BRCA1 Mutant Breast Cancer Cells Research Articles

1839: Stimulation of immune response to overcome radioresistance in BRCA1 mutant breast cancer cells

1839: Stimulation of immune response to overcome radioresistance in BRCA1 mutant breast cancer cells

Induction of the DNA-Repair Gene POLQ only in BRCA1-mutant Breast-Cancer Cells by Methionine Restriction.

BRCA1/2 mutations in breast cancer cells impair homologous recombination and promote alternative end joining (Alt-EJ) for DNA-damage repair. DNA polymerase theta, encoded by POLQ, plays a crucial role in Alt-EJ, making it a potential therapeutic target, particularly in BRCA1/2-mutant cancers. Methionine restriction is a promising approach to target cancer cells due to their addiction to this amino acid. The present study investigated the expression of POLQ in BRCA1/2 wild-type and BRCA1-mutant breast cancer cells under methionine restriction. POLQ mRNA expression was measured using qRT-PCR in BRCA1/2 wild-type (MDA-MB-231) and BRCA1- mutant (HCC1937 and MDA-MB-436) breast-cancer cells under normal, or serum-restricted, or serum- and methionine-restricted conditions. Compared to BRCA1/2 wild-type cells, BRCA1-mutant cells displayed significantly higher basal POLQ expression in normal medium. Methionine restriction further increased POLQ expression in the BRCA1-mutant cells but decreased it in the BRCA1/2 wild-type cells. The present findings suggest that methionine restriction showed differential effects on POLQ expression, potentially impacting Alt-EJ activity, in BRCA1/2 wild-type and BRCA1-mutant breast-cancer cells. Further investigation is needed to explore the potential of combining methionine restriction with DNA-repair inhibitors, such as PARP inhibitors, to overcome drug resistance in BRCA1/2 mutant cancers.

Self-Care: Seeking Queer Liberation from the Medical Gaze and Genetic Fatalism

Genetic diagnostics are radically and rapidly changing perceptions of health. Individuals with identifiable pathogenic genetic differences are now being made into cultural mutants. Unlike other forms of embodied difference, these diagnostics rely on a high statistical probability of developing a disease, known as genetic risk. As such, individuals with a mutation are often subjected to perform the same clinical labor as those who are already sick with a disease, which can involve invasive medical surveillance, preventative surgeries, and family planning. Self-Care is my artistic attempt to reckon with these biotechnological ruptures in identity caused by the rising use of genetic diagnostics in medicine. Using my body, Self-Care weaves a narrative about health, gender, and identity that seeks to resist the confines of the medical gaze. The work features a specially designed chest binder housing living BRCA1 mutant breast cancer cells, which allows the artist to take on the caring responsibilities of their cancer before it emerges in their body. Building off the artwork, this paper explores contemporary issues surrounding Hereditary Breast and Ovarian Cancer Syndrome in global healthcare systems. Traversing the boundaries between sick and healthy, male and female, and parent and child, this paper sets out to both present the scholarly research surrounding Self-Care and provide a platform of critical self-reflection for the artwork to question how best we can care for ourselves and others.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer.

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer.

Synthesis and evaluation of a new series of spiro aryl dioxolane compounds: A new scaffold as potential poly(ADP‐ribose)polymerase‐1 (PARP‐1) inhibitors

