BRCA1 Expression Research Articles

Abstract 1428: Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer

Abstract Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution. We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p<0.01). To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness. The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC. Citation Format: Anniina Farkkila, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Rainer Lehtonen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2017-1428

Abstract 1075: JQ1 induces DNA damage, inhibits expression of DNA repair proteins, and synergizes with PARP inhibitors in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. PDAC is a highly aggressive tumor with a 5-year survival of <6%. Surgery remains the only curative treatment for PDAC patients, but only 20% of newly diagnosed patients have resectable disease. The remaining 80% present with locally advanced or metastatic disease, and receive systemic chemotherapy with gemcitabine. The median survival of this patient population is ~6 months. Development of more effective therapy for this chemorefractory disease is imperative. The goal of the current study was to develop effective combination therapy for PDAC by identifying agents that might be combined with the BET bromodomain inhibitor JQ1, which we have shown to inhibit the growth in vivo of PDAC patient derived xenografts (PDX). Expression profile analysis of tumors from vehicle control and JQ1 treated mice revealed that JQ1 inhibited the expression of multiple gene products involved in DNA repair. Notably, JQ1 inhibited expression of DNA double-strand break (DSB) repair proteins BRCA2 and Ku80. Immunohistochemical staining confirmed down-regulation of expression of both proteins in tumors of mice treated with JQ1. Further, immunoblot and immunofluorescence analyses demonstrated that decreased expression of BRCA2 and Ku80 was coincident with increased levels of DNA damage, as reflected by expression of the DNA DSB marker gamma-H2AX. Data generated in vivo in three independent PDX models corroborated in vitro data generated using pancreatic cancer cell lines BxPC3 and Panc1. The data suggest that JQ1 induces DNA damage by inhibiting DNA repair. Because DNA repair deficiency sensitizes cells to PARP inhibitors, we hypothesized that JQ1-induced DNA repair deficiency would sensitize PDAC cells to PARP inhibitors. To address this hypothesis, we exposed Panc1 and BxPC3 to JQ1 or to a PARP 1/2 inhibitor (veliparib or olaparib) or to the combinations, and assessed the efficacy of each. Growth inhibition data, analyzed using Compusyn software and reported as combination indices, demonstrated that the combinations of JQ1 + veliparib or olaparib exert synergistic cytotoxicity. Further, the combination of JQ1 + a PARP inhibitor increased the accumulation of DNA damage in vitro, compared to either agent alone. We conclude that JQ1 induces DNA damage due at least in part to DNA repair deficiency, and propose that this mechanism sensitizes PDAC cells to PARP inhibitors. Citation Format: Aubrey Lynn Miller, Tracy Gamblin, Leona Council, Robert van Waardenburg, Eddy Yang, James Bradner, Karina Yoon. JQ1 induces DNA damage, inhibits expression of DNA repair proteins, and synergizes with PARP inhibitors in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1075. doi:10.1158/1538-7445.AM2017-1075

Abstract 5056: The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib

Abstract Introduction: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. Aims: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. Methods: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). Results: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. Conclusions: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. The bromodomain inhibitor JQ1 sensitizes homologous recombination proficient ovarian cancer cells to the PARP inhibitor olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2017-5056

Comparison of BRCA1 Expression between Triple-Negative and Luminal Breast Tumors

Background:Previous studies have suggested that BRCA1 dysregulation has been shown to have a role in triple-negative phenotypic manifestation. However, differences of BRCA1 expression, as a tumor suppressor gene, have rarely been investigated between luminal and triple-negative breast tumors. Therefore, the present study attempted to compare the BRCA1 expression in triple-negative with luminal breast tumors and its association with the clinicopathologic characteristics of patients.Methods:BRCA1 expression was evaluated by real-time PCR in 26 triple-negative and 27 luminal breast tumors.Results:The results revealed that there is a high frequency of BRCA1 underexpression in both triple-negative and luminal breast tumors. The BRCA1 underexpression was related to young age at diagnosis, lymph node involvement, and grade III tumors.Conclusion:The observations suggest that decreased BRCA1 expression, regardless of tumor subtype, has a general role in breast malignancy and associated with poor prognostic features in breast tumors.

