Jennifer E. Quinn

BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents

BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an effect that was preceded by an acute arrest at the G2/M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adriamycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these effects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds.

BRCA1: mechanisms of inactivation and implications for management of patients.

The BRCA1 gene was cloned in 1994 as one of the genes that conferred genetic predisposition to early-onset breast and ovarian cancer. Since then, a genetic test for identification of high-risk individuals has been developed. Despite being implicated in many important cellular pathways, including DNA repair and regulation of transcription, the exact mechanism by which inactivation of BRCA1 might lead to malignant transformation of cells remains unknown. We examine the mechanisms that underlie inactivation of BRCA1 and assess how they affect management of patients, in terms of both primary and secondary cancer prevention strategies. Furthermore, we look at the potential usefulness of BRCA1 as a prognostic tool and as a predictive marker of response to different classes of drugs. Finally, throughout this review, we draw links between the functional consequences of BRCA1 inactivation, in terms of key cellular signalling pathways, and how they might explain specific clinical observations in individuals who carry mutations in the gene.

BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage–Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

BRCA1 and implications for response to chemotherapy in ovarian cancer

OBJECTIVES Treatment of epithelial ovarian cancer (EOC) remains a challenge, despite advances in surgery and chemotherapy. Hereditary ovarian cancer is primarily due to germline mutations in the BRCA1 tumour suppressor gene. In addition, sporadic EOC tumours display significant of loss of BRCA1 function due to epigenetic inactivation of the BRCA1 gene. This article reviews the preclinical and clinical evidence to support a role for BRCA1 as a potential predictive biomarker of response to both platinum and taxane based chemotherapy in EOC. METHODS We conducted a Medline and Pubmed search for reports between 1990 and 2008 using the search terms: BRCA1 and hereditary ovarian cancer, BRCA1 and sporadic ovarian cancer, ovarian cancer and chemotherapy, ovarian cancer and taxanes, ovarian cancer and platinums, ovarian cancer and clinical response, BRCA1 and DNA damage, BRCA1 and DNA repair, BRCA1 and mitotic checkpoint. If reports identified by these criteria referred to other papers not in the initial search, then these were also reviewed if relevant to BRCA1 and ovarian cancer. RESULTS The BRCA1 pathway plays a significant role in the development of both hereditary and sporadic EOC. Evidence suggests that BRCA1 is a potential biomarker of response to platinum chemotherapy in EOC with BRCA1 deficiency predicting for enhanced response. In contrast, initial evidence suggests that loss of BRCA1 function results in reduced response to antimicrotubule-based chemotherapy. The ability of BRCA1 to differentially modulate response to these agents involves loss of BRCA1 mediated DNA repair and mitotic checkpoint control, respectively. CONCLUSIONS Standard first line treatment of EOC consists of a combination of platinum and taxane chemotherapy, however clinically useful biomarkers for predicting response to these agents have yet to be established. BRCA1 may prove useful as a biomarker in EOC for assigning chemotherapy treatments based on the presence or absence of BRCA1 function.

BRCA1 transcriptionally regulates genes associated with the basal-like phenotype in breast cancer

Expression profiling of BRCA1-deficient tumours has identified a pattern of gene expression similar to basal-like breast tumours. In this study, we examine whether a BRCA1-dependent transcriptional mechanism may underpin the link between BRCA1 and basal-like phenotype. In methods section, the mRNA and protein were harvested from a number of BRCA1 mutant and wild-type breast cancer cell lines and from matched isogenic controls. Microarray-based expression profiling was used to identify potential BRCA1-regulated transcripts. These gene targets were then validated (by in silico analysis of tumour samples) by real-time PCR and Western blot analysis. Chromatin immunoprecipitation (ChIP) assays were used to confirm recruitment of BRCA1 to specific promoters. In results, we demonstrate that functional BRCA1 represses the expression of cytokeratins 5(KRT5) and 17(KRT17) and p-Cadherin (CDH3) in HCC1937 and T47D breast cancer cell lines at both mRNA and protein level. ChIP assays demonstrate that BRCA1 is recruited to the promoters of KRT5, KRT17 and CDH3, and re-ChIP assays confirm that BRCA1 is recruited independently to form c-Myc and Sp1 complexes on the CDH3 promoter. We show that siRNA-mediated inhibition of endogenous c-Myc (and not Sp1) results in a marked increase in CDH3 expression analogous to that observed following the inhibition of endogenous BRCA1. The data provided suggest a model whereby BRCA1 and c-Myc form a repressor complex on the promoters of specific basal genes and represent a potential mechanism to explain the observed overexpression of key basal markers in BRCA1-deficient tumours.

