Breast Cancer Susceptibility Protein Research Articles

Exogenous Insulin Effect on the Initiation of Breast Cancer in Type 2 Diabetes Mellitus Patients

Background: Growing evidence has indicated that women with type 2 diabetes mellitus are at an increased risk of developing breast cancer. However, as a major adjuvant treatment, the influence of hormone therapy such as exogenous insulin on type 2 diabetes mellitus in primary breast-cancer remains controversial. Objective: To explore the relationship between different types of hypoglycemic medications for diabetes and the factors that may contribute to the initiation of breast cancer. Methods: The study included 80 blood samples taken from 80 females recruited from Ibn Al-Bitar Center for Cardiac Surgery Hospital in Baghdad, Iraq, between November 2022 and February 2023. They were within the age range of 40 to 70 years. They were divided into three groups according to the treatment strategy and duration of disease as follows: Group 1: (20) patients who take insulin only, Group 2: (20) patients who take metformin with insulin, and G 3: (40) patients on metformin only for less than one year. An enzymatic oxidation technique was used to test the following biochemical parameters for all research groups: Total cholesterol, triglycerides, fasting blood glucose, and high-density lipoprotein cholesterol. Using the Friedewald formula, the very low-density lipoprotein cholesterol and low-density lipoprotein cholesterol were determined. Using an enzymatic process, Insulin levels and Insulin like growth factor-1(IGF-1), Estrogen receptor alpha (ERα) levels, and breast cancer susceptibility protein 1 (BRCA1) were measured with an enzyme-linked immunosorbent assay (ELISA). Glycated hemoglobin (HbA1c) level was measured with a sandwich immunodetection method. Finally, The Homeostasis Model Assessment for insulin resistance (HOMA-IR) was calculated according to the specific formula. Results: The values for HbA1C%, FBS, and lipid profile showed non-significant differences when compared between study groups. While BRCA-1, estrogen receptor alpha (ERα), insulin, insulin growth factor IGF-1, and HOMA IR All showed significant differences among all groups. Conclusion: Taking metformin only for less than one year seemed to contribute to higher levels of BRCA-1.

Na+/H+-exchange inhibition by cariporide is compensated via Na+,HCO3−-cotransport and has no net growth consequences for ErbB2-driven breast carcinomas

Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3−-cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays—with some discrepancies—link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and—in some molecular subtypes—improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 μM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3−-dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by ∼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.

Mechanism of Astragali Radix-Curcumae Rhizoma in treating gastric cancer based on network pharmacology and experimental verification

This study aims to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in the treatment of gastric cancer based on network pharmacology. Further, the SGC7901 cell model of gastric cancer was employed to validate the efficacy and key targets of the herb pair. Firstly, the CCK-8 assay was employed to evaluate the direct effect of HQEZ on the proliferation of gastric cancer SGC7901 cells. Then, network pharmacology methods were employed to investigate the active ingredients, key targets, and key signaling pathways involved in the treatment of gastric cancer with HQEZ. The results showed that HQEZ contained 18 potential active ingredients, such as quercetin, naringenin, and curcumin. The results of gene ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment suggested that the main targets of HQEZ in treating gastric cancer were involved in the regulation of protein serine/threonine kinase activity, activation of mitogen-activated protein kinase(MAPK) activity, cysteine-type endopeptidase activity, and negative regulation of protein serine/threonine kinase activity. The hypoxia-inducible factor-1(HIF-1) signaling pathway, ATP-binding cassette(ABC) transporters, cytochrome P450-mediated metabolism of xenobiotics, p53 signaling pathway, and cell apoptosis were key signaling pathways of HQEZ in treating gastric cancer. The cell experiments demonstrated that HQEZ significantly downregulated the expression of ATP-binding cassette subfamily B member 1(ABCB1), epidermal growth factor receptor(EGFR), phosphorylated serine/threonine kinase(p-AKT), hypoxia inducible factor 1 subunit alpha(HIF1A), B-cell lymphoma 2(BCL2), breast cancer susceptibility protein 1(BRCA1), DNA polymerase theta(POLH), ribonucleotide reductase M1(RRM1), and excision repair cross-complementation group 1(ERCC1), and upregulated the expression of tumor protein P53(TP53) and cysteinyl aspartate-specific proteinase(CAPS3). Finally, a multivariate COX regression model was adopted to study the relationship between gene expression and clinical information data of gastric cancer patients in the TCGA database, which demonstrated that the key targets of HQEZ were associated with the poor prognosis in gastric cancer patients. Further feature selection using the LASSO algorithm showed that EGFR, HIF1A, TP53, POLH, RRM1, and ERCC1 were closely associated with the survival of gastric can-cer patients. In conclusion, HQEZ regulates the expression of genes involved in DNA repair, survival, and apoptosis in gastric cancer cells via multiple targets and pathways, assisting the treatment of gastric cancer.