Chronic treatment with cisplatin induces chemoresistance through the TIP60-mediated Fanconi anemia and homologous recombination repair pathways

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Deciphering the role of microRNA – A step by step guide

MicroRNAs (miRNAs), are small non-coding RNAs of approximately 22 nucleotides in length, playing an important role in regulating gene expression post-transcriptionally. Understanding the effect of miRNA regulation in a pathway-specific manner unravels the approaches adopted to apprehend biological mechanisms, the information, which is scanty for researchers, not primed already for miR related research. Here, we describe a quick perspective in 5 steps with probable approaches and assays at every level to unravel the specific role of a microRNA, miR-145a-5p, as an example. This perspective as a guide would help in identifying novel targets for a microRNA, as shown for miR-145a-5p, which down-regulated the mRNA expression of ADD3 and BRCA2, using bioinformatic tools and experimental assays.

Intrinsic LINE-1 Hypomethylation and Decreased Brca1 Expression are Associated with DNA Repair Delay in Irradiated Thyroid Cells.

Exposure to ionizing radiation greatly increases the risk of developing papillary thyroid carcinoma (PTC), especially during childhood, mainly due to gradual inactivation of DNA repair genes and DNA damages. Recent molecular characterization of PTC revealed DNA methylation deregulation of several promoters of DNA repair genes. Thus, epigenetic silencing might be a plausible mechanism for the activity loss of tumor suppressor genes in radiation-induced thyroid tumors. Herein, we investigated the impact of ionizing radiation on global methylation and CpG islands within promoter regions of homologous recombination (HR) and non-homologous end joining (NHEJ) genes, as well as its effects on gene expression, using two well-established normal differentiated thyroid cell lines (FRTL5 and PCCL3). Our data reveal that X-ray exposure promoted G2/M arrest in normal thyroid cell lines. The FRTL5 cells displayed a slower kinetics of double-strand breaks (DSB) repair and a lower long interspersed nuclear element-1 (LINE-1) methylation than the PCCL3 cells. Nevertheless, acute X-ray exposure does not alter the expression of genes involved in HR and NHEJ pathways, apart from the downregulation of Brca1 in thyroid cells. On the other hand, HR and NHEJ gene expressions were upregulated in radiation-induced senescent thyroid cells. Taken together, these data suggest that FRTL5 cells intrinsically have less efficient DNA DSB repair machinery than PCCL3 cells, as well as genomic instability, which could predispose the FRTL5 cells to unrepaired DSB lesions and, therefore, gene mutations.

Genistein Prevents BRCA1 CpG Methylation and Proliferation in Human Breast Cancer Cells with Activated Aromatic Hydrocarbon Receptor

