Breast Cancer BRCA2 Research Articles

MDC1 and RNF8 function in a pathway that directs BRCA1-dependent localization of PALB2 required for homologous recombination

The PALB2 protein is associated with breast cancer susceptibility and Fanconi anemia. Notably, PALB2 is also required for DNA repair by homologous recombination (HR). However, the mechanisms that regulate PALB2, and the functional significance of its interaction with the BRCA1 breast cancer susceptibility protein, are poorly understood. Here, to better understand these processes, we fused PALB2, or the PALB2(L21P) mutant which cannot bind to BRCA1, with the BRCT repeats that are present in, and which localize, BRCA1. Our results yield important insights into the regulation of PALB2 function. Both fusion proteins can bypass BRCA1 to localize to sites of DNA damage. Further, the localized fusion proteins are functional, as determined by their ability to support the assembly of RAD51 foci, even in the absence of the capacity of PALB2 to bind BRCA1. Strikingly, the localized fusion proteins mediate DNA double-strand break (DSB)-initiated HR and resistance to mitomycin C in PALB2-deficient cells. Additionally, we show that the BRCA1-PALB2 heterodimer, rather than the PALB2-PALB2 homodimer, mediates these responses. Importantly, we offer the first insight into how BRCA1-dependent recruitment of PALB2 is integrated with other DNA damage signaling pathways. We find that PALB2 localization depends on the presence of MDC1, RNF8, RAP80 and Abraxas upstream of BRCA1. Thus, PALB2 may link HR to a key ubiquitin-related signaling pathway that responds to DSBs.

Why choose the treatment with cisplatin for BRCA1 breast cancers patients?

ted with cisplatin 75 mg/m 2 every three weeks for four cycles, followed by mastectomy and conventional chemotherapy. Eight patients had prior chemotherapy for a previous cancer diagnosis. Three patients received prior chemotherapy for their current cancer diagnosis but received protocol therapy and thus were followed for toxicity and evaluated for response. Information was collected on clinical stage, grade, hormone receptor status and Her2neu (HER2) status prior to treatment. Pathologic complete response was determined by review of surgical specimens. Results 47 patients were enrolled in the study, including 39 patients for whom this was the first primary breast cancer and who had no previous chemotherapy and 8 patients who had previously received chemotherapy. A pathologic complete response (pCR) was observed in 34 patients (68%) including 82% of women who had no previous chemotherapy and 18% of women who had previously received chemotherapy. Conclusions Platinum-based chemotherapy is effective in a high proportion of patients with BRCA1-associated breast cancer, but may be less effective in women who have been previously been treated with another form of chemotherapy.

An ultrasensitive electrochemical impedance sensor for a special BRCA1 breast cancer gene sequence based on lambda exonuclease assisted target recycling amplification.

A label-free, target recycling electrochemical impedance spectroscopy (EIS) DNA sensor has been developed for detection of a model related to the BRCA1 breast cancer gene with a detection limit of 0.05 nM.

P3-06-03: Hypodiploidy, 1pter Loss and Inactive X Chromosome Retention Are Associated with BRCA1 Somatic or Germline Inactivation in Basal-Like Breast Carcinomas: Proposal for a New BRCAness Genomic Signature.

