9 gene promoter results in a higher transcription activity of the T-allelic promoter trough the loss of binding site for a repressor protein. The aim of this work was to investigate the influence of 1G/2G and C→T polymorphisms on the MMP-1 and MMP-9 level and therefore on the occurrence and progression of breast cancer.Experimental designWe investigated the distribution of genotypes and frequency of alleles of the 1G/2G and C→T polymorphisms for 270 patients with breast cancer and 300 healthy women served as control. The genotypes were determined by RFLP-PCR. Additionally, we estimated the level of MMP-1 and MMP-9 antigens in tumor samples and normal breast tissue using ELISA.ResultsThe levels of MMP-1 in tumor samples of node positive patients ware significantly higher than in samples of node negative patients (p<0.05). Increased level of MMP-9 correlates with Bloom-Richardson grading III (p<0.05), increased tumor size (p<0.05) and absence of estrogen and progesterone receptors (p<0.01). Additionally, both MMP-1 and MMP-9 levels were higher in tumor than in the normal breast tissue. We showed the higher risk of metastasis development in lymph node for the 2G/2G genotype (OR=2.14; CI 95% 1.24;3.69) and the 2G allele carriers (OR=1.68; CI 95% 1.19;2.39). We found correlation between the T allele (OR=2.61; CI 95% 1.33;4.87), 2G (OR=2.58; CI 95% 1.35;4.91) and malignance.ConclusionThe results suggest that MMP-1 is responsible for the local invasion and MMP-9 is associated with the malignance and the growth of the tumor. We suggest that the 2G allele of the 1G/2G MMP-1 gene polymorphism may be associated with the lymph node metastasis in patients with breast cancer and therefore it can be considered as a progression marker in this disease.

Association between DNA damage, DNA repair genes variability and clinical characteristics in breast cancer patients.

The cells susceptibility to DNA damage and its ability to repair this damage are important for cancer induction, promotion and progression. In the present work we determined the level of basal (total endogenous) and endogenous oxidative DNA damage as well as polymorphism of the DNA repair genes: RAD51 (135 G/C), XRCC3 (Thr241Met), OGG1 (Ser326Cys) and XPD (Lys751Gln) in peripheral blood lymphocytes of 41 breast cancer patients and 48 healthy individuals. DNA damage was evaluated by alkaline comet assay with DNA repair enzymes: Endo III and Fpg, preferentially recognizing oxidized DNA bases. The genotypes of the polymorphisms were determined by restriction fragment length polymorphism PCR. We observed a strong association between breast cancer occurrence and the genotypes C/C of the RAD51-135G/C polymorphism, Ser/Ser of the OGG1-Ser326Cys and Lys/Gln of the XPD-Lys751Gln, whereas the genotypes G/C of the RAD51-135G/C and Lys/Lys of the XPD-Lys751Gln exerted a protective effect against breast cancer. We also found that individuals with the G/C genotype of the RAD51-135G/C polymorphism and with the Lys/Lys genotype of the XPD-Lys751Gln polymorphism displayed a lower extent of basal and oxidative DNA damage. A strong association between higher level of oxidative DNA damage and the Lys/Gln genotype of the latter polymorphism was found. We also correlated genotypes with clinical characteristics of breast cancer patients. We observed a strong association between the G/C genotype of the RAD51-135 G/C polymorphism and the expression of the progesterone receptor and between both alleles of the OGG1-Ser326Cys polymorphism and lymph node metastasis. Our results suggest that the polymorphism of the RAD51, OGG1 and XPD genes may be linked with breast cancer by the modulation of the cellular response to oxidative stress and these polymorphisms may be considered as markers in breast cancer along with the genetic or/and environmental indicators of oxidative stress.

Polymorphism of the homologous recombination repair genes RAD51 and XRCC3 in breast cancer.