BackgroundPrevious studies have suggested a causative role for agonists of the aromatic hydrocarbon receptor (AhR) in the etiology of breast cancer 1, early-onset (BRCA-1)–silenced breast tumors, for which prospects for treatment remain poor. ObjectivesWe investigated the regulation of BRCA1 by the soy isoflavone genistein (GEN) in human estrogen receptor α (ERα)–positive Michigan Cancer Foundation-7 (MCF-7) and ERα-negative sporadic University of Arizona Cell Culture-3199 (UACC-3199) breast cancer cells, respectively, with inducible and constitutively active AhR. MethodsIn MCF-7 cells, we analyzed the dose- and time-dependent effects of GEN and (–)-epigallocatechin-3-gallate (EGCG) control, selected as prototype dietary DNA methyltransferase (DNMT) inhibitors, on BRCA-1 expression after AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in TCDD-washout experiments. We compared the effects of GEN and EGCG on BRCA1 cytosine-phosphate-guanine (CpG) methylation and cell proliferation. Controls for DNA methylation and proliferation were changes in expression of DNMT-1, cyclin D1, and p53, respectively. In UACC-3199 cells, we compared the effects of GEN and α-naphthoflavone (αNF; 7,8-benzoflavone), a synthetic flavone and AhR antagonist, on BRCA1 expression and CpG methylation, cyclin D1, and cell growth. Finally, we examined the effects of GEN and αNF on BRCA1, AhR-inducible cytochrome P450 (CYP)-1A1 (CYP1A1) and CYP1B1, and AhR mRNA expression. ResultsIn MCF-7 cells, GEN exerted dose- and time-dependent preventative effects against TCDD-dependent downregulation of BRCA-1. After TCDD washout, GEN rescued BRCA-1 protein expression while reducing DNMT-1 and cyclin D1. GEN and EGCG reduced BRCA1 CpG methylation and cell proliferation associated with increased p53. In UACC-3199 cells, GEN reduced BRCA1 and estrogen receptor-1 (ESR1) CpG methylation, cyclin D1, and cell growth while inducing BRCA-1 and CYP1A1. ConclusionsResults suggest preventative effects for GEN and EGCG against BRCA1 CpG methylation and downregulation in ERα-positive breast cancer cells with activated AhR. GEN and flavone antagonists of AhR may be useful for reactivation of BRCA1 and ERα via CpG demethylation in ERα-negative breast cancer cells harboring constitutively active AhR.

A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability

SummaryMutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.

Abstract NTOC-092: THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB

Abstract INTRODUCTION: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair pathways is a significant clinical problem. HR-proficient HGSOC tumors, for example those harboring cyclin E amplifications, have poorer clinical outcomes and show relative resistance to DNA-damaging platinum agents and newer poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional down-regulation of HR pathway components. An emerging class of epigenetic mediators of pro-tumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical anti-tumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA damaging agents. AIMS: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. METHODS: The HR-proficient ovarian cancer cell lines, OVCAR-3 (cyclin E-amplified) and SKOV3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi JQ1 or with the olaparib/JQ1 combination. Sulforhodamine B (SRB) assays assessed cell growth and viability (72 hours treatment). Immunofluorescence (IF) assays assessed markers of DNA damage (pH2AX), apoptosis (cleaved caspase-3), and HR efficiency (RAD51 foci, and GFP expression in cells co-transfected with I-Sce1 endonuclease and DRGFP HR reporter plasmids) (24 hours treatment). Steady state levels of the HR protein BRCA1, pH2AX and cleaved caspase-3 were assessed by western blot (24 hours treatment). RESULTS: The combination of JQ1 and olaparib synergistically reduced cancer cell viability following isobologram analyses of SRB experiments. Compared to either drug alone, the JQ1/olaparib combination also significantly reduced BRCA1 expression and increased protein levels of cleaved caspase-3 and pH2AX in western blots, and also increased the number of cells displaying DNA damage and apoptosis in IF assays. Finally, JQ1 and olaparib combined to significantly reduce HR efficiency in our RAD51 foci formation and DRGFP assays compared to olaparib alone. CONCLUSIONS: Our results suggest that BRDi sensitize HR-proficient cells to DNA damaging drugs, in part by reducing efficiency of HR DNA repair. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. Citation Format: Andrew J. Wilson, Janese Thompson, Abdirahman Osman, Jeanette Saskowski, Dineo Khabele. THE BROMODOMAIN INHIBITOR JQ1 SENSITIZES HOMOLOGOUS RECOMBINATION PROFICIENT OVARIAN CANCER CELLS TO THE PARP INHIBITOR OLAPARIB [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-092.

DNA repair gene expressions are related to bone marrow cellularity in myelodysplastic syndrome

ObjectiveTo evaluate the expression of genes related to nuclear excision (ERCC8, XPA and XPC), homologous recombination and non-homologous end-joining (ATM, BRCA1, BRCA2 and LIG4) repair mechanisms, using quantitative PCR methodologies,...