Abstract Basal-Like Carcinomas (BLCs) are high grade ductal carcinomas identified by large scale transcriptomic analyses among carcinomas with triple-negative phenotype (ER- PR- HER2−). A particular relationship between this phenotype and the BRCA1 breast cancer susceptibility gene has been described based on several observations including high genomic similarities of sporadic and BRCA1 BLCs. BRCA1 mutated patients have been shown to strongly benefit from treatments using PARP inhibitors. However, significant benefit of this treatment was not demonstrated in sporadic triple-negative breast cancers in association to standard chemotherapy in phase III clinical trials, which emphasizes the necessity for a better selection of DNA repair deficient tumors. The goal of this study was to determine whether BRCA1 inactivation leads to particular genomic alterations that could be used to identify BRCA1 deficient sporadic BLCs. Genomic profiling was performed using SNP-arrays (Affymetrix and Illumina) and high quality profiles were obtained for 60 BLCs with more than 35% of tumor cells. The series contained 28 BRCA1 BLCs and 32 sporadic BLCs consisting in 10 sporadic BLCs with BRCA1 somatic inactivation by promoter methylation (BRCA1-like BLCs) and 22 sporadic tumors without BRCA1 methylation (nonBRCA1 BLCs). Genomic data was mined with GAP methodology (Popova et al, Genome Biol 2009), which allows absolute copy-number evaluation with DNA content calculation and clarification of Loss Of Heterozygosity (LOH) status. Genomic patterns of BLCs were characterized by frequent and highly recurrent allelic losses. Comparison between BRCA1, BRCA1-like and nonBRCA1 BLCs confirmed their overall similarity, and identified few significant differences enhanced when BRCA1 and BRCA1-like BLCs were considered as one group. Firstly, hypodiploidy characterized BLCs with BRCA1 germline and/or somatic inactivation. Secondly, the lpter region was found more often lost in BRCA1 and BRCA1-like BLCs than in nonBRCA1 BLCs. Thirdly, retention of inactive X chromosome was a characteristic of BLCs with BRCA1 germline and/or somatic inactivation. Finally, a BRCA1ness score based on the above described difference is proposed. In conclusion, despite a strong similarity of genomic patterns in BLCs, BRCA1 inactivation leads to few key genomic aberrations which constitute a new BRCA1 molecular signature namely hypodiploidy, 1pter loss and inactive X chromosome retention. This signature needs to be further evaluated in terms of response to PARP inhibitors in order to evaluate its capacity to identify patients that will benefit from this treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-03.

P2-13-02: Parent of Origin of BRCA Mutation May Determine Age at Breast Cancer Diagnosis.

Abstract Background: Genetic diseases may display parent-of-origin effects. In such cases, the risk depends on the specific parent or origin allele. Imprinting effect is evident in autosomal dominant hereditary paraganglioma leads to tumors only if inherited from paternal germline. Cancer penetrance in mutations carriers may be determined by the parent origin of BRCA mutation. Methods: From 2007–2010 we analyzed 1889 consecutive (136 ovarian + 1753 breast) breast (BrCa) or ovarian cancer (OvCa) patients presenting for treatment at our outpatient facility. In 130 patients with BRCA 1 or 2 mutations the parent of origin for the mutation was known. Of the 130 patients 2 had both BRCA1 and BRCA2 mutated paternally inherited and were excluded from this analysis. Of the breast cancer patients: 28 patients had paternal and 29 had maternal BRCA1 mutations, 24 had paternal and 21 had maternal BRCA 2 mutations. Of the ovarian cancer patients 6 had paternal and 10 had maternal BRCA1 mutations, 7 had paternal and 3 had maternal BRCA2 mutations. In carriers of BRCA mutations the mean age at diagnosis for ovarian cancer was 51 (range 21–70) and for breast cancer was 43 (range 24–78). Two-sample t-test was used to compare the mean age at diagnosis in patients with BRCA 1 or 2 mutations of paternal or maternal inheritance. For breast cancer maternal allele versus paternal allele 2-sample t-test and p-value were compared for the age at first diagnosis. For breast cancer patients BRCA1 maternal inheritance (mean+SD yrs) 45.73+11.22 versus paternal inheritance 38.04+7.14 2-sample t-test p-value p<0.0020. For breast cancer BRCA2 maternal inheritance (mean+SD yrs) 50.65+10.44 versus paternal inheritance 41.68+6.16, 2-sample t-test p-value p<0.0008. Results: Significantly younger age at breast cancer diagnosis was observed in paternal vs. maternal inheritance of BRCA1 mutation (38 vs 46, respectively, p<0.0020) and BRCA2 mutation (42 vs 51 respectively, p<0.0008). There was no significant difference between paternal and maternal age of ovarian cancer diagnosis of BRCA1 (p<0.1415) or BRCA2 mutation (p<0.3470). Conclusion: The restrospective nature of the study may introduce ascertainment bias. However, the breast and ovarian cancers cases in BRCA1 & 2 carriers with maternal or paternal inheritance mirror the Mendelian autosomal dominant pattern in our unselected consecutive cohort of patients. Maternal and paternal inherited BRCA alleles may not be exchangeable. Women with paternally inherited mutations in BRCA gene mutations develop breast cancer at younger age compared with women who inherit the gene mutations from their mothers. In this small sample, clear differences at age of cancer diagnosis are apparent in paternal inheritance of BRCA gene mutation. If this observation duplicates in larger cohorts results will have important implications for recommendation of surgical risk reduction in BRCA mutation carriers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-02.