AbstractA new series of spiro aryl dioxolane derivatives were synthesized and evaluated to find a new scaffold as potential poly(ADP‐ribose)polymerases‐1 (PARP‐1) inhibitors. This key starting compound, 2,6‐bis(methoxybenzylidene)cyclohexanone, was functionalized with different nucleophilic reagents via cyclocondensation reactions to obtain new benzylidene scaffolds containing diverse aryl groups such as spiroindazoleorquinazoline [1,3]dioxolane compounds. Furthermore, the Michael addition reaction of bis‐benzylidene with some active methylene compounds afforded spirochromene‐ or naphthalene‐[1,3]dioxolane compounds. All the synthesized compounds revealed promising inhibition with IC50 values in the nanomolar range (0.997–2.698 nM), not significantly different from that of Olaparib (IC50 = 0.861 nM). Compounds 5b and 15 (IC50 = 1.009 and 0.997 nM) showed the highest potency among all the prepared compounds and accordingly their ability to inhibit the growth of BRCA1 mutated breast cancer cell line MDA‐MB‐436 was tested, where compound 5b (IC50 = 0.67 μM) showed a potency ten folds higher than that of Olaparib but compound 15 (IC50 = 7.47 μM) exerted lower activity but still comparable to that of Olaparib (IC50 = 6.84 μM). Both resulted in the arrest of cell cycle at S phase and caused cell apoptosis. In silico studies including absorption, distribution, metabolism, excretion (ADME) properties, drug‐likeness, and molecular docking were carried out to support the above‐mentioned findings. This work presents compounds 5b and 15 as promising scaffolds as PARP‐1 inhibitors.

Pregnancy Hormone Mediated Tumorigenesis in BRCA1 Defective Breast Cancers

Background and ObjectiveBRCA1 mutations predispose to breast and ovarian cancers, the exact reason for the tissue specificity is unknown. The role of hormonal factors estrogen/estrogen receptor α (ER‐α) was hypothesized to be a major contributing factor for this tissue specificity. However, tumor progression in BRCA1 defective condition could not be controlled by inhibiting ER‐α as they either lack or exhibit very low expression of ER‐α, progesterone receptor, and human epidermal growth factor receptor. In addition to these hormone receptors, the prominent hormone known to be important for the development and differentiation of breast tissue is the Human Chorionic Gonadotropin (hCG). Thus, we speculated the role of hCG, especially the β‐hCG, to be the key molecule actively contributing to the tumorigenesis in TNBCs.In our previous study, we have identified for the first time that β‐hCG expression is linked to BRCA1 status. β‐hCG overexpression was observed in BRCA1 mutated breast cancer cells and conditional knockout mouse models. ChIP and luciferase assay also demonstrated the transcriptional regulation of BRCA1 over β‐hCG. β‐hCG mediate signaling through TGFβRII and promotes migration and invasion predominantly in BRCA1 mutated breast cancer cells (Sengodan et al., 2017). In accordance with the above findings, the present study aims to analyze the effectiveness of β‐hCG as a potential diagnostic marker for BRCA1 mutated cancers. This involves the analysis of β‐hCG expression in cancer tissues, followed by a detailed investigation of its link with BRCA1 mutation and analysis of its protein level in the serum of BRCA1 defective cancer patients also, to understand the mechanism of transcriptional regulation of BRCA1 over β‐hCG.MethodsImmunohistochemical analysis, ELISA, Cloning, Protein expression and purification, Electrophoretic Mobility shift Assay (EMSA)ResultsImmunohistochemical analysis of β‐hCG in tumor tissue microarray showed a strong positive expression of β‐hCG in BRCA1mutated breast cancer. Significant levels of β‐hCG in serum was not observed in breast cancer patients when compared to age matched normal control group probably because detectable levels of β‐hCG may not be present in blood. This analysis has to be carried out in a larger cohort of patient samples. To understand the transcriptional regulation of BRCA1 over β‐hCG, the DNA binding domains of BRCA1were cloned into pET‐28b vector, expressed in BL21DE3 cells and purified by Ni‐NTA affinity chromatography. Purified proteins were used for electrophoretic mobility shift assay (EMSA). Results of EMSA showed that BRCA1 did not exhibit any specific binding with β‐hCG promoter. These results suggest that BRCA1 regulation could be due to protein‐protein association with additional DNA binding transcription factors which has to be elucidated in future experiments.ConclusionThe present study highlights the significance of the regulation of β‐hCG in BRCA1 mutated cancers, since a very high expression of β‐hCG in BRCA1 mutated breast cancer is observed, further a detailed study on the same will provide more insights into the exact role of this molecule during breast cancer progression.