Identification of direct target genes of miR-7, miR-9, miR-96, and miR-182 in the human breast cancer cell lines MCF-7 and MDA-MB-231

Some microRNAs have carcinogenic or tumor suppressive effects in breast cancer, which is the most common cancer in women worldwide. MiR-7 and miR-9 are tumor suppressor microRNAs, which induce apoptosis and inhibit proliferation in breast cancer cells. Moreover, miR-96 and miR-182 are onco-microRNAs that increase proliferation, migration, and tumorigenesis in breast cancer cells. This study aimed to identify the direct target genes of these four microRNAs in the human breast cancer cell lines MCF-7 and MDA-MB-231. Initially, bioinformatics tools were used to identify the target genes that have binding sites for miR-7, MiR-9, MiR-96, and miR-182 and are also associated with breast cancer. Subsequently, the findings of the bioinformatics analysis relating to the effects of these four microRNAs on the 3′-UTR activity of the potential target genes were confirmed using the dual luciferase assay in MCF-7 and MDA-MB-231 cells co-transfected with the vectors containing 3′-UTR segments of the target genes downstream of a luciferase coding gene and each of the microRNAs. Finally, the effects of microRNAs on the endogenous expression of potential target genes were assessed by the overexpression of each of the four microRNAs in MCF-7 and MDA-MB-231 cells. Respectively, three, three, three, and seven genes were found to have binding sites for miR-7, miR-9, miR-96, and miR-182 and were associated with breast cancer. The results of empirical studies including dual luciferase assays and real-time PCR confirmed that miR-7 regulates the expression of BRCA1 and LASP1; MiR-9 regulates the expression of AR; miR-96 regulates the expression of ABCA1; and miR-182 regulates the expression of NBN, TOX3, and LASP1. Taken together, our results suggest that the tumor suppressive effects of miR-7 may be mediated partly by regulating the expression of BRCA1 as a tumor suppressor gene in breast cancer. In addition, this microRNA and miR-182 may have effects on the nodal-positivity and tumor size of breast carcinoma through the regulation of LASP1. The tumor suppressive functions of miR-9 may be mediated partly by suppressing the expression of AR—an oncogene in breast cancer. Moreover, miR-96 may play an oncogenic role in breast cancer by suppressing the apoptosis through the regulation of ABCA1.

Impact of DNA repair deficiency signature on outcomes in triple negative breast cancer (TNBC) patients treated with AC chemotherapy (SWOG S9313).

529 Background: Biomarkers of response and resistance to adjuvant chemotherapy for TNBC are needed. Deficiency in DNA damage response (DDR) and repair pathways have been reported in TNBC and may impact response to chemotherapy. Aims: To investigate DNA damage response deficiency (DDRD) molecular signature, BRCA1mRNA expression and Tumor Infiltrating Lymphocytes (TILs) as prognostic markers in TNBC patients treated with adjuvant AC on S9313. Methods: S9313 accrued 3125 early stage BC patients to two alternative schedules of AC with no difference in outcomes between the two arms. We identified 425 (14%) patients with centrally determined TNBC with tissue availability. DDRD signature (44 gene signature, Almac Inc.) and BRCA1expression (NanoString nCounter) were performed on RNA isolated from pre-treatment FFPE tumor tissue. DDRD score was classified in quartiles. TILs evaluation was performed using previously described criteria. Markers were tested for prognostic effect on DFS and OS using Cox regression model with adjustment for randomized treatment assignment. Results: For 425 TNBC patients median age: 45 yrs, and 5 year DFS and OS = 74% and 82%, respectively. DDRD signature was successfully evaluated in 89.6% (381/425) but only 267 (62.8%) met 60% tumor content criterion for inclusion. DDRD score quartiles were associated with DFS (5 year DFS 59% & 82% in the lowest & highest quartiles respectively, p = 0.0005) and OS (5 year OS 74% and 86% in lowest and highest quartiles respectively, p = 0.008). BRCA1 expression and TILs were successfully determined in 78% and 99% samples, respectively. BRCA1expression was not associated with DFS. TILs were associated with DFS (10% increase HR = 0.88; 95% CI 0.79-0.97; p = 0.016) and OS (HR = 0.84; 95% CI 0.74-0.94; p = 0.0005). DDRD score and TILs were highly correlated (Pearson = 0.62). In multivariate model of DFS including TILs and DDRD quartiles, only DDRD remains significant (p = 0.018). Conclusions: DDRD signature was prognostic in TNBC patients treated with AC chemotherapy and has the potential to be used as a selection criterion to identify TNBC patients whose prognosis is sufficiently poor to justify evaluation of alternative treatment.