BRCA1 is both a prognostic and predictive biomarker of response to chemotherapy in sporadic epithelial ovarian cancer

OBJECTIVES We investigated the relationship between BRCA1 protein expression by immunohistochemistry (IHC) and clinical outcome following platinum and platinum/taxane chemotherapy in sporadic epithelial ovarian cancer (EOC). METHODS BRCA1 IHC was performed on a cohort of 292 ovarian tumours from two UK oncology centres. BRCA1 protein expression levels were correlated with overall survival (OS), progression free survival (PFS) and clinical response to chemotherapy by multivariate analysis. RESULTS EOC patients with absent/low BRCA1 protein expression (41%) had a better chance of clinical response following chemotherapy as compared to patients with high BRCA1 expression (odds ratio 2.47: 95%CI 1.10-5.55, p=0.029). Patients with absent/low BRCA1 had a higher probability of clinical response following single agent platinum compared to high BRCA1 expressing patients (68.5% vs. 46.8%), while addition of a taxane increased response rates independent of BRCA1. Overall, patients with absent/low BRCA1 had a better clinical outcome compared to patients with high BRCA1 protein expression in terms of both OS (HR=0.65: 95%CI 0.48-0.88, p=0.006) and PFS (HR=0.74, 95%CI 0.55-0.98, p=0.040). CONCLUSIONS We confirm that absent/low BRCA1 protein expression is a favourable prognostic marker. However, we also provide the first evidence that absent/low BRCA1 protein expression in sporadic EOC patients predicts for an improved clinical response to chemotherapy.

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Background There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection. Methods DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided. Results In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population. Conclusions A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

The 2,5 oligoadenylate synthetase/RNaseL pathway is a novel effector of BRCA1- and interferon-gamma-mediated apoptosis.

BRCA1 has been reported to have roles in DNA damage repair, cell cycle checkpoint control, transcriptional regulation and ubiquitination. We have previously demonstrated that BRCA1 is a potent activator of a subset of interferon (IFN)-regulated genes and that BRCA1 synergistically activated a number of these genes in the presence of IFN-γ, but not type I IFNs. Here we report that one of these targets, 2,5 oligoadenylate synthetase (2,5 OAS), is a mediator of BRCA1/IFN-γ-induced apoptosis. We show that the induction of 2,5 OAS in response to IFN-γ is BRCA1 and STAT1 dependent. Consistent with a role as a negative regulator of proliferation, transient transfection of 2,5 OAS into breast cancer cell lines results in decreased colony growth and apoptosis. Furthermore we show that IFN-γ-induced apoptosis is dependent on functional BRCA1 and STAT1 and we demonstrate that IFN-γ-induced apoptosis is dependent on 2,5 OAS induction. 2,5 OAS is the only known upstream regulator of RNaseL, a recently identified hereditary prostate tumour suppressor gene implicated in apoptosis. We propose that BRCA1 may be an upstream regulator of RNaseL, acting in concert with IFN-γ to transcriptionally activate 2,5 OAS, leading to the downstream activation of RNaseL and apoptosis.

BRCA1 Interacts with and Is Required for Paclitaxel-Induced Activation of Mitogen-Activated Protein Kinase Kinase Kinase 3

BRCA1 has been implicated in a number of cellular processes, including transcriptional regulation, DNA damage repair, cell cycle arrest, and apoptosis. We identified mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3), an upstream regulator of the c-Jun NH2-terminal kinase/stress-activated protein kinase and p38/MAPK pathways, as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by coimmunoprecipitation in mammalian cells. Deletion mapping demonstrated that amino acids 1611–1863 are required to mediate the interaction with MEKK3 in yeast. BRCA1 disease-associated mutations abrogated the interaction in yeast, and BRCA1 failed to interact with MEKK3 in BRCA1 mutant HCC1937 breast cancer cells. We demonstrate that small interfering RNA-based inhibition of endogenous BRCA1 reduces MEKK3 kinase activity and conversely that inducible expression of BRCA1 activates MEKK3 and p38/MAPK. Finally, we demonstrate using complementary approaches that BRCA1 is required for paclitaxel-induced activation of MEKK3. These data indicate that BRCA1 is a key regulator of the paclitaxel-induced stress response pathway and suggest that the ability of BRCA1 to associate with, and mediate the activation of, MEKK3 represents a potential mechanism through which this pathway is regulated.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer.

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non‐small cell lung carcinoma (NSCLC). The pro‐apoptotic BCL‐2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria‐independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi‐mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co‐depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1‐deficient subgroup (11–19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1‐immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1‐immunodeficient, platinum‐resistant tumours. Copyright