Knockdown Of BRCA1 Research Articles

Abstract 3508: The role of BRCA1 in the regulation of telomeres: Implications for genomic stability and malignant transformation

Abstract Background The precise role of the BRCA1 gene product in breast carcinogenesis is not fully understood. BRCA1 protein is a part of the DNA damage repair machinery and may have a role in telomere biology. Telomere maintenance is crucial for malignant transformation. The connection between BRCA1 and telomeres was reported in very few studies so far, mainly in cancer cells. Here we study the crosstalk between telomeres and BRCA1 by knocking down its expression in breast epithelial cells. We also assessed telomere/telomerase parameters in BRCA1 mutation carriers compared to normal age-matched controls. Methods BRCA1 knockdown was performed in HB-2 cells and validated by Western blot. Telomere length was measured by Southern blot. Telomerase activity was detected by the Q-TRAP assay and its gene expression was followed by Q-PCR. The binding of the shelterin proteins to telomeres was evaluated by the ChIP assay; their cellular levels were assessed by Western blot. DNA damage was indicated by following the levels of γH2AX. Pyro-sequencing was employed to decipher the methylation status of the sub-telomeric regions. Telomeres G-strand overhang length was measured by the G-tail assay. Total gene expression was evaluated by Affymetrix whole exome microarrays. Similar methods were employed to study the telomere-telomerase parameters in the BRCA1/2 carriers. Results Telomeres became incrementally longer and dysfunction, as γH2AX protein's expression increased, after silencing of BRCA1. Telomerase activity or hTERT expression remained unchanged, implicating their involvement in our setting. The binding of 5 shelterin members to telomeres did not change; however TRF1 increased its binding to telomeres after BRCA1 knock-down, an increase which preceded telomeres elongation. While the methylation of chromosomes 5 was similar in the knock-downs and controls cells, it incrementally increased in the sub-telomere region of chromosome 10. The G overhang was shorter after BRCA1 knockdown. The knock-down of BRCA1 promoted the expression of a distinct set of genes, some of which may relate to the pre-disposition to malignancy after BRCA1 silencing. BRCA1/2 mutations carriers have different telomere dynamics compared with to that of the normal population. They possess a constant DNA damage, differences in methylation pattern of the sub-telomeric regions and a shorter telomeric G- overhang length. Conclusions BRCA1 has a role in telomere length homeostasis in breast epithelial cells. Its knockdown causes telomeres elongation accompanied by their loss of function which promoted DNA damage in the cells. TRF1 increased its binding to telomeres, contributing to their elongation by a yet unknown mechanism. BRCA1 mutation carriers are more prone to malignant transformation probably due to luck of telomere protection which together with BRCA1 loss of function may promote the accumulation of cancerous mutations. Citation Format: Orit Uziel, Lital Zuriano, Rinat Yerushalmi, Einat Beery, Yardena Nordenberg, Dan Frumkin, Meir Lahav. The role of BRCA1 in the regulation of telomeres: Implications for genomic stability and malignant transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3508. doi:10.1158/1538-7445.AM2014-3508

BRCA1 as a nicotinamide adenine dinucleotide (NAD)-dependent metabolic switch in ovarian cancer

Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer.

Glucocorticoid receptor repression mediated by BRCA1 inactivation in ovarian cancer

