Downregulation Of BRCA1 Expression Research Articles

BRCA1 Gene Expression is Down Regulated in Both Familial and Sporadic Breast Cancer Cases in Baghdad- Iraq

Breast cancer is the commonest cancer and the leading cause of malignancies-related mortality in women worldwide. Understanding the underlying biology of the disease could improve patients’ stratification and may offer novel therapeutic targets and strategies. This study was set to investigate the association between BRCA1 gene expression and some of the clinical features of breast cancer patients in Baghdad-Iraq. Eighty peripheral blood samples were collected from sixty patients diagnosed with breast cancer and twenty healthy age-matched controls for BRCA1 qPCR gene expression analysis.
 The results showed a significant reduction in BRCA1 gene expression in all of the breast cancer patients with the vast majority of them (75%) having BRCA1expression below 25%. The down regulation of BRCA1 expression also showed consistency in breast cancer patients of both sporadic (n=45) and family history (n=15) cases,with expression averages of 18% and 20.19%, respectively. Moreover, the redcuation in BRCA1 expression was negatively associated with the disease’s grades,asbreast cancer patients with the advanced stage III (n=19) showed the lowest expression average of BRCA1 (13.8%) as compared to those in stages II (n=29) and I (n=12) of the disease (17.7% and 19.8%, respectively). 
 Overall, the study highlights the key role of BRCA1gene expression in the development of breast cancer and suggests its potential utility in the diagnosis strategies and preventing the progression of the disease, especially the sporadic type.

BRCA1 promoter hypermethylation and protein expression in ovarian carcinoma—an Indian study

Mounting evidences suggest that aberrant methylation of CpG islands is a major pathway leading to the inactivation of tumour suppressor genes and the development of cancer. The aim of the current study was to examine the prevalence of the promoter hypermethylation and protein expression of the BRCA1 gene in epithelial ovarian carcinoma (EOC) to understand the role of epigenetic silencing in ovarian carcinogenesis. We studied the promoter methylation of the BRCA1 gene by methylation-specific PCR in a cohort of 88 patients with EOC, 14 low malignant potential (LMP) tumours and 20 patients with benign tumours of the ovary. The expression of the BRCA1 protein by immunohistochemical analysis was carried out in a subset of 64 EOCs, 10 LMP tumours, 10 benign tumours and 5 normal ovarian tissues. The frequencies of methylation in EOCs and LMP tumours were 51.2 and 57%, respectively, significantly higher (p = 0.000 and p = 0.001) in comparison to benign tumours and normal ovarian tissue where no methylation was seen. Expression of BRCA1 was significantly lower in EOCs (p = 0.003). Lack of protein expression correlated with tumour grade and type. The methylation status correlated well with downregulation of BRCA1 expression. Our results clearly demonstrate that hypermethylation of BRCA1 promoter is a frequent event in ovarian cancer. These data support the hypothesis that BRCA1 promoter methylation plays an important role in the functional inactivation of BRCA1. Follow-up clinical data will reveal the impact of BRCA1 methylation on survival.

Abstract 3505: Energy restriction-mimetic agents (ERMAs) decrease BRCA1 expression levels in triple-negative breast cancer cells: A rationale for combining ERMAs with poly(ADP ribose) polymerase

