Colin R. James

BRCA1 mRNA Expression Levels Predict for Overall Survival in Ovarian Cancer after Chemotherapy

Purpose: We investigated whether BRCA1 mRNA expression levels may represent a biomarker of survival in sporadic epithelial ovarian cancer following chemotherapy treatment. Experimental Design: The effect of loss of BRCA1 expression on chemotherapy response in ovarian cancer was measured in vitro using dose inhibition assays and Annexin V flow cytometry. Univariate and multivariate analyses were done to evaluate the relationship between BRCA1 mRNA expression levels and survival after chemotherapy treatment in 70 fresh frozen ovarian tumors. Results: We show that inhibition of endogenous BRCA1 expression in ovarian cancer cell lines results in increased sensitivity to platinum therapy and decreased sensitivity to antimicrotubule agents. In addition, we show that patients with low/intermediate levels of BRCA1 mRNA have a significantly improved overall survival following treatment with platinum-based chemotherapy in comparison with patients with high levels of BRCA1 mRNA (57.2 versus 18.2 months; P = 0.0017; hazard ratio, 2.9). Furthermore, overall median survival for higher-BRCA1-expressing patients was found to increase following taxane-containing chemotherapy (23.0 versus 18.2 months; P = 0.12; hazard ratio, 0.53). Conclusions: We provide evidence to support a role for BRCA1 mRNA expression as a predictive marker of survival in sporadic epithelial ovarian cancer.

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.

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.

Abstract OT3-02-07: Neo-DDRD: A biomarker-driven neoadjuvant feasibility study in breast cancer

Background Anthracycline-based chemotherapy reduces the risk of early breast cancer recurrence but to date it has not been possible to predict which patients specifically benefit from this treatment. The Almac DDRD assay, a 44 gene signature, has been developed to detect DNA damage response deficiency (DDRD) within breast and other cancer types. The DDRD assay has been shown to predict benefit from DNA damaging chemotherapy in retrospective analyses in neoadjuvant and adjuvant settings (Mulligan et al, JNCI 2014). This pilot study aims to prospectively evaluate the feasibility and utility of this signature in the neoadjuvant setting in breast cancer. It will inform the design of a phase II randomised study where the DDRD assay will be utilised to guide neoadjuvant chemotherapy selection. Study design A single-centre, non-randomised feasibility study of 30 women with a histologically confirmed diagnosis of breast cancer, where neoadjuvant chemotherapy is the treatment modality of choice. Inclusion criteria: Age >18 years Early (T1-2, N0-1), locally advanced or inflammatory breast cancer Normal left ventricular function, haematological and biochemical parameters ECOG PS 0-1 Exclusion criteria: Metastatic disease Bilateral breast cancer Pregnancy/breastfeeding Inability to give informed consent At diagnosis, patients consented to providing two core needle biopsies in addition to a diagnostic biopsy. Axillary nodal status was determined by axillary ultrasound, fine needle aspiration cytology, and pre-treatment sentinel node biopsy if pre-operative axillary staging was negative. Chemotherapy was administered as per standard institutional practice: 6 cycles fluourouracil, epirubicin and cyclophosphamide (FEC) in node-negative patients; 3 cycles of FEC followed by 3 cycles of docetaxel in node positive patients. Neoadjuvant trastuzumab was given in Her2 positive patients. Imaging (mammography and ultrasound) was repeated after three cycles, and repeat FFPE and snap frozen core biopsies undertaken. At conclusion of treatment, patients underwent surgery as appropriate (mastectomy or breast conservation at the discretion of the operating surgeon), with axillary node clearance for patients who were node positive at diagnosis. Further biopsies (FFPE and fresh frozen) were taken intra-operatively. Plasma samples were obtained at diagnosis, mid-treatment and surgery. Pathological reporting of the surgical specimen was standardised using the residual cancer burden (RCB) reporting system. Aims · To assess the feasibility of carrying out the DDRD assay on diagnostic core biopsy specimens · To evaluate the turn-around time for the assay to assess feasibility of integration into the breast cancer treatment pathway · To assess the correlation of DDRD assay scores and pathological tumour response · Exploratory biomarker analysis will be carried out within Queen9s University, Belfast, including: o correlation of DDRD score with changes in Ki67 protein level after three cycles of chemotherapy o exploratory analysis of chemokine expression in peripheral plasma samples, and correlation with pathological response to treatment Accrual The study opened in April 2014. Accrual to date is 14 patients. Citation Format: McIntosh SA, Parkes EE, James CR, Lioe T, Lowry K, Keating KE, Khambata-Ford S, Kennedy RD. Neo-DDRD: A biomarker-driven neoadjuvant feasibility study in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT3-02-07.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs)