FIGL1 coordinates with dosage-sensitive BRCA2 in modulating meiotic recombination in maize.

Meiotic crossover (CO) formation between homologous chromosomes ensures their subsequent proper segregation and generates genetic diversity among offspring. In maize, however, the mechanisms that modulate crossover formation remain poorly characterized. Here, we found that both maize BREAST CANCER SUSCEPTIBILITY PROTEIN 2 (BRCA2) and AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) act as positive factors of crossover formation by controlling the assembly or/and stability of two conserved DNA recombinases RAD51 and DMC1 filaments. Our results revealed that ZmBRCA2 not only is involved in repair of DNA double-strand breaks (DSBs), but also regulates crossover formation in a dosage-dependent manner. In addition, ZmFIGL1 interacts with RAD51 and DMC1, and Zmfigl1 mutants had a significantly reduced number of RAD51/DMC1 foci and COs. Further, simultaneous loss of ZmFIGL1 and ZmBRCA2 abolished RAD51/DMC1 foci and exacerbated meiotic defects compared with the single mutant Zmbrca2 or Zmfigl1. Together, our data demonstrate that ZmBRCA2 and ZmFIGL1 act coordinately to regulate the dynamics of RAD51/DMC1-dependent DSB repair to promote crossover formation in maize. This conclusion is surprisingly different from the antagonistic roles of BRCA2 and FIGL1 in Arabidopsis, implying that although key factors that control crossover formation are evolutionarily conserved, specific characteristics have been adopted in diverse plant species. This article is protected by copyright. All rights reserved.

Deubiquitinase BAP1 regulates stability of BRCA1 protein and inactivates the NF-κB signaling to protect mice from sepsis-induced acute kidney injury

Sepsis and its associated organ dysfunction syndrome is a leading cause of death in critically ill patients. Breast cancer susceptibility protein 1 (BRCA1)-associated protein 1 (BAP1) is a potential regulator in immune regulation and inflammatory responses. This study aims to investigate the function of BAP1 in sepsis-induced acute kidney injury (AKI). A mouse model with sepsis-induced AKI was induced by cecal ligation and puncture, and renal tubular epithelial cells (RTECs) were treated with lipopolysaccharide (LPS) to mimic an AKI condition in vitro. BAP1 was significantly poorly expressed in the kidney tissues of model mice and the LPS-treated RTECs. Artificial upregulation of BAP1 ameliorated the pathological changes, tissue injury and inflammatory responses in kidney tissues of the mice, and it reduced the LPS-induced injury and apoptosis of the RTECs. BAP1 was found to interact with BRCA1 and enhance stability of BRCA1 protein through deubiquitination modification. Further downregulation of BRCA1 activated the nuclear factor-kappa B (NF-κB) signaling pathway and blocked the protective roles of BAP1 in sepsis-induced AKI. In conclusion, this study demonstrates that BAP1 protects mice from sepsis-induced AKI through enhancing stability of BRCA1 protein and inactivating the NF-κB signaling.

POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes

BackgroundMalaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium.MethodsA Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony.ResultsThe candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites.ConclusionsBrca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.Graphical

Natural Compounds or Their Derivatives against Breast Cancer: A Computational Study.