BackgroundBRCA mutations are the main known hereditary factor for ovarian cancer. Notably, emerging evidence indicates that the glucocorticoid receptor (GR) has drawn considerable interest in ovarian cancer development. However, dynamic cross-talk between BRCA1 and GR signaling pathways are poorly understood.MethodsThe regulatory effects of BRCA on GR were assessed in 146 serous ovarian cancer patients (28 pairs of BRCA1-mutated or not, 23 pairs of BRCA2-mutated or not, and 22 pairs with hypermethylated BRCA1 promoter or not). BRCA1 promoter methylation was analyzed by bisulfite sequencing using primers flanking the core promoter region. Expression levels of BRCA1 and GR were assessed by immunohistochemistry and real-time PCR. Regression analysis was used to examine the possible relationship between BRCA1 and GR expression levels. The knockdown and overexpression of BRCA1 were achieved using a lentiviral vector in 293 T cells, SKOV3 ovarian cancer cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells.ResultsGR expression levels were unchanged in non-BRCA1-mutated, non-BRCA2-mutated and BRCA2-mutated ovarian cancer compared to their normal tissues; BRCA1 repression (BRCA1 mutation or BRCA1 promoter hypermethylation) ovarian cancer showed decreased GR levels compared to normal tissue; there was a positive correlation between BRCA1 and GR expression in human ovarian cancer specimens; BRCA1 knockdown was effective at inhibiting GR expression, and overexpression of BRCA1 induces an increase in GR levels in ovarian cancer cells.ConclusionsThese results suggest that GR may be a potential target for BRCA1 in ovarian cancer progression.

High prevalence ofGPRC5Agermline mutations inBRCA1-mutant breast cancer patients

In a search for new breast cancer (BC) predisposing genes, we performed a whole exome sequencing analysis using six patient samples of familial BC and identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor GPRC5A. An extended case-control study revealed a tenfold enrichment for this mutation in BC patients carrying the 5382insC allele of BRCA1, the major founder mutation in the Russian population, compared to wild-type BRCA1 BC cases [6/117 (5.1%) vs. 8/1578 (0.5%), p = 0.0002]. In mammary tumors (n = 60), the mRNA expression of GPRC5A significantly correlated with that of BRCA1 (p = 0.00018). In addition, the amount of GPRC5A transcript was significantly lower in BC obtained from BRCA1 mutation carriers (n = 17) compared to noncarriers (n = 93) (p = 0.026). Accordingly, a siRNA-mediated knockdown of either BRCA1 or GPRC5A in the MDA-MB-231 human BC cell line reduced expression of GPRC5A or BRCA1, respectively. Knockdown of GPRC5A also attenuated radiation-induced BRCA1- and RAD51-containing nuclear DNA repair foci. Taken together, these data suggest that GPRC5A is a modifier of BC risk in BRCA1 mutation carriers and reveals a functional interaction of these genes.

Effect of BRCA1 on epidermal growth factor receptor in ovarian cancer

BackgroundBoth BRCA1 and epidermal growth factor receptor (EGFR) play a critical role in ovarian cancer progression. However, the crosstalk between BRCA1 and EGFR signaling pathways in ovarian cancer remains largely unknown.MethodsThe effect of BRCA1 on EGFR was assessed in 146 serous ovarian cancer patients (28 pairs of BRCA1-mutated or not, 23 pairs of BRCA2-mutated or not, and 22 pairs with hypermethylated BRCA1 promoter or not). BRCA1 promoter methylation was analyzed by bisulfite sequencing using primers flanking the core promoter region. Expression levels of BRCA1 and EGFR were assessed by immunohistochemistry and real-time PCR. The knockdown and overexpression of BRCA1 were achieved using a lentiviral vector in 293 T cells, SKOV3 ovarian cancer cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells.ResultsEGFR expression was increased in all cancer tissues compared to normal tissues. Additionally, EGFR expression was higher in normal tissues of BRCA1-mutated patients, and was further increased in cancer tissues; EGFR levels were also significantly elevated in ovarian cancer with promoter hypermethylation-mediated inactivation of BRCA1. BRCA1 knockdown was an effective way to activate EGFR expression in ovarian cancer cells.ConclusionsThese results indicate that BRCA1 may be a potential trigger in transcriptional regulation of EGFR in the development of ovarian cancer.