Tetraploidy in BRCA2 breast tumours

Tetraploidy and aneuploidy can be caused by cell division errors and are frequently observed in many human carcinomas. We have recently reported delayed cytokinesis in primary human fibroblasts from BRCA2 mutation carriers, implying a function for the BRCA2 tumour suppressor in completion of cell division. Here, we address ploidy aberrations in breast tumours derived from BRCA2 germline mutation carriers. Ploidy aberrations were evaluated from flow cytometry histograms on selected breast tumour samples (n=236), previously screened for local BRCA mutations. The ploidy between BRCA2-mutated (n=71) and matched sporadic (n=165) cancers was compared. Differences in ploidy distribution were examined with respect to molecular tumour subtypes, previously defined by immunohistochemistry on tissue microarray sections. Tetraploidy was significantly 3 times more common in BRCA2 breast cancers than sporadic. However, no differences were found in the overall ploidy distribution between BRCA2-mutation carriers and non-carriers. In BRCA2 cancers, tetraploidy was associated with luminal characteristics. The increased frequency of tetraploidy in BRCA2 associated cancers may be linked to cell division errors, particularly cytokinesis. Additionally, tetraploidy emerges predominantly in BRCA2 breast cancers displaying luminal rather than triple-negative phenotypes.

Evaluation of the BOADICEA risk assessment model in women with a family history of breast cancer

The ability of the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) model to predict BRCA1 and BRCA2 mutations and breast cancer incidence in women with a family history of breast cancer was evaluated. Observed mutations in 263 screened families were compared to retrospective predictions. Similarly, observed breast cancers in 640 women were compared to retrospective predictions of breast cancer incidence. The ratios of observed to expected number of BRCA1- , BRCA2- and BRCA(1 or 2) mutations were 1.43 (95% CI 1.05–1.90), 0.63 (95% CI 0.34–1.08), and 1.12 (95% CI 0.86–1.44), showing a significant underestimation of BRCA1 mutations. Discrimination between carriers and non-carriers as measured by area under the receiver operating characteristic (ROC) curve was 0.83 (95% CI 0.76–0.88). The ratio of observed to expected number of invasive breast cancers was 1.41 (0.91–2.08). The corresponding area under the ROC curve for prediction of invasive breast cancer at individual level was 0.62 (95% CI 0.52–0.73). In conclusion, the BOADICEA model can predict the total prevalence of BRCA(1 or 2) mutations and the incidence of invasive breast cancers. The mutation probability as generated by BOADICEA can be used clinically as a guideline for screening, and thus decrease the proportion of negative mutation analyses. Likewise, individual breast cancer risks can be used for selecting women whose risk of breast cancer indicates follow-up. Application of local mutation frequencies of BRCA1 and BRCA2 could improve the ability to distinguish between the two genes.

Biosensor-compatible encapsulation for pre-functionalized nanofluidic channels using asymmetric plasma treatment

Using asymmetric oxygen plasma treatment, we demonstrate the compatibility of a room-temperature sealing process with the use of functional biosensors embedded in nanofluidic channels. This method relies on the permanent bond between a plasma-treated polysilsesquioxane (PSQ) layer and a glass chip bearing nanochannels pre-biofunctionalized in an open-top fashion. The key feature of our method is to achieve selective surface modification of the fluidic chip right after a cleaning step in order to preserve the functionality of the silanol groups at the chip surface. For subsequent bonding, the oxygen plasma applies only to the PSQ gasket layer but not the pre-functionalized substrate to preserve its biosensing capability. We have used this technique to seal nanoslits previously grafted with oligonucleotides and biotins and demonstrated sensing of breast cancer BRCA1 genes and proteins, respectively, using fluorescence imaging. This encapsulation technique, along with our selective surface modification strategy, provides a viable solution for the fabrication of nanofluidic devices integrating biofunctional parts for miniaturized biosensing platforms where most conventional bonding processes are often incompatible with pre-biofunctionalized nanofluidic sensors.

BRCA2 (breast cancer 2, early onset)

Review on BRCA2 (breast cancer 2, early onset), with data on DNA, on the protein encoded, and where the gene is implicated.

The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.

