BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase alpha (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be over-expressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers.     

Risk-reducing salpingo-oophorectomy in BRCA1 and BRCA2 mutated patients: An evidence-based approach on what women should know

This review is focused on the ovarian cancer risk reduction management in BRCA mutation carriers and is intended to assist with clinical decision-making. Obviously, treatment decisions must be based on the available evidence. Despite risk-reducing salpingo-oophorectomy is firmly recommended, several separate questions can be raised to address the variety of intense controversy of this approach. A special emphasis lies in the effective preventive surgical measure against ovarian cancer risk, in an attempt to detect the optimal timing and mitigate the impact on patients. The long term implications of risk-reducing salpingo-oophorectomy as well as hormone replacement therapy are also actively debated. This is expected to represent an opportunity for improved management modelling of BRCA mutated patients.     

A common Greenlandic Inuit BRCA1 RING domain founder mutation

Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1 exon 3 nucleotide 234 T > G mutation, which has not previously been reported in the breast cancer information core (BIC) database. The mutation changes a conserved cysteine 39 to a glycine in the Zn²⁺ site II of the RING domain, which is essential for BRCA1 ubiquitin ligase activity. Eight of the families had members with ovarian cancer, suggesting that the RING domain may be an ovarian cancer hotspot. By SNP array analysis, we find that all 13 families share a 4.5 Mb genomic fragment containing the BRCA1 gene, showing that the mutation originates from a founder. Finally, analysis of 1152 Inuit, representing almost ~2% of the total Greenlandic Inuit population, showed that the frequency of the mutation was 1.0%. We conclude that the BRCA1 nucleotide 234 T > G is a common Greenlandic Inuit founder mutation. The relative high frequency in the general population, together with the ease of screening and possibility to reduce mortality in gene carriers, may warrant screening of the Greenlandic Inuit population. Provided screening is efficient, about 5% of breast- and 13% of ovarian cancers, respectively, may be prevented. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

BRCA1基因反义寡核苷酸对人骨肉瘤细胞的影响

 目的　探索抑癌基因BRCA1基因反义寡核苷酸对人骨肉瘤细胞SOSP 96 0 7的影响。方法　通过细胞培养 ,测量细胞生长曲线、计算细胞集落形成率、检测细胞周期等 ,观察BRCA1基因反义寡核苷酸对人骨肉瘤细胞系SOSP 96 0 7的影响。结果　BRCA1基因反义寡核苷酸浓度为 1 0 μmol/L时 ,骨肉瘤细胞的生长速度变快 ,集落形成率明显增高 ;流式细胞仪检测发现其引起细胞周期变化 ,细胞增殖活性提高。结论　BRCA1基因反义寡核苷酸对人骨肉瘤细胞SOSP 96 0 7有明显的影响 ,使得骨肉瘤细胞恶性生物学特性更加显著 ,提示BRCA1基因在人骨肉瘤发展过程中起作用     

A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining

Heterozygosity for mutations in the BRCA1 gene in humans confers high risk for developing breast cancer, but a biochemical basis for this phenotype has not yet been determined. Evidence has accumulated implicating BRCA1, in the maintenance of genomic integrity and the protection of cells against DNA double strand breaks (DSB). Here we present evidence that human cells heterozygous for BRCA1 mutations exhibit impaired DNA end-joining, which is the major DSB repair pathway in mammalian somatic cells. Using an in vivo host cell end-joining assay, we observed that the fidelity of DNA end-joining is strongly reduced in three BRCA1(+/-) cell lines in comparison to two control cell lines. Moreover, cell-free BRCA1(+/-) extracts are unable to promote accurate DNA end-joining in an in vitro reaction. The steady-state level of the wild type BRCA1 protein was significantly lower than the 50% expected in BRCA1(+/-) cells and thus may underlie the observed end-joining defect. Together, these data strongly suggest that BRCA1 is necessary for faithful rejoining of broken DNA ends and that a single mutated BRCA1 allele is sufficient to impair this process. This defect will compromise genomic stability in BRCA1 germ-line mutation carriers, triggering the genetic changes necessary for the initiation of neoplastic transformation.     

Identification of a gamma-tubulin-binding domain in BRCA1.

We previously reported (L-C. Hsu and R. L. White, Proc. Natl. Acad. Sci. USA, 95: 12983-12988, 1998) that hypophosphorylated BRCA1 is associated with mitotic centrosomes in vivo, perhaps through its interaction with gamma-tubulin. In vitro evidence presented here indicates that full-length BRCA1 protein generated by in vitro translation interacts with gamma-tubulin. A specific domain of BRCA1 protein, BRCA1 fragment no. 3 (BF3; amino acids 504-803), is both necessary and sufficient to bind gamma-tubulin. BF3 and gamma-tubulin coimmunoprecipitated when coexpressed in cells. In addition, expression of BF3 interfered with the interaction between BRCA1 and gamma-tubulin. Stable transformants of COS-7 cells that overexpressed BF3 showed a reduced growth rate partly because of increased apoptosis. Furthermore, overexpression of BF3 in COS-7 cells results in the accumulation of mitotic cells with multiple centrosomes and abnormal spindles. Okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, induces hyperphosphorylation of BRCA1, a reduction of both BRCA1 and gamma-tubulin associated with mitotic centrosomes, and an accumulation of abnormal spindle formation. Thus, attenuating the interaction between BRCA1 and gamma-tubulin, and their association with mitotic centrosomes, may induce an increase of aneuploid cell population and contribute to tumorigenesis.     