Abstract Purpose: Triple-negative (ER-negative, PR-negative, HER2/neu-negative) breast cancer (TNBC) is a clinical challenge because of the lack of a specific therapeutic target. Poly (ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in the detection and repair of DNA damage. In DNA repair-defective tumors, inhibition of PARP can cause genomic instability and cell death. Among the PARP inhibitors currently being assessed in clinical trials, recent results showed that, in a randomized phase II clinical trial of TNBC patients, BSI-201 when added to platinum-containing chemotherapy significantly improved the outcome of metastatic breast cancer patients relative to chemotherapy alone. Previously, we reported that the thiazolidinedione family of peroxisome proliferation-activated receptor-γ agonist could induce apoptosis in cancer cells by eliciting cellular responses indicative of energy restriction. Consequently, we used ciglitazone as a scaffold to develop novel energy restriction-mimetic agents (ERMAs) through structural optimization, which has yielded two lead compounds, CG-5 and CG-12. In prostate cancer cells, ERMAs could induce cell death and reduce the expression of BRCA1 protein. BRCA1 helps repair DNA double-stranded breaks through homologous recombination. BRCA1 deficiency has been reported to sensitize cells to PARP inhibition through synthetic lethality. We hypothesize that ERMAs can potentiate the anti-proliferative activity of PARP inhibitors (AZD-2281, AG-014699, ABT-888 and BSI-201) through the down-regulation of BRCA1 expression in TNBC cells. Methods: TNBC cell lines, MDA-MB-468, MDA-MB-231 and Cal-51 (all of which have functional BRCA1) were obtained from ATCC and DSMZ and maintained in low-glucose complete growth medium. Drug effects on cell viability were assessed by MTT assays. The expression of BRCA1, phosphorylated AMP-activated kinase (AMPK), and PARP cleavage in treated cells were examined by western blotting. Results: The ERMAs CG-5 and CG-12 exhibited dose-dependent anti-proliferative activity against all TNBC cell lines tested with IC50 values of about 3 and 4 μM, respectively, after 72 h of treatment. These anti-proliferative effects were attributed to apoptosis as both compounds induced the cleavage of PARP. Moreover, AMPK was activated by both drugs indicative of energy restriction. BRCA1 levels were reduced dose-dependently in all three TNBC cell lines after treatment with CG-5 or CG-12. Conclusion: The ERMAs CG-5 and CG-12 can reduce BRCA1 levels in TNBC cells. These findings provide a rationale for combining the ERMA-mediated ablation of BRCA1 with PARP inhibitors to induce TNBC cell death through synthetic lethality. Further study to address this hypothesis could lead to a new therapeutic approach for TNBC patients for whom available options are limited. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2011-3505

Targeting the Akt/mTOR pathway in Brca1-deficient cancers.

The breast cancer susceptibility gene 1 (Brca1) has a key role in both hereditary and sporadic mammary tumorigenesis. However, the reasons why Brca1-deficiency leads to the development of cancer are not clearly understood. Activation of Akt kinase is one of the most common molecular alterations associated with human malignancy. Increased Akt kinase activity has been reported in most breast cancers. We previously found that downregulation of Brca1 expression or mutations of the Brca1 gene activate the Akt oncogenic pathway. To further investigate the role of Brca1/Akt in tumorigenesis, we analyzed Brca1/Akt expression in human breast cancer samples and found that reduced expression of Brca1 was highly correlated with increased phosphorylation of Akt. Consistent with the clinical data, knockdown of Akt1 by short-hairpin RNA inhibited cellular proliferation of Brca1 mutant cells. Importantly, depletion of Akt1 significantly reduced tumor formation induced by Brca1-deficiency in mice. The third generation inhibitor of mammalian target of rapamycin (mTOR), Palomid 529, significantly suppressed Brca1-deficient tumor growth in mice through inhibition of both Akt and mTOR signaling. Our results indicate that activation of Akt is involved in Brca1-deficiency mediated tumorigenesis and that the mTOR pathway can be used as a novel target for treatment of Brca1-deficient cancers.

Abstract 757: Identification and characterization of small molecule activators of BRCA1 expression

Abstract Breast cancer susceptibility gene 1 (BRCA1) has been intensely investigated in normal and cancerous cells and mutations in the BRCA1 gene have been found to account for half of the hereditary breast cancer cases. Although the role of BRCA1 in sporadic breast cancer is still uncertain, 30-40% of such cases show downregulation of BRCA1 expression. Because BRCA1 is a tumor suppressor gene, decreased expression of BRCA1 accelerates growth of mammary tumor cells, while increased expression leads to growth arrest and apoptosis. Furthermore, overexpression of BRCA1 in the murine mammary gland provides protection against mutagen-induced mammary neoplasia. In 2009, the American Cancer Society estimates that 254,650 new cases of invasive and in situ breast cancer will be diagnosed among women. Based on estimates of BRCA1 dysfunction in sporadic breast cancers, over 80,000 newly diagnosed malignancies would have decreased BRCA1 expression. Therefore, a novel prevention or therapeutic strategy targeting BRCA1 could have significant impact on incidence and/or survival. We developed an MCF-7 breast cancer cell line containing stably integrated copies of a BRCA1 promoter-driven firefly luciferase reporter plasmid and screened over 100,000 compounds for their ability to increase BRCA1-firefly luciferase expression. Six hundred and sixty two compounds (hits) increased BRCA1-firefly luciferase activity greater than 2 standard deviations above the control, DMSO. Secondary dose-response BRCA1-firefly luciferase assays confirmed 27 of the top 32 hits as true active compounds. Potency and efficacy of the active compounds were characterized quantitatively as effective concentration (EC50) and area under the curve (AUC), respectively. Cell viability assays determined that the active compounds had little cytotoxic effects. Non-cytotoxic compounds that increased BRCA1-firefly luciferase activity in a dose-dependent manner with a potency (EC50) and efficacy (AUC) greater than or equal to that of genistein, the positive control, were selected to move forward as lead compounds. Thus far, several lead compounds have been identified. Western blot analysis indicates lead compound 17 increases endogenous BRCA1 expression. Furthermore, DNA foci formation assays indicate compound 17 enhances the ability of BRCA1 to localize to sites of ionizing radiation-induced DNA damage. Because BRCA1 appears to play a role as a growth inhibitor and tumor suppressor in adult breast tissues, identification and characterization of a plausible active compound that enhances BRCA1 expression could potentially translate into a novel prevention or therapeutic option for patients afflicted with breast cancer. 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 757.