Various cyclin-dependent kinase (Cdk) complexes have been implicated in the regulation of transcription. In this study, we identified a 70-kDa Cyclin K (CycK) that binds Cdk12 and Cdk13 to form two different complexes (CycK/Cdk12 or CycK/Cdk13) in human cells. The CycK/Cdk12 complex regulates phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and expression of a small subset of human genes, as revealed in expression microarrays. Depletion of CycK/Cdk12 results in decreased expression of predominantly long genes with high numbers of exons. The most prominent group of down-regulated genes are the DNA damage response genes, including the critical regulators of genomic stability: BRCA1 (breast and ovarian cancer type 1 susceptibility protein 1), ATR (ataxia telangiectasia and Rad3-related), FANCI, and FANCD2. We show that CycK/Cdk12, rather than CycK/Cdk13, is necessary for their expression. Nuclear run-on assays and chromatin immunoprecipitations with RNA polymerase II on the BRCA1 and FANCI genes suggest a transcriptional defect in the absence of CycK/Cdk12. Consistent with these findings, cells without CycK/Cdk12 induce spontaneous DNA damage and are sensitive to a variety of DNA damage agents. We conclude that through regulation of expression of DNA damage response genes, CycK/Cdk12 protects cells from genomic instability. The essential role of CycK for organisms in vivo is further supported by the result that genetic inactivation of CycK in mice causes early embryonic lethality.

Abstract A65: Does the lifetime number of ovulatory cycles predict the risk of breast cancer in BRCA1 and BRCA2 mutation carriers?

Abstract Background: In the general population, various reproductive factors are believed to influence breast cancer risk via their effects on the number of ovulatory cycles and subsequently on cumulative endogenous hormonal exposure. It is not clear to what extent this is true for women with a BRCA1 or BRCA2 mutation. We undertook a case-control study to evaluate the effect of the cumulative number of ovulatory cycles and each of the contributing components) on the risk of developing breast cancer among BRCA mutation carriers. Methods: A matched case-control study was conducted in 2,632 pairs of women with a deleterious mutation in either BRCA1 (n = 1,915 pairs) or BRCA2 (n = 717 pairs). Women diagnosed with breast cancer were matched to unaffected control subjects by year of birth, mutation status, and country of residence. Information about reproductive factors, hormonal exposures and menopausal status was collected from a routinely administered questionnaire and was used to estimate the cumulative number of ovulatory cycles. Conditional logistic regression was used to estimate the association between various exposures and the risk of breast cancer. Results: The risk of breast cancer decreased with the number of ovulatory cycles (ρ = −0.69;P = 0.03). Age at menarche and breastfeeding were inversely related with risk of breast cancer among BRCA1 (P-trend < 0.0001) but not BRCA2 (P-trend ≥ 0.36) mutation carriers. Parity, oral contraceptive use, and the number of ovulatory cycles, were not associated with risk (P-trend ≥ 0.21). The reduction in breast cancer risk associated with surgical menopause (OR = 0.49; 95%CI 0.37–0.64; P-trend < 0.001) was stronger than that conferred by natural menopause (OR = 0.78; 95%CI 0.58–1.06; P-trend = 0.11). There was a significant reduction in breast cancer risk among women who had an oophorectomy after natural menopause (OR = 0.13; 95%CI 0.02–0.54; P = 0.006). Conclusions: These data challenge the prevailing hypothesis that the risk of breast cancer in a woman can be predicted by her lifetime number of ovulatory cycles in women with a BRCA1 or BRCA2 mutation. Reproductive risk factors, such as age at menarche and breastfeeding are important in BRCA1, but not BRCA2 carriers. The observation that a postmenopausal oophorectomy protects against breast cancer is consistent with either a direct carcinogenic activity of ovarian androgens or through aromatization of androgen to estrogen. It is important to distinguish between these two possibilities because of the different implications for chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A65.

BRCA in breast cancer: ESMO Clinical Practice Guidelines

BRCA in breast cancer: ESMO Clinical Practice Guidelines

Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR).