Background Breast cancer is one of the most common types of cancer diagnosed and the second leading cause of death among women. Breast cancer susceptibility proteins of type 1 and 2 are human tumor suppressor genes. Genetic variations/mutations in these two genes lead to overexpression of human breast tumor suppressor genes (e.g., BRCA1, BRCA2), which triggers uncontrolled duplication of cells in humans. In addition, multidrug resistance protein 1 (MDR1), an important cell membrane protein that pumps many foreign substances from cells, is also responsible for developing resistance to cancer chemotherapy. Aim of the Study. The aim of this study was to analyze some natural compounds or their derivatives as part of the development of strong inhibitors for breast cancer. Methodology. Molecular docking studies were performed using compounds known in the literature to be effective against BRCA1 and BRCA2 and MDR1, with positive control being 5-fluorouracil, an antineoplastic drug as a positive control. Results The binding affinity of the compounds was analyzed, and it was observed that they had a better binding affinity for the target proteins than the standard drug 5-fluorouracil. Among the compounds analyzed, α-hederin, andrographolide, apigenin, asiatic acid, auricular acid, sinularin, curcumin, citrinin, hispolon, nerol, phytol, retinol palmitate, and sclareol showed the best binding affinity energy to the BRCA1, BRCA2, and MDR1 proteins, respectively. Conclusions α-Hederin, andrographolide, apigenin, asiatic acid, auricular acid, hispolon, sclareol, curcumin, citrinin, and sinularin or their derivatives can be a good source of anticancer agents in breast cancer.

RETRACTED: Cervical cancer treatment of Co(II) coordination polymer through miR-9-5p-regulated BRCA1-OCT1-GADD45 pathways

Fresh Co(II) concluding coordination polymer based on {[Co(μ-ppda)(μ-pbmeix)]∙0.5pbmeix·H 2 O} n ( 1 ) was formed smoothly through the reaction of Co(II) salt with p -phenylenediacetic acid (H 2 ppda) under the conditions of N-donor co-ligand semirigid 1,4-bis(2-methylimidazol-1-ylmethyl)benzene (pbmeix) for cervical cancer therapy. In our biological research, we explored the pathogenesis of cervical cancer and provided new targets for cervical cancer treatment. In patients with cervical cancer and the cervical cancer rat model, the expression of Breast Cancer Susceptibility Protein-1 (BRCA1) was aberrantly upregulated. This phenomenon may play a role in the occurrence and development of cervical cancer. Then, bioinformatics prediction was conducted, and miR-9-5p was speculated as the up-regulator of BRCA1 in cervical cancer cells. The influence of miR-9-5p on the vascular endothelial growth factor and pigment epithelium-derived factor contents of cervical cancer lesions was determined via enzyme‐linked immunosorbent (ELISA) assay. Then, the proliferation of cervical cancer cells was determined via CCK-8 assay after miR-9-5p transfection. Finally, we proved that complex 1 is an excellent candidate for cervical cancer therapy through miR-9-5p-regulated BRCA1-OCT1-GADD45 pathways.

Structural Insight into the Mechanism of PALB2 Interaction with MRG15.

The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

POTENTIAL COUMARIN THIOSEMICARBAZONE HYBRIDS AS BRCA-1 MIMETICS FOR ER POSITIVE BREAST CANCER THERAPY: AN IN-SILICO APPROACH

The goal of this study is to find a novel class of BRCA-1 mimics for Estrogen Receptor α (ERα) breast cancer that works differently from conventional anti-estrogens. Breast Cancer Susceptibility Protein-1 (BRCA-1) is a protein was discovered to bind to ERα and decrease its function by a direct contact between regions inside BRCA1's amino terminus and the carboxy terminus of ERα. A novel class of hybrids with coumate and thiosemicarbazone scaffolds was created with the premise of developing small compounds that imitate the function of BRCA-1 to down regulate the ERα and inhibit the tumor activity of breast cancer cells. Using Schrodinger 2020-2, ADMET and in silico molecular docking tests of the proposed hybrids were performed on the BRCA-1 binding cavity of ERα. TSCO-XIV and TSCO-III are developed hybrids that have high docking scores and good binding interactions with important residues.