The RAD51 protein and its paralog, XRCC3, play an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Since DSBs may contribute to the pathogenesis of breast cancer and variability in DNA repair genes may be linked with some cancers, we performed a case-control study (135 cases and 175 controls) to check the association between the genotypes of the Thr241Met polymorphism of the XRCC3 gene and the 135G>C polymorphism of the RAD51 gene and breast cancer occurrence and progression. Genotypes were determined in peripheral blood lymphocytes by RFLP-PCR. We did not find any association between either polymorphism singly and breast cancer occurrence. Both polymorphisms were not related to tumor size, estrogen and progesterone receptors status, cancer type and grade. However, the Thr241Met genotype of the XRCC3 polymorphism slightly increased the risk of local metastasis in breast cancer patients (OR 2.56, 95% CI 1.27-5.17). The combined Thr241Met/135G>C genotype decreased the risk of breast cancer occurrence (OR 0.22, 95% CI 0.08-0.59). Our results suggest that the variability of the DNA homologous recombination repair genes RAD51 and XRCC3 may play a role in breast cancer occurrence and progression, but this role may be underlined by a mutual interaction between these genes.

Polymorphisms of the BRCA2 and RAD51 genes in breast cancer.

SummaryWe performed a case-control study (150 cases and 150 controls) to test the association between three polymorphisms in BRCA2 and RAD51 genes and breast cancer risk. Genotypes were determined in DNA from blood cells by PCR–RFLP. Cancer occurrence was strongly associated with the BRCA2 Met/1915Thr homozygous polymorphic variants, whereas heterozygous variant was associated with significant reduction in breast cancer risk. Gene-gene interaction between the BRCA2-Met1915Thr Thr/Thr and BRCA2-Met784Val Met/Met homozygous variants increased the risk. Therefore, the Met1915Thr polymorphism in the BRCA2 gene may be considered as an independent marker of breast cancer.

Polymorphisms of the DNA Mismatch Repair Gene HMSH2 in Breast Cancer Occurence and Progression

SummaryThe response of the cell to DNA damage and its ability to maintain genomic stability by DNA repair are crucial in preventing cancer initiation and progression. Therefore, polymorphism of DNA repair genes may affect the process of carcinogenesis. The importance of genetic variability of the components of mismatch repair (MMR) genes is well documented in colorectal cancer, but little is known about its role in breast cancer. hMSH2 is one of the crucial proteins of MMR. We performed a case-control study to test the association between two polymorphisms in the hMSH2 gene: an A → G transition at 127 position producing an Asn → Ser substitution at codon 127 (the Asn127Ser polymorphism) and a G → A transition at 1032 position resulting in a Gly → Asp change at codon 322 (the Gly322Asp polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 age-matched women (controls) by restriction fragment length polymorphism and allele-specific PCR. We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. A strong association between breast cancer occurrence and the Gly/Gly phenotype of the Gly322Asp polymorphism (odds ratio 8.39; 95% confidence interval 1.44–48.8) was found. Therefore, MMR may play a role in the breast carcinogenesis and the Gly322Asp polymorphism of the hMSH2 gene may be considered as a potential marker in breast cancer.

STI571 reduces NER activity in BCR/ABL-expressing cells

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.

Efficacy of DNA double-strand breaks repair in breast cancer is decreased in carriers of the variant allele of the UBC9 gene c.73G>A polymorphism

UBC9 (E2) SUMO conjugating enzyme plays an important role in the maintenance of genome stability and integrity. In the present work we examined the association between the c.73G>A (Val25Met) polymorphism of the UBC9 gene (rs11553473) and efficacy of DNA double-strand breaks (DSBs) repair (DRE) in breast cancer patients. We determined the level of endogenous (basal) and exogenous (induced by γ-irradiation) DSBs and efficacy of their repair in peripheral blood lymphocytes of 57 breast cancer patients and 70 healthy individuals. DNA damage and repair were studied by neutral comet assay. Genotypes were determined in DNA from peripheral blood lymphocytes by allele-specific PCR (ASO-PCR). We also correlated genotypes with the clinical characteristics of breast cancer patients. We observed a strong association between breast cancer occurrence and the variant allele carried genotypes in patients with elevated level of basal as well as induced DNA damage (OR 6.74, 95% CI 2.27-20.0 and OR 5.33, 95% CI 1.81-15.7, respectively). We also found statistically significant (p<0.05) difference in DRE related to the c.73G>A polymorphism of the UBC9 gene in breast cancer patients. Carriers of variant allele have decreased DNA DRE as compared to wild type genotype carriers. We did not find any association with the UBC9 gene polymorphism and estrogen and progesterone receptor status. The variant allele of the UBC9 gene polymorphism was strongly inversely related to HER negative breast cancer patients (OR 0.03, 95% CI 0.00-0.23). Our results suggest that the c.73G>A polymorphism of the UBC9 gene may affect DNA DSBs repair efficacy in breast cancer patients.