MicroRNA expression in CD44-positive BRCA1-mutant breast cancer cells after adding anticancer drug gemcitabine

MicroRNA expression in CD44-positive BRCA1-mutant breast cancer cells after adding anticancer drug gemcitabine

Interactions of BRCA1-mutated Breast Cancer Cell Lines with Adipose-derived Stromal Cells (ADSCs)

Lipofilling may constitute a technique to assist reconstruction of breasts following prophylactic mastectomy for patients with mutated BRCA1 or BRCA2 genes. However, to date it is not clear whether adipose-derived stromal cells (ADSCs) increase the risk of tumor initiation and progression in this situation. Therefore, the interactions of BRCA1 mutated breast cancer cell lines with normal ADSCs were investigated in the present study. Characteristics of MDA-MB-436 (BRCA1 c.5277 + 1G > A) and HCC1937 (BRCA1 p.Gln1756.Profs*74) were compared to MDA-MB-231 and T47D BRCA1/2 wild-type breast cancer cell lines. ADSCs were cultivated from lipoaspirates of a panel of BRCA1/2- wildtype patients. Interactions of conditioned medium (CM) of these cells with the breast cancer lines were studied using proliferation and migration assays as well as adipokine expression western blot arrays. CM of ADSCs exhibit a dose-dependent stimulation of the proliferation of the breast cancer cell lines. However, of the ADSC preparations tested, only 1 out of 18 samples showed a significant higher stimulation of BRCA1-mutated MDA-MB-436 versus wildtype MDA-MB-231 cells, and all CM revealed lower stimulatory activity for BRCA1-mutated HCC1937 versus wildtype T47D cells. Additionally, migration of breast cancer cells in response to CM of ADSCs proved to be equivalent or slower for BRCA1/2 mutated versus nonmutated cancer cells and, with exception of angiopoietin-like 2, induced expression of adipokines showed no major difference. Effects of media conditioned by normal ADSCs showed largely comparable effects on BRCA1-mutated and wildtype breast cancer cell lines thus advocating lipofilling, preferentially employing allogeneic non-mutated ADSCs.

Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.

Abstract B91: BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts

Abstract Tumor microenvironment plays an indispensable role in cancer development as it can be described as the territory where the interaction takes place between tumor and microenvironment elements such as the endothelial cells, epithelial cells, fibroblasts, immune cells, and ECM proteins. Among these structural stromal components, specifically cancer-associated fibroblasts (CAFs) have attracted substantial attention over recent decades. CAFs in the tumor microenvironment assist the accelerated growth demands of the cancer cells and enhance cancer progression through their paracrine activity by the enhanced secretion of growth factors and cytokines, which also help in remodeling the extracellular matrix (ECM). Studies reveal that CAFs have extended their role in cancer metastasis by accompanying the cancer cells to distant metastatic sites. From our in vitro studies, we have identified that coculturing the CAFs with the conditioned medium (CM) from BRCA1 mutated but not wild-type cancer cells results in the transformation of CAFs to an altered phenotype. Since these transformed CAFs showed increased migrative, invasive, and proliferative abilities, we named them “Metastasis Associated Fibroblasts” (MAF) (Sreelatha et al., 2018). The MAF thus generated showed a significant increase in the expression of the Ezrin, Radixin, and Moesin (ERM) protein complex, which may be attributed to the association of BRCA1 C-terminal (BRCT) domains of BRCA1 with the ERM protein superfamily. This association of BRCA1 with the key regulators of ERM complex can influence cell adhesion and motility, suggesting that BRCA1 may act upon their signaling pathways. To study the effect of BRCA1 mutation and ERM protein expression in CAFs in vivo, BRCA1 mutated breast cancer cells were orthotopically implanted to NOD-SCID mice. CAFs isolated from the tumor were analyzed for the expression of ERM. It was found that there is a significant increase in the expression of ERM protein in the tumors isolated from mice that were implanted with BRCA1 mutated breast cancer cells compared to the mouse group that received BRCA1 wild-type breast cancer cells. Inhibitors to ezrin and CCL5 could effectively reduce the metastatic potential of BRCA1 mutant cancer models. A combination of ezrin inhibitor (NSC 668394) and plumbagin, a plant-derived naphthoquinone that effectively reduces BRCA1 mutant breast cancer cell proliferation, is under investigation. Citation Format: Neetha Rajan Latha, Geetu Rose Varghese, Vishnu Sunil Jaikumar, Arathi Rajan, Revathy Nadhan, Arathy Warrier, Priya Srinivas. BRCA1 mutation in breast cancer cells and generation of metastasis associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B91.