Insights into the Molecular Basis of Human Hereditary Breast Cancer from Studies of the BRCA1 BRCT Domain

The C-terminal, BRCT repeats of BRCA1 are essential for the tumor suppressor function of this protein. Here we review structural and functional studies of this domain. Both repeats adopt similar folds and pack in an intimate, head-to-tail manner. The domain binds phosphorylated targets such as the DNA damage-associated kinase BACH1, with a specificity for pSer-X-X-Phe motifs. Structural studies reveal that the N-terminal repeat is responsible for pSer binding while a groove at the interface of the two repeats recognizes the Phe. Missense variants identified in breast cancer screening programs often disrupt these interactions and these molecular defects may lead to an increased cancer risk.     

Conserved BRCT regions of TopBP1 and of the tumor suppressor BRCA1 bind strand breaks and termini of DNA.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is a ubiquitous region homologous to regions in DNA repair enzymes and cell cycle regulators. We showed that the BRCT regions bound DNA fragments, using the TopBP1 protein (topoisomerase II binding protein 1), with eight BRCTs as a model protein. The bindings were independent of DNA sequences, forms of DNA termini and energy. The BRCT-DNA complex showed resistance to an exonuclease, indicating that BRCT bound DNA breaks. The BRCTs also bound DNA nicks, suggesting that BRCTs play an important role in detection of both single- and double-strand DNA breaks or ends. On the other hand, BRCTs did not bind circular intact DNA. BRCTs of BRCA1 also bound DNA termini. Since some BRCTs are unique general elements in some tumor suppressions, these findings will reveal novel aspects of the tumor suppression mechanism.     

A recombination-based method to characterize human BRCA1 missense variants

Many missense variants in BRCA1 are of unclear clinical significance. Functional and genetic approaches have been proposed for elucidating the clinical significance of such variants. The purpose of this study was to evaluate BRCA1 missense variants for their effect on both homologous recombination (HR) and non homologous end joining (NHEJ). HR frequency evaluation: HeLaG1 cells, containing a stably integrated plasmid that allows us to measure HR events by gene conversion events, were transfected with the pcDNA3β expression vector containing the BRCA1-wild-type (BRCA1 wild type) or the BRCA1-unclassified variants (BRCA1-UCVs). The NHEJ was measured by a random plasmid integration assay. The assays suggested a BRCA1 involvement mainly in the NHEJ. As a matter of fact, the Y179C and the A1789T variant significantly altered the NHEJ activity as compared to the wild type, suggesting that they may be related to BRCA1-associated pathogenicity by affecting this function. The variants N550H and I1766S, and the mutation M1775R did not alter the NHEJ frequency. These data, besides proposing a method for the study of BRCA1 variants' effect on HR and NHEJ, highlighted the need for a range of functional assays to be performed to identify variants with altered function.     

A multicenter study of supportive-expressive group therapy for women with BRCA1/BRCA2 mutations

BACKGROUND: Women with a BRCA1/BRCA2 mutation experience significant challenges. These include decision-making regarding surgical options and notification to offspring and family, along with a sense of isolation, which may lead to psychological and emotional distress. The current study developed, standardized, and conducted preliminary testing of a supportive-expressive group therapy intervention designed to address these challenges. METHODS: Seventy women with a BRCA1/BRCA2 mutation recruited from familial cancer risk clinics participated in 12 sessions of supportive-expressive group therapy that lasted 6 months. Before and after measures of psychosocial functioning, knowledge, and surveillance/surgery activities were completed. RESULTS: Sixty-seven women completed the intervention. Significant improvements were observed in psychosocial functioning: cancer worries (P = 0.005), anxiety (P = 0.033), and depression (P = 0.015). Knowledge level and surveillance levels were high at baseline and there were no significant changes postintervention. A significant number of women made decisions concerning prophylactic surgery (oophorectomy/mastectomy) during and after the intervention. CONCLUSIONS: The feasibility of a supportive-expressive group for BRCA1/BRCA2 mutation carriers was demonstrated. Findings from the study are consistent with an effective intervention. However, further research is required using a randomized controlled study design.     

Emotional impact on the results of BRCA1 and BRCA2 genetic test: an observational retrospective study

Background

BRCA1 and BRCA2 mutations are associated with a higher risk of breast and ovarian tumors. This study evaluated the emotional states of women 1 month after having received the results of the genetic test and assessed eventual associations with the type of outcome, personal/familiar disease history and major socio-demographic variables.

Methods

The study, an observational retrospective one, involved 91 women, evaluated 1 month after receiving their results. Patients were administered the Hospital Anxiety and Depression Scale, the Profile of Mood States and emotional Thermometers.