Regulation of angiotensin II type 1 receptor expression in ovarian cancer: a potential role for BRCA1

BackgroundBoth BRCA1 and angiotensin II type 1 receptor (AGTR1) play a critical role in ovarian cancer progression. However, the crosstalk between BRCA1 and AGTR1 signaling pathways remains largely unknown.MethodsBRCA1 promoter methylation was analyzed by bisulfite sequence using primers focused on the core promoter region. Expression levels of BRCA1 and AGTR1 were assessed by immunohistochemistry and real-time PCR. Regression analysis was used to examine the possible relationship between BRCA1 and AGTR1 protein levels. Knockdown or overexpression of BRCA1 was achieved by using a lentiviral vector in 293 T cells and SKOV3 ovarian carcinoma cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells.ResultsBRCA1 dysfunction (BRCA1 mutation or hypermethylated BRCA1 promoter) ovarian cancer showed decreased AGTR1 levels compared to normal tissue. In contrast, AGTR1 expression was increased in non-BRCA1-mutated ovarian cancer. Notably, BRCA1 activation was an effective way to induce AGTR1 expression in primary ovarian cancer cells and a positive correlation exists between BRCA1 and AGTR1 expression in human ovarian cancer specimens.ConclusionsThese results indicate that BRCA1 may be a potential trigger involved in the transcriptional regulation of AGTR1 in the development of ovarian cancer.

Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγnull mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells.

Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway

M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3% compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P<0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

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

Abstract 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.

Targeting Aberrant DNA Double-Strand Break Repair in Triple-Negative Breast Cancer with Alpha-Particle Emitter Radiolabeled Anti-EGFR Antibody

The higher potential efficacy of alpha-particle radiopharmaceutical therapy lies in the 3- to 8-fold greater relative biological effectiveness (RBE) of alpha particles relative to photon or beta-particle radiation. This greater RBE, however, also applies to normal tissue, thereby reducing the potential advantage of high RBE. As alpha particles typically cause DNA double-strand breaks (DSB), targeting tumors that are defective in DSB repair effectively increases the RBE, yielding a secondary, RBE-based differentiation between tumor and normal tissue that is complementary to conventional, receptor-mediated tumor targeting. In some triple-negative breast cancers (TNBC; ER(-)/PR(-)/HER-2(-)), germline mutation in BRCA-1, a key gene in homologous recombination DSB repair, predisposes patients to early onset of breast cancer. These patients have few treatment options once the cancer has metastasized. In this study, we investigated the efficacy of alpha-particle emitter, (213)Bi-labeled anti-EGF receptor antibody, cetuximab, in BRCA-1-defective TNBC. (213)Bi-cetuximab was found to be significantly more effective in the BRCA-1-mutated TNBC cell line HCC1937 than BRCA-1-competent TNBC cell MDA-MB-231. siRNA knockdown of BRCA-1 or DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key gene in non-homologous end-joining DSB repair pathway, also sensitized TNBC cells to (213)Bi-cetuximab. Furthermore, the small-molecule inhibitor of DNA-PKcs, NU7441, sensitized BRCA-1-competent TNBC cells to alpha-particle radiation. Immunofluorescent staining of γ-H2AX foci and comet assay confirmed that enhanced RBE is caused by impaired DSB repair. These data offer a novel strategy for enhancing conventional receptor-mediated targeting with an additional, potentially synergistic radiobiological targeting that could be applied to TNBC.

Cross-species synthetic lethal interaction screening as a strategy for the identification of novel therapeutic targets in cancer.