BRCA1 accumulates in the nucleus in response to hypoxia and TRAIL and enhances TRAIL‐induced apoptosis in breast cancer cells

A major contributing factor to the development of breast cancer is decreased functional expression of breast cancer susceptibility gene 1, BRCA1. Another key contributor to tumorigenesis is hypoxia. Here we show that hypoxia increased the nuclear localization of BRCA1 in MCF-7 and MDA-MB-468 human breast cancer cell lines without changing its steady-state expression level. Nuclear accumulation of BRCA1 was not evident in MCF-12A or HMEC (human mammary epithelial cell) nonmalignant mammary epithelial cells under the same conditions. Hypoxia also increased the cell surface expression of TRAIL on MDA-MB-468 cells. Neutralization of TRAIL precluded the hypoxia-induced accumulation of BRCA1 in the nucleus, whereas exogenously administered TRAIL mimicked the effect. Treatment of MDA-MB-468 cells with TRAIL resulted in a dose- and time-dependent increase in apoptosis. Furthermore, TRAIL-induced apoptosis in HCC1937 cells, which harbor a BRCA1 mutation, increased synergistically when wild-type BRCA1 was reconstituted in the cells, and downregulation of BRCA1 expression in MDA-MB-468 cells reduced the apoptotic response to TRAIL. These data provide a novel link between hypoxia, TRAIL and BRCA1, and suggest that this relationship may be especially relevant to the potential use of TRAIL as a chemotherapeutic agent.

Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells

Adipose tissue provides an important extragonadal source of estrogen. Obesity-associated elevation of estrogen production increases risk of breast cancer in postmenopausal women. Aromatase (CYP19), which converts androgen to estrogen, is a key enzyme in estrogen biosynthesis. In normal adipose tissue, transcription of the aromatase gene is initiated from a relatively weak adipose-specific promoter (I.4). However, in breast cancer, a switch of promoter utilization from I.4 to a strong ovary-specific promoter, PII, leads to increased aromatase expression and, hence, elevated estrogen production. Here, we report an intriguing relationship between the breast cancer susceptibility gene BRCA1 and aromatase expression in human adipose stromal cells (ASCs). Upon stimulation by phorbol ester or dexamethasone, increased aromatase expression in ASCs was accompanied by significant reduction of the BRCA1 level. In addition, adipogenesis-induced aromatase expression was also inversely correlated with BRCA1 abundance. Downregulation of BRCA1 expression in response to various stimuli was through distinct transcription or posttranscription mechanisms. Importantly, siRNA-mediated knockdown of BRCA1 led to specific activation of the breast cancer-associated PII promoter. Therefore, in addition to its well-characterized activities in breast epithelial cells, a role of BRCA1 in modulation of estrogen biosynthesis in ASCs may also contribute to its tissue-specific tumor suppressor function.

REDUCED EXPRESSION OF THE BRCA1 GENE AND INCREASED CHROMOSOMAL INSTABILITY IN MCF-7 CELL LINE

Using clonal cell cultures, a significant increase in chromosomal aberrations (aneuplolidy, dicentrics and chromatid breaks) were observed in MCF-7 cells compared with HeLa. BRCA1 expression was lower in MCF-7 cells than in HeLa cells. Since BRCA1 is known to play a role in the maintenance of chromosomal integrity, the increase in chromosomal aberrations in MCF-7 clones suggests that downregulation of BRCA1 expression could be one of the possible mechanisms for increased chromosomal instability in this cell line.