Results

Anxiety was significantly higher than depression (p < 0.001), and 21.3% and 21.3% of the sample were, respectively, possible and probable cases for anxiety, whereas 13.5% and 10.1% were possible and probable cases for depression. Within the six mood states, Confusion-Bewilderment (M = 48.5) was the lowest, whereas Fatigue-Inertia (M = 52.3) was the highest. Differences were recorded within the ten assessed emotions too. Being a proband/nonproband and being or not a cancer patient were associated with many tested variables.

Conclusion

The psycho-emotional screening of women undertaking genetic counseling is relevant and should cover a large range of dimensions.

     

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20-45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC(50)) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC(50) : 30-40 microM) compared with MCF-7 (IC(50) : 60-70 microM) and MDA-MB231 (IC(50) : 90-100 microM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC(50) : 45-50 microM) compared with MCF-7 (IC(50) : 1-5 microM) and MDA-MB231 (IC(50) : 5-10 microM) (P<0.02), as well as to paclitaxel (Tax) (IC(50) : >2 microM for HCC1937, 0.1-0.2 microM for MCF-7 and 0.01-0.02 microM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/(WT)BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.     

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/- and BRCA2+/- cells raises the question of the response of heterozygous women to radiation.     

Histopathological characteristics of BRCA1- and BRCA2-associated intraperitoneal cancer: a clinic-based study

The aim of the research was to assess possible histopathological differences between BRCA1- and BRCA2-associated malignant intraperitoneal (ovarian/fallopian tube/peritoneal) tumors and their sporadic counterparts. Dutch families harboring pathogenic BRCA1 or BRCA2 mutations were selected. Included were patients who had had malignant primary ovarian, fallopian tube or peritoneal tumors. Histopathological data was compared with data obtained from the Dutch cancer registry between 1989 and 1993 (reference group). A total of 63 with primary intraperitoneal malignant tumors were identified in 41 families. Non-epithelial malignant tumors were not observed in the study group versus 6% (n = 404) in the reference group (n = 6,789, P = 0.04). These tumors were excluded from further analysis, as were ovarian adenocarcinomas not otherwise specified, since these were detected in 22% of the study group, and in 19% of the reference group (P = 0.76). Serous carcinomas were detected in 94% (47/50) of the women in the study group in contrast to 62% (3,145/5,088) of the reference group (P < 0.01). In the study group, mucinous and endometrioid ovarian adenocarcinomas and serous ovarian borderline tumors each comprised 2.0% of the tumors. Clear cell ovarian carcinomas were not detected. In contrast, these percentages were 16% (P < 0.01), 10% (P = 0.07), 7% (P = 0.16) and 5% (P = 0.12), respectively, in the reference group. In the study group, 6.0% of the carcinomas arose in the fallopian tube versus 1.9% in the reference group (P = 0.03). Four percent of the study group developed primary serous peritoneal carcinomas, versus six percent in the reference group (P = 0.57). Serous carcinoma is the predominant type of intraperitoneal malignancy occurring in women harboring BRCA1 or BRCA2 mutations. Non-epithelial cancer does not seem to be part of the tumor spectrum of BRCA mutation carriers. This suggests, therefore, that serous tumors may be the only subtype related to a BRCA1 or BRCA2 mutation. Furthermore, fallopian tube carcinoma occurred more often in BRCA mutation carriers than in the reference population. This revised version was published online in August 2006 with corrections to the Cover Date.     

A population-based study of low-grade gliomas and mutated isocitrate dehydrogenase 1 (IDH1)

Low-grade gliomas (LGG) have a slow growth rate, but transformations into malignant gliomas with a rapid deterioration occur in many patients. The aim of this study was to evaluate clinical prognostic factors in a population-based cohort of patients with LGG. In addition we investigated the expression and prognostic value of the isocitrate dehydrogenase 1 (IDH1) R132H mutation. Seventy-four patients diagnosed between 2005 and 2009 in the Region of Southern Denmark were identified using the Danish Cancer Register and The Danish Pathology Databank. Survival analysis using Cox regression was performed in 52 patients with tumor samples useable for immunohistochemical evaluation of IDH1 status. Patients with a contrast enhancing tumor, neurological deficits, headache, an astrocytic tumor and PS 2–4 had an increased risk of recurrence. In univariate analysis age > 50 years (HR 2.14, 95 % CI 1.08–4.24), having neurological deficit (HR 2.28, 95 % CI 1.15–4.52), receiving post-surgical treatment (HR 2.52, 95 % CI 1.19–5.32), being in performance status 2–4 (HR 1.44, 95 % CI 1.15–1.81), and having an astrocytic tumor (HR 3.79, 95 % CI 1.64–8.73) were associated with poor survival. Mutated IDH1 (mIDH1) was identified in 46 % of the patients and was significantly correlated to a good survival in both univariate (HR 0.24, 95 % CI 0.11–0.53) and in multivariate analysis (HR 0.40, 95 % CI 0.17–0.91). The other clinical variables were not significant when adjusted for the effect of mIDH1 status. We find that young age, the absence of neurologic deficit, PS 0–1 and oligodendroglial histology were associated with better survival. IDH1 status showed independent prognostic information when adjusting for classical prognostic factors, and should be validated in a larger patient population.