11105 Background: Mutation, deletion, or epigenetic silencing of tumor suppressor genes is a near universal feature of malignant cells. However, therapeutic strategies for restoring the function of mutated or deleted genes have proven difficult. Synthetic lethality, an event in which the simultaneous perturbation of two genes results in cellular death, has been proposed as a method to selectively target cancer cells. Identifying and pharmacologically inhibiting proteins encoded by genes that are synthetic lethal with known tumor suppressor mutations should result in selective toxicity to tumor cells. Methods: To identify candidate target proteins we measured all pair-wise genetic interactions between all known orthologs of human tumor suppressor genes (162 genes) and all orthologs of druggable human proteins (~400 genes) in the model organism S. Cerevisiae. Analysis of the data uncovered 2,087 distinct synthetic lethal interactions between a tumor suppressor and druggable gene. A computational algorithm was then developed to identify those interactions which were likely to be conserved in humans based on conservation of the synthetic lethal relationship in the distant fission yeast S. pombe. Results: Our bioinformatic analysis suggested a high probability of conservation of the synthetic lethal interactions between the yeast RAD51 (ortholog of BRCA1) and RAD57 (ortholog of XRCC3) with HDA1 (a histone deacetylase; HDAC). We confirmed this by treating LN428 cells with stable lentiviral knockdown of BRCA1 or XRCC3 with the HDAC inhibitors vorinostat (SAHA) and entinostat (MS-275). Both the BRCA1 and XRCC3 knockdown cell lines were significantly more sensitive to HDAC inhibition relative to wild-type (non-silencing lentiviral control) cell line (Table). Conclusions: These results demonstrate that high-throughput approaches for screening synthetic lethal interactions in model organisms such as S. cerevisiae and S. pombecan serve as a valuable resource in helping to identify novel therapeutic targets in human cancer. [Table: see text]

Abstract 1605: Aurora-A promotes radio- and chemo- resistance through regulation of DNA damage response proteins.

Abstract Background: Poor prognosis of cancer is usually resulted from radio- and chemo-resistance. Aurora-A, a serine/threonine kinase, associated with spindle separation and centrosome duplication, is overexpressed in many cancers. Purposes: High expression of Aurora-A negatively regulates BRCA2, indicating that Aurora-A is probably associated with DNA damage repair, which may mediate resistance of radio- and chemo-therapy. This study was to investigate the role of Aurora-A in radio- and chemo-resistance in order to seek novel therapeutic methods for human cancer. Materials and methods: Cell lines from breast cancer, pancreatic cancer and ovarian cancer, including those with either low Aurora-A expression (MCF-7, PANC-1 and OVCA420), or high Aurora-A expression (MDA-MB-231, BXPC3 and OVCA429), were used to overexpress or silence Aurora-A, BRCA1, or BRCA2. Cell growth curve and colony formation were tested by cell counting and soft agar assay. Flow cytometry and MTT were used to examine cell apoptosis or cell viability (IC50) after treatment with radiation and cisplatin or VX680 (Aurora-A inhibitor). Western blotting was used to test the expression of DNA damage responsive proteins. Immunofluorescence was used to detect intracellular localization of above proteins. Results: Our data showed that introduction of Aurora-A or silencing of BRCA1 and BRCA2 increased cell growth and colony formation, while silencing of Aurora-A or introduction of BRCA1 and BRCA2 decreased cell proliferation and colony formation. Introduction of Aurora-A also reduced apoptosis in response to gamma-irradiation and cisplatin treatment, while silencing of Aurora-A or treatment of cells with VX680 enhanced apoptosis. Introduction of Aurora-A increased cell viability and IC50 in response to cisplatin treatment, while knockdown of Aurora-A or treatment with VX680 decreased cell viability and IC50. Western blot suggested that Aurora-A had a negative association with BRCA1 and BRCA2. Moreover, overexpression of Aurora-A either by introduction of Aurora-A cDNA or knockdown of BRCA1 or BRCA2 attenuated the expression of DNA damage responsive proteins including RAD51, H2AX, γH2AX, ATR, ERCC2, Chk1, pChk1, Chk2, pChk2,p53, pp53, DNApolζ, which was confirmed by Immunofluorescence. Conclusions: Aurora-A regulates DNA damage responsive proteins including BRCA1, BRCA2, RAD51, H2AX, ERCC2, Chk1, Chk2, ATR, DNA pol ζ to confer cancer radio- and chemo-resistance. Therefore, DNA damage pathway should be essentially considered in the targeted therapy against Aurora-A in cancer patients either with activated Aurora-A or with inactivated BRCA1 or BRCA2. Citation Format: Huizhen Sun, Ziliang Wang, Jiao Meng, Yan Wang, Zihao Qi, Han Xu, Fengjuan Lin, Wen Gao, Jianmin Sun, Jing Hou, Gong Yang. Aurora-A promotes radio- and chemo- resistance through regulation of DNA damage response proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2013-1605 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

A zinc finger nuclease assay to rapidly quantitate homologous recombination proficiency in human cell lines

Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.