Lipid peroxidation and glutathione peroxidase activity relationship in breast cancer depends on functional polymorphism of GPX1

BackgroundSince targeting oxidative stress markers has been recently recognized as a novel therapeutic target in cancer, it is interesting to investigate whether genetic susceptibility may modify oxidative stress response in cancer. The aim of this study was to elucidate whether genetic polymorphism in the antioxidant enzymes is associated with lipid peroxidation in breast cancer.MethodsWe conducted a study among Polish women, including 136 breast cancer cases and 183 healthy controls. The analysis included genetic polymorphisms in five redox related genes: GPX1 (rs1050450), GPX4 (rs713041), SOD2 (rs4880), SEPP1 (rs3877899) and SEP15 (rs5859), lipid peroxidation, the activities of antioxidant enzymes determined in blood compartments as well as plasma concentration of selenium – an antioxidant trace element involved in cancer. Genotyping was performed using the Real Time PCR. Lipid peroxidation was expressed as plasma concentration of thiobarbituric acid reactive substances (TBARS) and measured with the spectrofluorometric method. Glutathione peroxidase activity was spectrophotometrically determined in erythrocytes (GPx1) and plasma (GPx3) by the use of Paglia and Valentine method. Spectrophotometric methods were employed to measure activity of cytosolic superoxide dismutase (SOD1) in erythrocytes (Beauchamp and Fridovich method) and ceruloplasmin (Cp) in plasma (Sunderman and Nomoto method). Plasma selenium concentration was determined using graphite furnace atomic absorption spectrophotometry.ResultsBreast cancer risk was significantly associated with GPX1 rs1050450 (Pro198Leu) polymorphism, showing a protective effect of variant (Leu) allele. As compared to the control subjects, lipid peroxidation and GPx1 activity were significantly higher in the breast cancer cases, whereas ceruloplasmin activity was decreased. After genotype stratification, both GPx1 activity and TBARS concentration were the highest in GPX1 Pro/Pro homozygotes affected by breast cancer. At the same time, there was a significant correlation between the level of lipid peroxidation and GPx1 activity among the cancer subjects possessing GPX1 Pro/Pro genotype (r = 0.3043; p = 0.0089), whereas such a correlation was completely absent in the cases carrying at least one GPX1 Leu allele as well as in the controls (regardless of GPX1 genotype).ConclusionsGPX1 polymorphism may be an important factor modifying oxidative stress response in breast cancer subjects. Further studies are needed to elucidate its potential clinical significance.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey s HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

BLM and RAD51 Genes Polymorphism and Susceptibility to Breast Cancer

DNA repair by homologous recombination is one of the main processes of DNA double strand breaks repair. In the present work we performed a case-control study (304 cases and 319 controls) to check an association between the genotypes of the c.-61 G>T and the g.38922 C>G polymorphisms of the RAD51 gene and the g.96267 A>C and the g.85394 A>G polymorphisms of the BLM gene and breast cancer occurrence. Genotypes were determined in DNA from peripheral blood by PCR-RLFP and by PCR-CTPP. We observed an association between breast cancer occurrence and the T/G genotype (OR 4.41) of the c.-61 G>T-RAD51 polymorphism, the A/A genotype (OR 1.69) of the g.85394 A>G-BLM polymorphism and the A/A genotype (OR 2.49) of the g.96267 A>C-BLM polymorphism. Moreover, we demonstrated a correlation between intra- and intergenes genotypes combinations and breast cancer occurrence. We found a correlation between progesterone receptor expression and the T/G genotype (OR 0.57) of the c.-61 G>T- RAD51 polymorphism. We also found a correlation between the T/G genotype (OR 1.86) and the T/T genotype (OR 0.56) of the c.-61 G>T- RAD51 polymorphism and the lymph node metastasis. We showed an association between the A/A genotype (OR 2.45) and the A/C genotype (OR 0.41) of the g.96267 A>C-BLM polymorphism and G3 grade of tumor. Our results suggest that the variability of the RAD51 and BLM genes may play a role in breast cancer occurrence. This role may be underlined by a common interaction between these genes.