Identification of differentially expressed genes and their upstream regulators in colorectal cancer.

To identify the differentially expressed genes (DEGs) and their transcription factors (TFs) in colorectal cancer (CRC). We performed an integrated analysis of microarray studies. Functional annotation and CRC-specific transcriptional regulatory network construction were performed. Expression of selected DEGs and TFs was verified with The Cancer Genome Atlas (TCGA) data sets and qRT-PCR. SurvMicro was used to analyze the correlation between the overall survival time of CRC patients and the expression of DEGs and TFs. Seven data sets were obtained and 2014 DEGs in CRC were identified. Pathways in cancer and fatty acid metabolism were significantly enriched pathways of upregulated and downregulated DEGs, respectively. Expression of five DEGs (RERGL, ESM1, CA1, ANGPTL7 and TMEFF2) and their five TFs (ZNF354C, ARID3A, NFIC, BRCA1 and ZEB1) was verified by TCGA data sets and qRT-PCR. Their expression in TCGA data sets was same as that in our integrated analysis. Only the expression of EMS1, NFIC, BRCA1 and ZEB1 was inconsistent with integrated analysis and TCGA data sets. Expression of RERGL and BRCA1 was significantly correlated with the overall survival time of CRC patients. These five DEGs may have roles in CRC regulated by their five upstream TFs, which may make a contribution in uncovering the mechanism and providing new strategy of diagnosis and therapies for CRC.

Decreased expression of BRCA2 accelerates sporadic breast cancer progression

Decreased expression of BRCA2 accelerates sporadic breast cancer progression

Let-7e sensitizes epithelial ovarian cancer to cisplatin through repressing DNA double strand break repair

BackgroundResistance to platinum-based chemotherapy remains a great challenge for ovarian cancer treatment. The human let-7 family contains 13 members located on nine different chromosomes, and most members have been implicated in the modulation of drug sensitivity in cancers. Our previous study showed that deregulation of let-7e in epithelial ovarian cancer (EOC) promoted the development of resistance to cisplatin. In the present study, we aimed to investigate the underlying mechanism and further evaluate the clinical value of let-7e in predicting chemo-response and prognosis in EOC.ResultsIn situ hybridization assays revealed a significantly decreased expression of let-7e in chemo-resistant EOC tissues compared with chemo-sensitive cases. Transfection with let-7e agomir sensitized EOC cells to cisplatin, down-regulated BRCA1 and Rad51 expression, and repressed the repair of cisplatin-induced DNA double strand break, while let-7e inhibitor exerted the opposite effects. In human EOC tissues, BRCA1 and Rad51 levels were increased in the chemo-resistant group compared with the sensitive group and were negatively correlated with let-7e. Low let-7e and high Rad51 were significantly associated with poor progression-free survival and overall survival and multivariate regression analyses showed that let-7e was an independent predictor for overall survival and chemotherapy response in EOC. Receiver operating characteristic analysis indicated that let-7e level was highly predictive of resistance to platinum-taxane chemotherapy with an area under the curve of 0.826.ConclusionsIn EOC, low let-7e leads to activation of BRCA1 and Rad51 expression and subsequent enhancement of DSB repair, which in turn results in cisplatin-resistance. Let-7e is a potential predictor for survival and chemo-response in EOC and re-expression of let-7e might be an effective strategy for overcoming chemo-resistance.