High Expression of Stem Cell Marker ALDH1 is Associated with Reduced BRCA1 in Invasive Breast Carcinomas

Cancer stem cells (CSC) have been described in a variety of malignancies, including breast carcinomas. Among several markers, aldehyde dehydrogenase 1 (ALDH1) has been identified as reliable for breast cancer stem cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells expressing ALDH1. We examined 127 breast carcinomas for expression of ALDH1, using immunohistochemistry and correlated with clinicopathological parameters as well as the BRAC1 status. Comparing the results for both ALDH1 and BRCA1 expression showed a significant inverse association between the two, indicating that reduced BRCA1 was more often seen in breast cancer cells expressing ALDH1 (p-value = 0.044). A total of 24/110 (22%) of tumours displayed the ALDH1 + / BRCA1 -/low phenotype, which showed a trend for a relation with a high grade (p-value= 0.056). Cytoplasmic expression of ALDH1 was not correlated with tumour characteristics. Taken together, our findings suggest that increased ALDH1 is inversely correlated with decreased BRCA1 in a series of unselected breast carcinomas. Therefore, ALDH1 positive (cancer stem) cells with reduced BRCA1 phenotype may indicate a subset of patients for whom specific targeting of the CSC marker ALDH1 and more aggressive adjuvant treatment is appropriate.

Abstract 2739: Panobinostat potentiates the cytotoxic effects of ara-C and daunorubicin on acute myeloid leukemia cells through suppressing the DNA damage response

Abstract Acute myeloid leukemia (AML) remains a challenging disease to treat. Resistance to anthracycline [e.g., daunorubicin (DNR)] and ara-C-based chemotherapy is a major cause of treatment failure in this disease. Therefore, new therapies are urgently needed to improve treatment outcome of AML. Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising new class of anti-cancer drugs, which induce differentiation, cell cycle arrest, and apoptosis in human leukemic cells, but not in normal cells. We previously demonstrated strikingly synergistic antileukemic interactions between valproic acid and ara-C in pediatric AML, accompanied by cooperative induction of DNA double-strand breaks (DSBs) and apoptosis. However, the precise molecular mechanisms remain to be identified. Recent studies with solid tumor cell lines established that HDACIs down-regulate expression of genes encoding critical DNA DSB repair proteins, which play critical roles in the DNA damage response (DDR), an important mechanism limiting chemotherapeutic efficacy. These results led to our hypothesis that HDACIs suppress the DDR in AML cells to enhance the cytotoxic effects of the DNA damaging chemotherapeutic drugs, DNR and ara-C. In four AML cell lines and ten diagnostic AML blast samples, treatment with the novel pan-HDACI, panobinostat, resulted in suppressed expression of BRCA1, CHK1, and RAD51 in a dose-dependent fashion, accompanied by dose-dependent induction of apoptosis or proliferation arrest. Treatments of the AML cell lines with ara-C or DNR resulted in induction of RAD51, BRCA1, and CHK1, which was abolished by co-administration of panobinostat. Importantly, panobinostat substantially enhanced ara-C- or DNR-induced DNA DSBs, as reflected in the induction of γH2AX, an established biomarker for DNA DSBs. This was accompanied by a dramatic synergistic induction of apoptosis. Whereas panobinostat alone resulted in G1 arrest, ara-C and DNR alone caused S-G2/M and G2/M arrest, respectively. The latter effects were abrogated when these agents were simultaneously administered with panobinostat. Interestingly, shRNA knockdown of BRCA1, CHK1, or RAD51 caused delayed repair of DNA DSBs induced by ara-C or DNR. Further, shRNA knockdown of BRCA1 or CHK1 resulted in significantly increased basal and ara-C- or DNR-induced apoptosis, along with substantially increased DNA DSBs and abrogated S and G2 checkpoints. In contrast, shRNA knockdown of RAD51 significantly enhanced DNR-induced DSBs and apoptosis, but had no impact on S and G2 checkpoints. These results support our hypothesis that suppression of the DDR by HDACIs represents a novel mechanism underlying the antileukemic activities of this class of drugs, and the synergy between HDACIs and DNA damaging agents in AML. Studies are underway evaluating in vivo efficacies of these drug combinations using xenograft NSG Mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2739. doi:1538-7445.AM2012-2739