Abstract A57: βhCG regulates immune cell population in BRCA1 mutated breast cancers

Abstract BRCA1 mutation predisposes to tumors mainly to the breast and ovaries, though the exact reason for the said tissue specificity still remains a mystery. We have shown that when breast cancer cells become defective in BRCA1, hCG is overexpressed. Also, we have demonstrated that β-hCG can promote tumor progression by inducing TGFβRII, specifically and selectively in BRCA1 defective breast cancer cells in vitro (Sengodan et al., 2017). β-hCG induces expression of hemoglobin genes and protects the cancer cells during oxidative stress, resulting in drug resistance (Sengodan et al., 2017). Since hCG has immunosuppressive action, we hypothesized that hCG might be skewing the immune response of the BRCA1 defective cells towards tumorigenesis. To examine the immunologic response of βhCG in vivo, we performed studies using NOD-SCID and Balb/C mouse models by orthotopically implanting BRCA1 wild-type, BRCA1 mutated, and βhCG overexpressed BRCA1 mutated breast cancer cells in to the fourth mammary fat pad of the mice. We used flow cytometry to characterize immune cell populations in lymphoid tissues and infiltrating the tumor. Myeloid-derived suppressor cells (MDSC), which promote tumor growth by inhibiting T-cell immunity, M1 and M2 macrophages, which are involved in antitumorigenic and protumorigenic immune responses respectively, CD4/CD8 T cells, and FoxP3 T regulatory cells, which play an important role in T cell-mediated immune response, were analyzed. Comparison of MDSC from spleen and tumor done in various studies has demonstrated that tumor MDSC have more potent suppressive activity than that from other peripheral lymphoid organs (Maenhout et al., 2013; Corzo et al., 2018). In our study there was a significant increase in the percentage of tumor MDSCs and M2 macrophages in the order, BRCA1 wild-type < BRCA1 mutant < βhCG overexpressed BRCA1 mutant breast cancers. Correspondingly, there was a significant decrease in CD8+/FOXP3+ ratio in the order, in the tumors derived from mouse models that received BRCA1 wild-type < BRCA1 mutated < βhCG overexpressed BRCA1 mutated cancers. This reflects a marked decrease in the proportion of tumor-infiltrating cytotoxic CD8+ T cells, coincident with an increase in the proportion of FOXP3+ Treg cells, which adds to the suppressive action of βhCG in BRCA1 mutated breast cancers. Collectively, these findings raise the possibility that induction of βhCG found in BRCA1-mutated tumors increases the population of MDSC and M2 macrophages, thus inhibiting the host antitumor immune response and promoting tumor growth. It was also proved for the first time in our study that βhCG causes a decrease in the proportion of cytotoxic CD8+ T cells in BRCA1 mutant cancer cells. This study shows that resistance to immunotherapy shown by BRCA1 mutated breast cancers could be reverted by modulating βhCG in BRCA1-defective cancer patients. Citation Format: Geetu Rose Varghese, Vishnu Sunil Jaikumar, Arathi Rajan, Neetha Rajan Latha, Dipyaman Patra, Priya Srinivas. βhCG regulates immune cell population in BRCA1 mutated breast cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A57.

CRISPR/Cas9-mediated mutagenesis to validate the synergy between PARP1 inhibition and chemotherapy in BRCA1-mutated breast cancer cells.