BRCA1-BARD1 Regulates Axon Regeneration in Concert with the Gqα-DAG Signaling Network.

The breast cancer susceptibility protein BRCA1 and its partner BRCA1-associated RING domain protein 1 (BARD1) form an E3-ubiquitin (Ub) ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1–BARD1 in postmitotic cells, such as neurons, remain poorly defined. Here, we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα–diacylglycerol (DAG) signaling pathway. This pathway is downregulated by DAG kinase (DGK), which converts DAG to phosphatidic acid (PA). We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1–BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1–BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1–BRD-1 complex regulates axon regeneration in concert with the Gqα–DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.SIGNIFICANCE STATEMENT BRCA1–BRCA1-associated RING domain protein 1 (BARD1) is an E3-ubiquitin (Ub) ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1–BARD1 in postmitotic cells, such as neurons, remain poorly defined. We show here that Caenorhabditis elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase (DGK)-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1–BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1–BARD1 in fully-differentiated neurons.

Dissecting the molecular determinants of clinical PARP1 inhibitor selectivity for tankyrase1

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/β-catenin pathway–related cancers, such as colorectal cancers.

Roles of OB-Fold Proteins in Replication Stress.

Accurate DNA replication is essential for maintaining genome stability. However, this stability becomes vulnerable when replication fork progression is stalled or slowed – a condition known as replication stress. Prolonged fork stalling can cause DNA damage, leading to genome instabilities. Thus, cells have developed several pathways and a complex set of proteins to overcome the challenge at stalled replication forks. Oligonucleotide/oligosaccharide binding (OB)-fold containing proteins are a group of proteins that play a crucial role in fork protection and fork restart. These proteins bind to single-stranded DNA with high affinity and prevent premature annealing and unwanted nuclease digestion. Among these OB-fold containing proteins, the best studied in eukaryotic cells are replication protein A (RPA) and breast cancer susceptibility protein 2 (BRCA2). Recently, another RPA-like protein complex CTC1-STN1-TEN1 (CST) complex has been found to counter replication perturbation. In this review, we discuss the latest findings on how these OB-fold containing proteins (RPA, BRCA2, CST) cooperate to safeguard DNA replication and maintain genome stability.

Abstract 5682: Synergism of PARP inhibitor and MET inhibitor in multiple cancer types with intrinsic and acquired PARP inhibitor resistances

