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BACKGROUND Breast cancer (BC) is one of the leading causes of cancer mortality in women. Glutathione S-transferase (GSTT1) is involved in activation of detoxification reactions and catalysis of chemicals conjugation with glutathione. GSTT1 genotype is a limiting factor for some environmental diseases. Epigenetic changes have an essential role in BC through inappropriate interaction between genomic and environmental risk factors. AIM This study was directed to explore the association of BC risk with GSTT1 genetic variations and its methylation status in Egyptian women. DESIGN AND METHODS This study included 100 healthy women as the control group and 100 patients were clinically and histologically diagnosed with breast cancer. All blood samples were used for genomic DNA extraction. GSTT1 genotyping was accomplished by multiplex PCR and methylation-specific PCR was used to analyze the GSTT1 promoter methylation status. RESULTS Breast cancer patients showed significant incidence of null GSTT1 in relation to controls (p = 0.004). GSTT1 gene promoter methylation status showed significant difference between hypermethylated and unmethylated patients when compared with healthy subjects (p = 0.005). GSTT1 promoter methylation status was not significantly associated with null genotype. There was no significant association between GSTT1-null genotypes and BC stage in cases with or without family history, but for promotor methylation, there was significant association with stage III and IV breast cancer disease. CONCLUSION GSTT1 null genetic variant and promoter hypermethylation in the GSTT region of the gene may be considered as critical risk factors for BC in Egyptian women.

It has become clear that the initiation and progression of carcinomas depend not only on alterations in epithelial cells, but also on changes in their microenvironment. To identify these changes, we have undertaken cellular and molecular characterization of carcinoma-associated fibroblasts (CAF) and their tumor counterpart fibroblasts (TCF) isolated from 12 breast cancer patients. Normal breast fibroblasts (NBF) from plastic surgery were used as normal control. We present evidence that both CAFs and TCFs are myofibroblasts and show tumor-associated features. Indeed, the p53/p21 response pathway to γ-rays was defective in 70% CAFs, whereas it was normal in all the TCF and NBF cells. In addition, the basal levels of the p53 and p21 proteins were significantly low in 83% of CAFs and modulated in the majority of TCFs compared with NBFs. Interestingly, both TCFs and CAFs expressed high levels of the cancer marker survivin and consequently exhibited high resistance to cisplatin and UV light. Moreover, most CAFs were positive for the proliferation marker Ki-67 and exhibited high proliferation rate compared with NBFs and TCFs. However, proliferating cell nuclear antigen was highly expressed in both CAFs and TCFs. Using the two-dimensional gel electrophoresis technique, we have also shown that CAF, TCF, and NBF cells present different proteome profiles, with many proteins differentially expressed between these cells. Taken together these results indicate that different genetic alterations can occur in breast CAFs and their corresponding adjacent counterparts, showing the important role that stroma could play in breast carcinogenesis and treatment. [Cancer Res 2008;68(8):2717–25]

Objective: A SNP in the NQO1 gene has been implicated in the response of patients with breast cancer to anthracycline containing regimens. NQO1, and its homologue NQO2, share many substrates yet retain distinct functional differences, with NQO2 being a more permissive molecule for electron accepting substrates. We aimed to determine whether functional NQO2 variants are associated with altered response to adjuvant doxorubicin and cyclophosphamide therapy, with or without tamoxifen, in the treatment of breast cancer. Methods: Genomic DNA samples from 227 women with early breast cancer were genotyped for NQO1 and NQO2 polymorphisms. All participants were treated with an AC adjuvant therapy regimen. The functional implications of NQO2 polymorphisms were validated in in-vitro ectopic expression models. Results: The NQO1 SNP (rs1800566) was associated with a poorer outcome and a lower likelihood of having a treatment delay. Patients who had ER and PR negative disease and were wild type for both the NQO1 and an NQO2 SNP (rs1143684) had 100% 5-year overall survival compared with 88% for carriers of one minor allele and 70% for carriers of two or more minor alleles (P=0.018, log rank). Carriers of minor alleles of a triallelic NQO2 promoter polymorphism were more likely to be withdrawn from tamoxifen therapy prematurely due to intolerance (P=0.009, log rank). MCF-7 cells were sensitized to growth inhibition by doxorubicin and 4OH tamoxifen, but not cyclophosphamide, by ectopic expression of NQO2. Conclusion: This study suggests that both NQO1 and NQO2 modulate the efficacy of AC therapy and that NQO2 is associated with tamoxifen toxicity. Refereed/Peer-reviewed

The purpose of this study is to understand the relative importance of the two chemokine receptors CXCR4 and CXCR7 in the response of estrogen-responsive and estrogen-nonresponsive breast cancer cells to the chemokine CXCL12. CXCR4 has long been accepted, whereas CXCR7 was identified more recently as a CXCL12 receptor. CXCL12 is expressed by stromal and epithelial cells in breast tumors and has been shown to be regulated by estrogen in estrogen-responsive breast cancer epithelial cells. CXCL12 activates the two seven transmembrane G protein-coupled receptors, CXCR4 and CXCR7, that transduce signals via heterotrimeric G-proteins. Methods The expression of CXCL12, CXCR4 and CXCR7 was measured in five estrogen-responsive and five estrogen-nonresponsive breast cancer cell lines by qRT-PCR and western transfer analysis. The ability of CXCL12 to activate the CXCR4 receptors was measured by western transfer analysis with a phospho-specific antibody. The motogenic responses of different breast cancer cell lines to CXCL12 were measured in modified Boyden chamber and in vitro wounding assays. The relative importance of CXCR4 in the CXCL12 response was probed with a specific inhibitor of CXCR4 receptor. Results The expression of CXCL12, CXCR4 and CXCR7 varied greatly between the ten breast cancer cell lines analyzed. CXCL12 expression was highest in estrogen-responsive T-47D cells whereas CXCR4 and CXCR7 expression was highest in estrogen-nonresponsive MDA-MB-231 cells. CXCL12 protein expression was not detected in protein extracts from breast cancer cells. CXCL12 was detected however after secretion into medium conditioned by T-47D and MCF-7 breast cancer cells. Chemotaxis assays showed that CXCL12 stimulated breast cancer cell migration in a concentration-dependent manner and cells that expressed higher concentrations of the receptors were more responsive to CXCL12. CXCR4 phosphorylation was stimulated by CXCL12 in some but not all breast cancer cells. AMD3100, a competitive inhibitor of CXCL12 binding to CXCR4, inhibited CXCL12-induced migration. Maximum inhibition of migration stimulated by 12.5 nM CXCL12 was achieved with 5 µM AMD3100. Conclusion This study shows that CXCL12 is an autocrine motogen in estrogen-responsive breast cancer cells. CXCL12 stimulates migration of both estrogen-responsive and estrogen-nonresponsive breast cancer cells. The sensitivity of the migratory response of breast cancer cells to CXCL12 depends on the relative concentrations of CXCR4 and CXCR7. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3175.