For patients carrying BRCA1 mutations, at least one‐third develop triple negative breast cancer (TNBC). Not only is TNBC difficult to treat due to the lack of molecular target receptors, but BRCA1 mutations (BRCA1m) also result in chemotherapeutic resistance, making disease recurrence more likely. Although BRCA1m are highly heterogeneous and therefore difficult to target, BRCA1 gene's synthetic lethal pair, PARP1, is conserved in BRCA1m cancer cells. Therefore, we hypothesize that targeting PARP1 might be a fruitful direction to sensitize BRCA1m cancer cells to chemotherapy. We used CRISPR/Cas9 technology to generate PARP1 deficiency in two TNBC cell lines, MDA‐MB‐231 (BRCA1 wild‐type) and MDA‐MB‐436 (BRCA1m). We explored whether this PARP1 disruption (PARP1m) could significantly lower the chemotherapeutic dose necessary to achieve therapeutic efficacy in both a 2D and 3D tumor‐on‐a‐chip model. With both BRCA1m and PARP1m, the TNBC cells were more sensitive to three representative chemotherapeutic breast cancer drugs, doxorubicin, gemcitabine and docetaxel, compared with the PARP1 wild‐type counterpart in the 2D culture environment. However, PARP1m did not result in this synergy in the 3D tumor‐on‐a‐chip model, suggesting that drug dosing in the tumor microenvironment may influence the synergy. Taken together, our results highlight a discrepancy in the efficacy of the combination of PARP1 inhibition and chemotherapy for TNBC treatment, which should be clarified to justify further clinical testing.

Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target.

BackgroundBRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1.MethodsExpression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.ResultsThe expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G2/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression.ConclusionsLack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

Abstract 1851: Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells

Abstract As single agents, small molecule inhibitors of poly-ADP-ribose polymerase (PARP) have shown remarkable efficacy in BRCA1/2-mutant tumors due to their synthetic lethality in DNA damage response-deficient cells. While PARP inhibitors, such as olaparib, have gained FDA-approval for gynecological malignancies, recurrent disease remains a challenge. To identify drug combinations with the potential to improve the initial response to PARP inhibition, we carried out a high-throughput drug screen in which we examined the ability of olaparib to synergize with more than 500 small molecules in BRCA1-mutant breast cancer cells. The compounds investigated included not only conventional “anti-cancer” agents but also lesser-studied metabolic and stress-inducing drugs. All compounds were tested across 10-point dose curves using an image-based readout to avoid false positives/negatives associated with more traditional metabolic readouts of cell viability (i.e. Cell Titer Glo). This screening approach revealed that inhibition of the unfolded protein response (UPR) sensitizes breast cancer cells to PARP inhibition. We have further characterized interaction of one class of inhibitors that target IRE1 (inositol-requiring enzyme 1), which initiates adaptive responses to ER stress through unconventional mRNA splicing of the transcription factor X-box binding protein 1 (XBP1). Combining olaparib with an inhibitor of IRE1-mediated XBP1 splicing (STF-0831010) synergistically reduced cancer cell viability in BRCA1-mutant SUM149 breast cancer cells. This combination led to increased DNA damage, indicated by increased γ-H2AX staining. This study provides evidence that UPR inhibition sensitizes cells to PARP inhibition, suggesting a previously un-recognized connection between PARP inhibition and protein homeostasis. Furthermore, it identifies a potential new drug combination to increase the efficacy of PARP inhibitors in the clinic. Citation Format: Jennifer E. Endress, Isaac S. Harris, Jacob Stewart-Ornstein, Galit Lahav, Joan S. Brugge. Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1851.

Proteomics Analysis to Assess the Role of Mitochondria in BRCA1-Mediated Breast Tumorigenesis.

Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca2+ homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer. Germline mutations of the BRCA1 gene account for 5–10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher than in non-carriers. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in BRCA1 driven tumorigenesis, we herein based our study on the hypothesis that BRCA1 haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell line (MCF7) and BRCA1 mutated breast cancer cell line (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty-three mitochondrial proteins whose expression resulted significantly altered in response to BRCA1 mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, and energy production and nucleic acid metabolism were, respectively, the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar mitochondrial compartmentalization of specific proteins such us HSP60 and HIF-1α. Particularly intriguing is the correlation between BRCA1 mutation status and HIF-1α localization into the mitochondria in a BRCA1 dependent manner. Data obtained led us to hypothesize an interesting connection between BRCA1 and mitochondria pathways, capable to trigger metabolic changes, which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype.