Abstract Leveraging compromised DNA damage repair (DDR) pathways commonly found in tumor cells, a classic strategy in cancer therapy is inducing excessive DNA damage to cause cancer cell death. Small molecule poly(ADP-ribose) polymerase (PARP) inhibitors (PARP-is) have been approved for clinical use in treating breast cancer and ovarian cancer patients bearing DDR-deficient tumors with mutations in breast cancer susceptibility proteins (BRCAm). However, accumulating evidences show that both intrinsic and acquired resistances to PARP-is exist in clinic and pre-clinical animal models. Therefore, we developed panels of cells with acquired PARP-is resistance from PARP-is-sensitive triple negative breast cancer (TNBC) and estrogen receptor positive breast cancer cell lines, and used these cells to screen for common traits that can be targeted with feasible therapeutic agent combinations to overcome PARP-is resistance. Since TNBC lacks of effective targeted therapy so far, we focused on using the panel of PARP-is-resistant TNBC cells in this study. Among the molecular mechanisms known contribute to PARP-is resistance, oncogenic kinase activations, including several hyper-activated receptor tyrosine kinases (RTKs), are involved in enhancing DNA damage repair and decreasing affinity of PARP-is to PARP1. In this study, we systematically screened for activated RTKs in the PARP-is-resistant cells we developed by antibody arrays. We then identified that activations of MET, Axl and EphA2 were common traits in TNBC cells with acquired PARP-is resistance, but not in estrogen receptor positive cells. Among the three RTKs, MET has more small molecules inhibitors that can target it, and thus, we made it a priority in investigating synergism between MET inhibitor and PARP inhibitor in multiple cancer types including TNBC with intrinsic and acquired PARP-is-resistance, high-grade serous ovarian cancer (HGSOC) and liver cancer cells. Here, we demonstrated that combinations of PARP-is and MET inhibitors (MET-is) possess moderate to strong synergism in the different cancer types we studied. As we previously reported that MET translocates into cell nucleus and phosphorylates PARP1 at tyrosine (Y) 907 residue in TNBC with intrinsic PARP-is resistance, we found that this MET-mediated PARP1-Y907 phosphorylation also exist in and can serve as marker to indicate PARP-is resistance among the HGSOC, liver cancer cells and the TNBC cells with acquired PARP-is resistance. We further found that MET phosphorylation is high (immunohistological staining H-score greater than 200) in breast cancer (23 out of 31, 70%) and ovarian cancer (8 out of 23, 35%) patient-derived xenograft mouse model tissue microarrays, suggesting that the combination of MET-is and PARP-is is likely to benefit a huge population of cancer patient in multiple cancer types. Citation Format: Mei-Kuang Chen, Weiya Xia, Qiongzhu Dong, Yi Du, Hung-Ling Wang, Coya Tapia, Yongkun Wei, Ye Han, Yu-Yi Chu, Clinton Yam, Yuan Gao, Yu-Han Wang, Funda Meric-Bernstam, Jinsong Liu, Shao-Chun Wang, Dihua Yu, Mien-Chie Hung. Synergism of PARP inhibitor and MET inhibitor in multiple cancer types with intrinsic and acquired PARP inhibitor resistances [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5682.

1H, 13C and 15N backbone resonance assignment of BRCA1 fragment 219\u2013504

The breast cancer susceptibility protein 1 (BRCA1) plays a central role in the suppression of human breast and ovarian cancer. Germ line mutations of the BRCA1 gene are responsible for the hereditary breast and ovarian cancer (HBOC) syndrome. Here were report 1H, 13C, and 15N resonance assignments for the intrinsically disordered BRCA1 fragment 219–504, which contains important interaction sites for the proto-oncogenic transcription factor MYC as well as for p53. A nuclear magnetic resonance assignment was achieved at 18.8 T magnetic field strength using a 5D HN(CA)CONH experiment and its associated 4D H(NCA)CONH and 4D (H)N(CA)CONH experiments. 13Cα and 13Cβ assignments were obtained using a 5D HabCabCONH experiment. With this strategy, 90% of 1H/15N backbone pairs could be assigned. Similarly, 264 C’ resonances were assigned corresponding to 86% of the total number of C’ atoms. In addition, 252 Cβ resonances (i.e. 85%) were assigned, together with 461 attached Hβ nuclei, as well as 264 (i.e. 86%) Cα resonances, together with 275 attached Hα nuclei.