IntroductionA common feature of neoplastic cells is that mutations in SMADs can contribute to the loss of sensitivity to the anti-tumor effects of transforming growth factor-β (TGF-β). However, germline mutation analysis of SMAD3 and SMAD4, the principle substrates of the TGF-β signaling pathway, has not yet been conducted in breast cancer. Thus, it is currently unknown whether germline SMAD3 and SMAD4 mutations are involved in breast cancer predisposition.MethodsWe performed mutation analysis of the highly conserved mad-homology 2 (MH2) domains for both genes in genomic DNA from 408 non-BRCA1/BRCA2 breast cancer cases and 710 population controls recruited by the Ontario site of the breast cancer family registry (CFR) using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. The results were interpreted in several ways. First, we adapted nucleotide diversity analysis to quantitatively assess whether the frequency of alterations differ between the two genes. Next, in silico tools were used to predict variants' effect on domain function and mRNA splicing. Finally, 37 cases or controls harboring alterations were tested for aberrant splicing using reverse-transcription polymerase chain reaction (PCR) and real-time PCR statistical comparison of germline expressions by non-parametric Mann-Whitney test of independent samples.ResultsWe identified 27 variants including 2 novel SMAD4 coding variants c.1350G > A (p.Gln450Gln), and c.1701A > G (p.Ile525Val). There were no inactivating mutations even though c.1350G > A was predicted to affect exonic splicing enhancers. However, several additional findings were of note: 1) nucleotide diversity estimate for SMAD3 but not SMAD4 indicated that coding variants of the MH2 domain were more infrequent than expected; 2) in breast cancer cases SMAD3 was significantly over-expressed relative to controls (P < 0.05) while the case harboring SMAD4 c.1350G > A was associated with elevated germline expression (> 5-fold); 3) separate analysis using tissue expression data showed statistically significant over-expression of SMAD3 and SMAD4 in breast carcinomas.ConclusionsThis study shows that inactivating germline alterations in SMAD3 and SMAD4 are rare, suggesting a limited role in driving tumorigenesis. Nevertheless, aberrant germline expressions of SMAD3 and SMAD4 may be more common in breast cancer than previously suspected and offer novel insight into their roles in predisposition and/or progression of breast cancer.

The efficacy of taxane chemotherapy for metastatic breast cancer in BRCA1 and BRCA2 mutation carriers

We assessed the efficacy of taxane chemotherapy in BRCA1- and BRCA2-associated patients compared with sporadic metastatic breast cancer patients. Response rates (RRs) to and progression-free survival (PFS) after taxane chemotherapy of 35 BRCA1-associated and 13 BRCA2-associated metastatic breast cancer patients were compared with those outcomes in 95 matched (1:2) sporadic patients. Matching was performed for age at and year of diagnosis of primary breast cancer, year of metastatic disease, and line of therapy (first vs second or third). Among BRCA1-associated patients, the RR was worse (objective response [OR], 23% vs 38%; progressive disease [PD], 60% vs 19%; P < 0.001); and the median PFS shorter (2.2 vs 4.9 months; P = 0.04) compared with sporadic patients. In the subgroup of hormone receptor (HRec)-negative patients, BRCA1-associated patients (n = 20) had a worse RR (OR, 20% vs 42%, respectively; PD, 70% vs 26%, respectively; P = 0.03) and a shorter PFS (1.8 vs 3.8 months; P = 0.004) compared with sporadic patients (n = 19). These outcomes in HRec-positive patients were similar in BRCA1-associated (n = 11) and sporadic (n = 61) patients (OR, 36% vs 38%; PD, 28% vs 20%; median PFS, both 5.7 months). In BRCA2-associated patients, who were mainly HRec-positive, the OR was higher than in sporadic patients (89% vs 38%, respectively; P = 0.02), whereas the median PFS was not significantly different (7.1 vs 5.7 months, respectively). BRCA1-associated, HRec-negative metastatic breast cancer patients were less sensitive to taxane chemotherapy than sporadic HRec-negative patients. HRec-positive BRCA1- and BRCA2-associated patients had a sensitivity to taxane chemotherapy similar to that of sporadic patients.

Hereditary breast cancer in Middle Eastern and North African (MENA) populations: identification of novel, recurrent and founder BRCA1 mutations in the Tunisian population

Germ-line mutations in BRCA1 breast cancer susceptibility gene account for a large proportion of hereditary breast cancer families and show considerable ethnic and geographical variations. The contribution of BRCA1 mutations to hereditary breast cancer has not yet been thoroughly investigated in Middle Eastern and North African populations. In this study, 16 Tunisian high-risk breast cancer families were screened for germline mutations in the entire BRCA1 coding region and exon–intron boundaries using direct sequencing. Six families were found to carry BRCA1 mutations with a prevalence of 37.5%. Four different deleterious mutations were detected. Three truncating mutations were previously described: c.798_799delTT (916 delTT), c.3331_3334delCAAG (3450 delCAAG), c.5266dupC (5382 insC) and one splice site mutation which seems to be specific to the Tunisian population: c.212 + 2insG (IVS5 + 2insG). We also identified 15 variants of unknown clinical significance. The c.798_799delTT mutation occurred at an 18% frequency and was shared by three apparently unrelated families. Analyzing five microsatellite markers in and flanking the BRCA1 locus showed a common haplotype associated with this mutation. This suggests that the c.798_799delTT mutation is a Tunisian founder mutation. Our findings indicate that the Tunisian population has a spectrum of prevalent BRCA1 mutations, some of which appear as recurrent and founding mutations.