RNA-Seq Analysis of Microglia Reveals Time-Dependent Activation of Specific Genetic Programs following Spinal Cord Injury.

Neurons have inherent competence to regrow following injury, although not spontaneously. Spinal cord injury (SCI) induces a pronounced neuroinflammation driven by resident microglia and infiltrating peripheral macrophages. Microglia are the first reactive glial population after SCI and participate in recruitment of monocyte-derived macrophages to the lesion site. Both positive and negative influence of microglia and macrophages on axonal regeneration had been reported after SCI, raising the issue whether their response depends on time post-lesion or different lesion severity. We analyzed molecular alterations in microglia at several time-points after different SCI severities using RNA-sequencing. We demonstrate that activation of microglia is time-dependent post-injury but is independent of lesion severity. Early transcriptomic response of microglia after SCI involves proliferation and neuroprotection, which is then switched to neuroinflammation at later stages. Moreover, SCI induces an autologous microglial expression of astrocytic markers with over 6% of microglia expressing glial fibrillary acidic protein and vimentin from as early as 72 h post-lesion and up to 6 weeks after injury. We also identified the potential involvement of DNA damage and in particular tumor suppressor gene breast cancer susceptibility gene 1 (Brca1) in microglia after SCI. Finally, we established that BRCA1 protein is specifically expressed in non-human primate spinal microglia and is upregulated after SCI. Our data provide the first transcriptomic analysis of microglia at multiple stages after different SCI severities. Injury-induced microglia expression of astrocytic markers at RNA and protein levels demonstrates novel insights into microglia plasticity. Finally, increased microglia expression of BRCA1 in rodents and non-human primate model of SCI, suggests the involvement of oncogenic proteins after CNS lesion.

Abstract B36: Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC

Abstract Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course, resistance to chemotherapy, and poor prognosis compared to other breast cancer subtypes. However, breast cancers arising in BRCA1 mutation carriers appear to be particularly sensitive to platinum-based chemotherapy agents. The tumor suppressor BRCA1 (breast cancer type 1 susceptibility protein) is part of a protein complex that repairs DNA damage by homologous recombination. Recent studies have shown that tumor cells expressing high levels of BRCA1 are resistant to both ionizing radiation (IR) and platinum-based chemotherapy agents, and that ablation of BRCA1 expression can restore sensitivity to these agents. We have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. However, established HSP90 inhibitors exhibit a high level of toxicity unrelated to HSP90. Recently, we observed that the natural compounds gedunin and celastrol modulate HSP90 activity without inducing side effects. We believe that combinatory use of these compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies. Study Design: The goal of these experiments is to demonstrate that combination treatment with the HSP90 inhibitors gedunin and celastrol will maximize BRCA1 degradation (via inhibition of HSP90), impair the DNA repair system, and allow carboplatin to effectively eradicate TNBC cells. We will confirm that this is a HSP90 mediated event that results in the impairment of DNA repair. Using in vivo models of breast cancer, we will demonstrate that natural compounds could sensitize BRCA1-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth. Results: Using Western blot analysis, we observed that the combination of gedunin and celastrol attenuates HSP90 activity, leading to a dose-dependent reduction in BRCA1 degradation in HCC1937BRCA1 TNBC cells. Additionally, we the combination facilitated carboplatin-mediated inhibition of proliferation in both BT-20 and HCC1937BRCA1 cells. Immunofluorescence staining for g-H2AX and RAD51 demonstrated that the two HSP90 inhibitors prevent BRCA1-mediated recruitment of DNA repair complexes following ionizing radiation-induced DNA damage. To test the effects of celastrol and gedunin in vivo, we generated mouse xenografts by performing fat pad injections of HCC1937BRCA1 cells. Mice were dosed I.P. every 5 days with celastrol (5 mg/kg), gedunin (5 mg/kg), and carboplatin (25 mg/kg) for 20 days. Treatment with the HSP90 inhibitors along with carboplatin resulted in significant reduction in both tumor size and weight. In contrast treatment with carboplatin alone resulted in tumors of size similar to vehicle-treated animals. Additionally, phospho-H3 and cleaved caspase 3 immunofluorescence staining indicated a corresponding decrease in cell proliferation and increased apoptosis in tumors from celastrol+gedunin+carboplatin -treated animals compared to either vehicle or carboplatin alone-treated. Future studies will focus on examining the effects of this combination treatment in TNBC xenografts created with patient specimens, and in defining the interaction between HSP90 and BRCA1. Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC. Citation Format: Kelli Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B36.