Reproductive epidemiology of glial tumors may reveal novel treatments: high-dose progestins or progesterone antagonists as endocrino-immune modifiers against glioma.

Female gender, contraceptives, and menopausal hormone replacement treatments containing progesterone analogues associate with higher risk of meningiomas yet with lower risk of gliomas. Progesterone receptor (PR) expression and mifepristone treatment was highly discussed for meningiomas. However, much less is known in regard to progesterone actions in gliomas despite PR expression strongly correlates with their grade. Meningiomas and gliomas may grow faster during gestation; but paradoxically, parousity reduces lifetime risk of gliomas which can be explained with dichotomous cell growth-stimulating and inhibitory actions of progesterone at low versus high levels. Progesterone levels gradually increase in gestation up to 200-fold and the incidence of highly angiogenic brain tumors decreases in the last trimester. Indeed, progesterone stimulates glial tumor cell growth at low doses (10nM) while induces cell kill at higher doses. During gestation, some immune pathways are activated to protect the mother and the fetus against microbial pathogens. In parallel, high-dose medroxyprogesterone acetate (MPA) used in treatment of endometrial carcinoma decreases tumoral expression of PR-B and increases infiltration of cytotoxic T lymphocytes and natural killer cells. MPA also synergies with IL-2 in clinical treatment of renal cancer. In both glioma and meningioma, the dominant cytosolic PR is PR-B which increases cell growth, while PR-A limits cell growth. This seems also paradoxical at the first glance due to opposite behavior of these tumors in diverse endocrine conditions. High-dose progestins may inhibit brain tumor growth by downregulating PR-B, yet the dosage thresholds may differ between glial and meningeal tumors due to higher total PR expression in meningiomas. Supporting this proposal, certain progestins were reported to stimulate meningioma growth in anecdotal reports, but same agents at much higher doses reduced meningioma cell proliferation in pilot clinical studies. PR antagonist mifepristone reduced meningioma growth in some clinical studies, but lacked efficacy in others. In fact, mifepristone also has partial PR agonist efficacy and acts in synergy with MPA to block EC growth. Hence, a similar mechanism of receptor downregulation may also account for mifepristone. Both MPA and mifepristone also harbor myeloprotective features against chemotherapy. Ulipristal is another contraceptive PR antagonist and exerts promising anticancer activity on drug-resistant ovarian cancer and BRCA1-mutant breast cancer cells, which can be tested in animal glioblastoma models. We propose that progestins strongly deserve to be investigated in experimental models of glioblastoma alone and in combination with immunostimulating agents.

Antioxydation And Cell Migration Genes Are Identified as Potential Therapeutic Targets in Basal-Like and BRCA1 Mutated Breast Cancer Cell Lines.

Basal-like breast cancers are among the most aggressive cancers and effective targeted therapies are still missing. In order to identify new therapeutic targets, we performed Methyl-Seq and RNA-Seq of 10 breast cancer cell lines with different phenotypes. We confirmed that breast cancer subtypes cluster the RNA-Seq data but not the Methyl-Seq data. Basal-like tumor hypermethylated phenotype was not confirmed in our study but RNA-Seq analysis allowed to identify 77 genes significantly overexpressed in basal-like breast cancer cell lines. Among them, 48 were overexpressed in triple negative breast cancers of TCGA data. Some molecular functions were overrepresented in this candidate gene list. Genes involved in antioxydation, such as SOD1, MGST3 and PRDX or cadherin-binding genes, such as PFN1, ITGB1 and ANXA1, could thus be considered as basal like breast cancer biomarkers. We then sought if these genes were linked to BRCA1, since this gene is often inactivated in basal-like breast cancers. Nine genes were identified overexpressed in both basal-like breast cancer cells and BRCA1 mutated cells. Amongst them, at least 3 genes code for proteins implicated in epithelial cell migration and epithelial to mesenchymal transition (VIM, ITGB1 and RhoA).Our study provided several potential therapeutic targets for triple negative and BRCA1 mutated breast cancers. It seems that migration and mesenchymal properties acquisition of basal-like breast cancer cells is a key functional pathway in these tumors with a high metastatic potential.