APOE and Alzheimer disease: a major gene with semi-dominant inheritance.

Apolipoprotein E (APOE) dependent lifetime risks (LTRs) for Alzheimer Disease (AD) are currently not accurately known and odds ratios alone are insufficient to assess these risks. We calculated AD LTR in 7351 cases and 10 132 controls from Caucasian ancestry using Rochester (USA) incidence data. At the age of 85 the LTR of AD without reference to APOE genotype was 11% in males and 14% in females. At the same age, this risk ranged from 51% for APOE44 male carriers to 60% for APOE44 female carriers, and from 23% for APOE34 male carriers to 30% for APOE34 female carriers, consistent with semi-dominant inheritance of a moderately penetrant gene. Using PAQUID (France) incidence data, estimates were globally similar except that at age 85 the LTRs reached 68 and 35% for APOE 44 and APOE 34 female carriers, respectively. These risks are more similar to those of major genes in Mendelian diseases, such as BRCA1 in breast cancer, than those of low-risk common alleles identified by recent GWAS in complex diseases. In addition, stratification of our data by age groups clearly demonstrates that APOE4 is a risk factor not only for late-onset but for early-onset AD as well. Together, these results urge a reappraisal of the impact of APOE in Alzheimer disease.

Skp2 Overexpression Is Associated with Loss of BRCA2 Protein in Human Prostate Cancer

BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.

Abstract 1188: BRCA1 regulates EGFR expression through microRNAs

Abstract Germline mutations in the tumor suppressor BRCA1 (breast cancer 1, early onset) are associated with a substantially higher risk of developing basal-like breast cancer (BLBC). Many sporadic breast cancers express decreased levels of BRCA1 and show a strong resemblance to BRCA1-hereditary tumors. BRCA1-associated tumors are triple-negative (TN), basal-like high grade ductal carcinomas that display an undifferentiated phenotype and frequently overexpress epidermal growth factor receptor (EGFR/ERBB1/HER1) and basal cytokeratins (CK5, CK6, CK14 and CK17) and confer a poor clinical prognosis. Although upregulation of EGFR is commonly observed in BRCA1-associated breast cancer, the mechanism of EGFR regulation by BRCA1 is not understood. EGFR upregulation could occur at multiple transcriptional and/or post-transcriptional levels. MicroRNAs (miRNAs) are an evolutionarily conserved class of small non-coding RNAs that regulate gene expression post-transcriptionally and/or translationally. Several miRNAs are found to be differentially expressed in breast cancer and studies show their involvement in stem cell differentiation, epithelial-to-mesenchymal transition (EMT), invasion and metastasis. Identifying the functional relationship between loss of BRCA1 and miRNAs will help us understand the post-transcriptional mechanism by which BRCA1 deficiency leads to aberrant EGFR expression. In the current study, we show for the first time, a direct inverse correlation between BRCA1 and EGFR protein expression levels in breast cancer cell lines representing the different intrinsic subtypes and in primary mammary epithelial cells. By overexpression and knockdown of BRCA1, we demonstrate that BRCA1 loss results in increased stability of EGFR protein. Using our in vitro system of immortalized primary mammary epithelial cells, we evaluated the effect of functional loss of BRCA1 on the expression of miRNAs in secondary mammospheres which are enriched for stem-like cells and in cells differentiated in vitro. We observed that BRCA1 deficiency leads to dysregulation of miRNAs linked to stem cell renewal, EGFR signaling and EMT. miRNA gain-of-function experiments using Pre-miR™ miRNA precursors and loss-of-function experiments using miRCURY LNA™ miRNA inhibitors were performed with the selected miRNAs to further confirm their effect on EGFR expression. Results suggest that miRNA mediated EGFR deregulation could be a critical step in tumor initiation and progression in BRCA1 deficiency. Since EGFR upregulation is an early event in breast cancer, additional functional studies will provide a novel molecular mechanism by which BRCA1 deficiency leads to the development of BLBC with aberrant EGFR expression mediated by miRNAs. The study will offer new therapeutic approaches for BLBC, a highly aggressive disease with limited therapeutic options. 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 1188. doi:10.1158/1538-7445.AM2011-1188