P08.18 Replication stress as a driver of genomic instability in malignant gliomas

AbstractGlioblastoma (GBM, WHO grade IV astrocytoma) is among the deadliest of solid tumors with median survival rates of only approximately 15 months. In spite of concerted efforts, molecular mechanisms/genes causing high recurrence and treatment resistance are poorly understood. The tumor suppressor BRCA1 plays a critical role in maintaining genomic stability, in general, and replication stress (RS), in particular. Numerous reports have demonstrated that wild type BRCA1 loss impairs the growth of several cancers (breast, ovarian, lung, prostate and colon). Our data show considerably higher level of RS in malignant gliomas compared to other solid tumors (breast, prostate ovarian) and identify a novel role for BRCA1, where high BRCA1 expression negatively associates with glioma patient survival. BRCA1 plays an unexpected tumor-promoting role in GBM, safeguarding a protective response to supraphysiological RS levels via transcriptional regulation of RRM2 expression. shRNA-mediated knockdown of BRCA1 or chemical inhibition of RRM2 both induce GBM cell death due to accumulation of RS-induced DNA damage in vitro and extend survival of tumor bearing mice in vivo, highlighting the clinical relevance of our findings. Acknowledgements: This work was supported by Danish Cancer Society Foundation.

BRCA1 expression, proliferative and apoptotic activities in ovarian epithelial inclusions

BackgroundThe purpose of this study was to examine proliferative and apoptotic activity in relation with BRCA1 expression in ovarian epithelial inclusions (OEIs), the putative precursor lesions of ovarian epithelial cancer (OEC).MethodsBenign ovaries from 95 patients were examined. Dual immunohistochemical staining for both BRCA1 and MIB-1 were performed to examine the relationship between BRCA1 and MIB-1 in OEI cells. Apoptotic activity was assessed on the parallel tissue sections by using TUNEL assay. Patients’ age, menstrual phase and menopausal status were analyzed.ResultsOEIs were present in the ovaries of 53% of the patients. OEIs were less frequently found in premenopausal (45%) than postmenopausal women (58%). BRCA1 and MIB-1 were found in 27 and 47% of the OEI-containing ovaries, respectively. All BRCA1 positive OEI cells are MIB-1 positive with dual staining method, although overall the percentage of positive cells was small. No significant difference was found for BRCA1 and MIB-1 expression in OEIs between menopausal status and menstrual phases. Apoptosis containing OEIs were seen in 70% of the ovaries. Compared to OEIs in proliferative menstrual phase and premenopausal status, significantly more apoptosis was found in OEIs from secretory phase and postmenopausal women. A small fraction of the epithelial cells within OEIs are proliferating or dying.ConclusionsLow estrogen and/or high progesterone levels may promote OEI cell turnover via induction of apoptosis. Imbalance between cell proliferation and death within OEIs under influence of hormones may play a role in the ovarian epithelial tumorigenesis.