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets. We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -‘good’ outcome no relapse > 3 years; ‘poor’ outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in ‘good’ outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression. We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

Abstract B051: The identification of the Thromboxane A2 receptor as an oncogenic driver in triple-negative breast cancer

Purpose of Study: To identify and characterise genes involved in driving the oncogenesis of Triple Negative Breast Cancer (TNBC), and assess the possibility of use as potential therapeutic targets. Triple Negative Breast Cancer (TNBC) is defined by the lack of the Estrogen Receptor α (ERα), Progesterone Receptor (PR) and low expression of the HER2 receptor and represents the breast cancer subtype with the poorest clinical outcomes. The majority of TNBCs are also basal-like breast cancers (BLBCs) which express basal cytokeratins found in the normal basal epithelia of the breast. BRCA1 mutant breast cancers closely resemble the TNBC/BLBC subtypes and BRCA1 expression is downregulated in up to 30% of sporadic invasive BLBCs. Whilst luminal or HER2-positive breast cancers can be targeted by endocrine and Herceptin therapies, respectively, TNBCs have no specific therapy designed against them as they lack targetable receptors. Our aim is to identify genes involved in TNBC pathogenesis which could represent novel therapeutic targets. We have performed microarray profiling of 16 triple negative breast tumors (8 good and 8 poor response tumors, following treatment with 5-fluorouracil, epirubicin and cyclophosphamide). Elevated levels of the thromboxane A2 receptor gene (TBXA2R) were observed in TNBCs, and siRNA knockdown of TBXA2R consistently showed dramatic growth inhibition in BLBC cell lines but not in ‘normal’ HME-1 basal cells indicating specificity for BLBC. TBXA2R is a G-protein coupled receptor with a well-established role in platelet activation and haemostasis but also regulates diverse cellular processes such as angiogenesis, cell survival and cytoskeletal arrangement. TBXA2R mRNA was higher in both Basal A and B cell lines than other subtypes and basal cell lines were sensitive to knockdowns suggesting that TBXA2R may represent a novel driver of TNBC proliferation. TBXA2R mRNA is upregulated following BRCA1 siRNA in MCF10-A and MCF7 cells and TBXA2R promoter activity is enhanced by knockdown of BRCA1 in T47D cells. We have mapped the minimal BRCA1-responsive region on the proximal TBXA2R promoter and have identified cMyc to be involved in TBXA2R repression. We are currently investigating pathways downstream of TBXA2R to determine the mechanism by which it enhances cell proliferation in TNBC and have shown that TBXA2R may also contribute to migration and invasion of TNBC via signalling through the Rho pathway. We are also currently determining the feasibility of using TBXA2R antagonists or inhibitors of the Rho-associated kinases in the treatment of TNBC. Conclusions: Our current understanding of the mechanisms underlying the pathogenesis of TNBC is lacking. Here we report a novel gene required for proliferation and migration of poor outcome TNBCs which could provide us with the opportunity to improve current therapy or develop novel, more effective treatments. Citation Format: K S. Orr, N E. Buckley, J E. Quinn, C R. James, J. Blainey, P. B. Mullan. The identification of the Thromboxane A2 receptor as an oncogenic driver in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B051.