Abstract 224: Novel potential precursor structures for BRCA1-associated breast cancer

Abstract Objectives: Female individuals with BRCA1 germline mutation have an 80-90% risk to develop breast cancer during their lifetime. BRCA1 cancers are distinct from sporadic breast cancers as they more frequently show high-grade histology and are typically estrogen receptor and HER2-negative. While sporadic breast ductal carcinoma is thought to be preceded by in-situ precursors, the presence of precursors is uncertain in BRCA1 cancers. To better understand breast carcinogenesis in BRCA1 patients, we developed an approach to perform unprecedently detailed structural analysis of breast tissue. Design: A prophylactic mastectomy product of a BRCA1 patient with history of contralateral breast cancer was obtained after routine pathological evaluation then trimmed into 0.3 × 1 x1.5 cm tissue blocks (n=94). Initial sections from each block were H&amp;E stained and microscopically evaluated. Any lobular or ductal structure with increased cellularity was recorded as hyperplastic event. Correlation of hyperplastic events on the surface of blocks and topographic information of events led to the identification of “hot” regions with larger numbers of these events. From the center of this region, the block was serially sectioned at 5μm-depth. H&amp;E staining of every tenth section allowed a three-dimensional (3D) reconstruction of all hyperplastic events. The same procedure was applied to a normal control tissue product of a prophylactic mastectomy in a non-BRCA1 patient with personal history of contralateral breast cancer. Results: We identified five novel “isolated epithelial structures” within BRAC1 breast tissue. On a single two dimensional (2D) slide, these isolated epithelial structures resemble regular lobular and ductal structures with hyperplastic features; while after 3D reconstruction with 2D slides, they revealed in isolation from connection to regular anatomic epithelial structures and were entirely separated by mesenchymal tissue. Importantly, we report in detail an isolated tubular epithelial structure in association with an incidental millimeter-sized, BRCA1-deficient cancer focus. This structure is 2mm in length, mostly surrounded by myoepithelial cells, and remarkable for simultaneous development of lobular and ductal hyperplasia including flat epithelial hyperplasia. The transition zone into cancer is characterized by loss of myoepithelial cells and significant cytologic atypia. Conclusions: Our thorough examination of BRCA1 breast tissue allowed for the first time the identification of isolated epithelial structures. Our identification of one isolated epithelial structure in connection with the development of an infiltrating carcinoma focus strongly suggests that isolated epithelial structures are potential precursors for BRCA1 breast cancers. Analysis of these structures may lead to the discovery of new preventive and therapeutic targets. 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 224. doi:10.1158/1538-7445.AM2011-224

Predictors of contralateral breast cancer in BRCA1 and BRCA2 mutation carriers

Purpose:The objective of this study was to estimate the risk of contralateral breast cancer in BRCA1 and BRCA2 carriers; and measure the extent to which host, family history, and cancer treatment-related factors modify the risk.Patients and methods:Patients were 810 women, with stage I or II breast cancer, for whom a BRCA1 or BRCA2 mutation had been identified in the family. Patients were followed from the initial diagnosis of cancer until contralateral mastectomy, contralateral breast cancer, death, or last follow-up.Results:Overall, 149 subjects (18.4%) developed a contralateral breast cancer. The 15-year actuarial risk of contralateral breast cancer was 36.1% for women with a BRCA1 mutation and was 28.5% for women with a BRCA2 mutation. Women younger than 50 years of age at the time of breast cancer diagnosis were significantly more likely to develop a contralateral breast cancer at 15 years, compared with those older than 50 years (37.6 vs 16.8% P=0.003). Women aged <50 years with two or more first-degree relatives with early-onset breast cancer were at high risk of contralateral breast cancer, compared with women with fewer, or no first-degree relatives with breast cancer (50 vs 36% P=0.005). The risk of contralateral breast cancer was reduced with oophorectomy (RR 0.47; 95% CI 0.30–0.76; P=0.002).Conclusion:The risk of contralateral breast cancer risk in BRCA mutation carriers declines with the age of diagnosis and increases with the number of first-degree relatives affected with breast cancer. Oophorectomy reduces the risk of contralateral breast cancer in young women with a BRCA mutation.