Abstract LB-245: Investigating the role of BRCA1 in sensitivity to GRN163L, a telomerase template antagonist

Abstract BRCA1 is a tumor suppressor gene with a variety of functions related to safeguarding genomic integrity. Regulation of telomere length is crucial in maintaining genomic stability, with critically short telomeres leading to telomere uncapping, end-to-end fusions, activation of the DNA damage response, and cell cycle arrest. Recent evidence suggests knockdown of BRCA1 in cell lines increases both telomerase activity and average telomere length, but likely results in more unstable telomeres compared to BRCA1 wild-type cell lines. The objective of this study was to determine whether GRN163L, a telomerase template antagonist currently in clinical trials, has enhanced activity in BRCA1 mutant breast or ovarian cancer cell lines compared to paired BRCA1 wild-type breast/ovarian cancer cell lines. In addition, we sought to determine whether BRCA1 cell lines have shorter average telomere lengths than other cancer cell lines, making them more amenable to inhibition of telomerase activity. We utilized a panel of breast and ovarian cancer cell lines harboring both N-terminal and C-terminal BRCA1 mutations, in addition to matched cells expressing exogenous wild-type BRCA1, and assessed the effects of GRN163L or a mismatch oligonucleotide on telomerase activity and telomere length using the TRAP (Telomere Repeat Amplification Protocol) and TeloTAGGG assays, respectively. All cell lines tested exhibited a dose-dependent response to treatment with GRN163L, but not a mismatch control oligonucleotide. In addition, we observed differential sensitivity between BRCA1 mutant and BRCA1 wild-type cell lines tested to clinically relevant concentrations of GRN163L in both TRAP and clonogenic survival assays. Of interest, the majority of the cell lines tested showed gross morphological changes as early as 12 hours following inhibitor treatment. We found UWB1.289 cells have a shorter average telomere length at baseline compared to their BRCA1 wild-type counterpart cells, and saw progressive telomere shortening over a three-week period of treatment with GRN163L, but not with mismatch oligonucleotide. Furthermore, long-term (12 week) treatment with GRN163L preferentially induced complete cell death in the HCC1937 (BRCA1 mutant) breast cancer cell population compared to the HCC1937 cells reconstituted with wild-type BRCA1. Finally, we demonstrate that pretreatment with GRN163L acts synergistically with cis-platin in UWB1.289 and UWB1.289+BRCA1 ovarian cancer cells. In summary, this work provides insight into the role of BRCA1 mutations in cancer cells and their impact on sensitivity to the cellular effects of telomerase antagonism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-245. doi:1538-7445.AM2012-LB-245

Inhibition of constitutively activated phosphoinositide 3‐kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1‐defective breast cancer cells

Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103, and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.

Detoxification: A Novel Function of BRCA1 in Tumor Suppression?