BRCA1 regulation on \u03b2-hCG: a mechanism for tumorigenicity in BRCA1 defective breast cancer

Human chorionic gonadotropin β (β-hCG) has been implicated in breast tumorigenesis. However, the role of this hormone is highly controversial as certain studies suggest it has anti-tumor properties while others have found it to be pro-tumorigenic. To unveil the truth, we have analyzed the expression of β-hCG in breast cancer. We identified for the first time that β-hCG expression is linked to BRCA1 status and its overexpression is seen in BRCA1 mutated breast cancer cells, BRCA1 conditional knockout mouse breast cancer tissues and BRCA1 floxed basal cell carcinoma (BCC) tissues. An analysis of three large, transcriptomic data sets from TCGA (The Cancer Genome Atlas) expression profile confirmed the inverse correlation between BRCA1 and β-hCG in human breast cancer. Using ChIP and luciferase assays, we also demonstrated that the cancer cells with wild-type but not mutant BRCA1 directly repress the expression of β-hCG by binding to its promoter. Further, β-hCG promotes migration and invasion predominantly in BRCA1 mutant breast cancer cells. Interestingly, stable overexpression of β-hCG in BRCA1 mutant but not wild-type breast cancer cells results in the formation of spheres even on monolayer cultures. The cells of these spheres show high expression of both EMT and stem cell markers. Since β-hCG belongs to a cysteine knot family of proteins like TGFβ and TGFβ signaling is deregulated in BRCA1 defective tumors, we checked whether β-hCG can mediate signaling through TGFβRII in BRCA1 mutated cells. We found for the first time that β-hCG can bind and phosphorylate TGFβRII, irrespective of LHCGR status and induce proliferation in BRCA1 defective cells. Our results confirmed that there exists a transcriptional regulation of BRCA1 on β-hCG and BRCA1 mutation promotes β-hCG mediated tumorigenesis through TGFβRII signaling. Thus inhibiting β-hCG-TGFβRII could prove an effective treatment strategy for BRCA1 mutated tumors.

Molecular trail for the anticancer behavior of a novel copper carbohydrazone complex in BRCA1 mutated breast cancer.

Novel chelated metal complexes were synthesized from carbohydrazones to thiocarbohydrazones using metal-based drug designing platforms and their combination effect with Pb, a naphthaquinone were analyzed for anticancer activity in breast cancer cell lines. A panel of BRCA1 wild-type and mutated breast cancer cells: MCF-7 (BRCA1+ /ER+ ), MDA-MB-231 (BRCA1+ /ERα- ), HCC-1937 (BRCA1- /ERα- ), HCC1937/wt BRCA1, MX1 (BRCA1- /ERα- ), and MDA-MB-436 (BRCA1- /ERα- ) were screened for anti-cancer activity. Cu2 (HL)(HSO4 ) · H2 O]SO4 · 6 H2 O (CS2) is the most potent anticancer agent among the copper carbohydrazone and thiocarbohydrazone complexes analyzed in this study. It can be suggested that the presence of sulphate, as pharmacologically active centre, can induce cytotoxicity more effectively when compared to chlorine, bromine, perchlorate, and methanol. This is the first report demonstrating that CS2 can bind to DNA by hindering BamH1 activity and could induce DNA double strand breaks as evidenced by γ-H2AX expression. In addition to this, CS2 could also act as a Topo II inhibitor at a much lower concentration than etoposide and induce apoptosis, making it a potent anticancer agent. In combination with Pb, a potent ROS inducer, CS2 could induce synergistic anti-cancer activity in HR/ BRCA1 defective breast cancer cells. This is the first study reporting the mechanism involved in the induction of apoptosis for a metal chelated copper carbohydrazone complex and its combination effects with Pb in HR defective, BRCA1 mutated breast cancer cells.