Our studies found that BRCA1 levels negatively correlate with DNA adducts induced by Benzo(a)pyrene (BaP). Pulse-chase experiments showed that the increase in BaP-induced DNA adducts in BRCA1 knockdown cells may not be associated with BRCA1’s function in nucleotide excision repair activity; rather, it may be associated with its function in modulating transcriptional regulation. BRCA1 knockdown in MCF-10A cells significantly attenuated the induction of CYP1A1 following BaP treatment indicating that the increase in BaP-induced adducts in BRCA1 knockdown cells is not CYP1A1 dependent. However, our study shows that BRCA1 defective cells may still be able to biotransform BaP by regulating other CYP enzymes, including CYP1B1. Knockdown of BRCA1 also severely affected the expression levels of two types of uridine diphosphate glucorunyltransferase (UGT1A1 and UGT1A9) and NRF2. Both UGTs are known as BaP-specific detoxification enzymes, and NRF2 is a master regulator of antioxidant and detoxification genes. Thus, we concluded that the increased amount of BaP-induced DNA adducts in BRCA1 knockdown cells is strongly associated with its loss of functional detoxification. Chromatin immunoprecipitation assay revealed that BRCA1 is recruited to the promoter/enhancer sequences of UGT1A1, UGT1A9, and NRF2. Regulation of UGT1A1 and UGT1A9 expression showed that the induction of DNA adducts by BaP is directly affected by their expression levels. Finally, overexpression of UGTs, NRF2, or ARNT significantly decreased the amount of BaP-induced adducts in BRCA1-deficient cells. Overall, our results suggest that BRCA1 protects cells by reducing the amount of BaP-induced DNA adducts possibly via transcriptional activation of detoxification gene expression.

Abstract 229: A cross-species approach to identifying genetic modifiers of BRCA1-associated breast cancer risk

Abstract Background: Although carriers of germline mutations of BRCA1 have a high risk of developing breast and ovarian cancers, penetrance is not complete, and it is likely that unidentified genes modify the risk for individual mutation carriers. We hypothesize that genes that modify the penetrance of cancer in BRCA1 carriers interact with BRCA1 mutations to confer increased sensitivity to DNA damage at the cellular level, and that cellular sensitivity to DNA damage can be used as an intermediate phenotype to predict breast cancer susceptibility in humans. Objective: Our goal is to test a cross-species systems biology approach to predict gene-gene interactions underlying breast cancer susceptibility, using sensitivity to DNA damage as an intermediate phenotype. Experiment Procedure: The yeast RAD9 gene is one putative homolog of the human breast tumor suppressor gene BRCA1. We conducted a genome-wide screen to identify genes that interact with a rad9 mutation to confer enhanced sensitivity to DNA damage. The yeast screen identified 25 interactions, from which we selected 10 candidate gene-gene interactions to be tested in human breast epithelial cells. To determine if the gene-gene interactions discovered in yeast are conserved in human mammary epithelial cells, we built stable human mammary epithelial cells (MCF10A) capable of single- or double-gene knockdown via lentivirus-mediated RNA interference. Cells confirmed for intended gene knockdown were subject to a clonogenic assay to determine if increased sensitivity to DNA damage can be observed in double gene vs. single gene knockdowns. Results: Among the 10 pairs of interactions tested in MCF10A cells, two genes, HLTF and MGMT, were found to interact with BRCA1 to confer enhanced DNA damage sensitivity, based on a clonogenic assay. Cells with combined knockdown of BRCA1 and HLTF show increased sensitivity to a chronic low dose of MMS treatment compared with those with only single knockdown of BRCA1 or HLTF. In addition, distinct morphological features were observed in double knockdown cells with MMS treatment. Cells became enlarged and showed a flat, senescence-like morphology. Even a low concentration of 0.001% MMS induced a strong and irreversible growth arrest in cells with combined knockdown of BRCA1 and HLTF, but not in the single knockdown cells. Conclusion: Our results suggest that it is feasible to use a cross-species (yeast-to-human) comparative systems genetics strategy to identify gene-gene and pathway-pathway interactions. Ongoing studies are aimed at determining whether any of the observed gene-gene interactions contribute to breast cancer penetrance. 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 229. doi:10.1158/1538-7445.AM2011-229