Abstract P1-05-13: Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer

Abstract Background and Aims: Triple-negative breast cancer (TNBC) is the most aggressive subtype, and is often associated with mutations in the breast cancer susceptibility protein (BRCA) genes, which are known tumor-suppressor genes involved in DNA damage repair. Chromosomal instability (CIN) has been added to the hallmarks of cancer and is characterized by a frequent gain or loss of chromosomes during mitosis (i.e. chromosomal aberrations). BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene profiles and genome aberration spectra differ from each other and from those in sporadic BC. The lncRNA Non-Coding RNA Activated by DNA Damage (NORAD) has been demonstrated to contribute to the progression of several cancer entities by regulating genomic stability. Therefore, we wanted to explore a potential additive anti-cancerous effect by treating TNBC cells with a combination of NORAD silencing and PARP inhibition. Material and Methods: To clarify the potential clinical significance of NORAD in human TNBC, patient data from publicly available databases were analyzed. In order to further characterize NORAD, we performed phenotypic experiments after establishing an siRNA-mediated knock-down approach. Expression levels of NORAD as well as DNA-damage repair proteins were analyzed by qRT-PCR and Western Blot after combined cell treatment with NORAD silencing and PARP inhibition. To further unravel the molecular mechanism behind, we performed immunofluorescent experiments against markers for DNA-double strand breaks and homologous recombination efficiency (i.e. Rad51 and yH2AX) after TNBC treatment with NORAD siRNAs and PARP inhibitors. Results: High NORAD levels were identified to be a negative prognostic factor for disease-free survival of TNBC patients (HR with 95% CI: 2.4 (1.35-4.25); n=161; p=0.002). Furthermore, the NORAD gene is amplified in up to 3% of BC cases and mRNA levels are higher in tumor tissue when compared to healthy tissue. A qPCR based screening of several breast cancer cell lines showed an at least 4-times increased expression of NORAD when compared to normal mammary cells (i.e. MCF 12A). Knock-down of NORAD in TNBC cells resulted in a slightly reduced proliferation as well as to a decreased expression of several DNA repair proteins. Immunofluorescent experiments of TNBC cells treated with the PARP inhibitor olaparib and/or after NORAD silencing revealed increased yH2AX-dependent nuclear foci formation when compared to control conditions, indicating augmented DNA double-strand breaks after treatment. Conclusion: Our first data let us hypothesize that a combination of NORAD knock-down and PARPi treatment increases inhibition of DNA repair mechanisms and leads to a more pronounced “anti-tumorigenic” phenotype in TNBC cells. This approach could lead to the optimization of current treatment concepts. Furthermore, as increased NORAD expression is associated with an unfavorable patient survival, this lncRNA could serve as potential prognostic biomarker. Citation Format: Christiane Klec, Anita Kapeller, Felix Prinz, Christoph Trenk, Herbert Stöger, Martin Pichler. Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-13.

1H, 13C and 15N backbone resonance assignment of the human BRCA2 N-terminal region.

The Breast Cancer susceptibility protein 2 (BRCA2) is involved in mechanisms that maintain genome stability, including DNA repair, replication and cell division. These functions are ensured by the folded C-terminal DNA binding domain of BRCA2 but also by its large regions predicted to be disordered. Several studies have shown that disordered regions of BRCA2 are subjected to phosphorylation, thus regulating BRCA2 interactions through the cell cycle. The N-terminal region of BRCA2 contains two highly conserved clusters of phosphorylation sites between amino acids 75 and 210. Upon phosphorylation by CDK, the cluster 1 is known to become a docking site for the kinase PLK1. The cluster 2 is phosphorylated by PLK1 at least at two positions. Both of these phosphorylation clusters are important for mitosis progression, in particular for chromosome segregation and cytokinesis. In order to identify the phosphorylated residues and to characterize the phosphorylation sites preferences and their functional consequences within BRCA2 N-terminus, we have produced and analyzed the BRCA2 fragment from amino acid 48 to amino acid 284 (BRCA248-284). Here, we report the assignment of 1H, 15N, 13CO, 13Cα and 13Cβ NMR chemical shifts of this region. Analysis of these chemical shifts confirmed that BRCA248-284 shows no stable fold: it is intrinsically disordered, with only short, transient α-helices.

A New Class of Coumate Benzimidazole Hybrids as BRCA 1 Mimetics Through Unconventional Binding Mode; Synthesis and Preliminary Cytotoxicity Screening

It was found that breast cancer susceptibility protein 1 (BRCA 1) binds to estrogen receptor alpha (ERα) and inhibits its activity by direct interaction between domains within the amino terminus of BRCA 1 and the carboxy terminus of ER alpha. The present work focuses to identify a new class of BRCA 1 mimetics that work differently from conventional anti-estrogens. A novel class of hybrids having coumate and benzimidazolone scaffolds were designed to mimic BRCA 1 protein, suppressing the tumor activity of breast cancer cells. In silico molecular docking studies of the designed ligands were performed on BRCA 1 binding cavity of ER alpha. The designed hybrids which gave significant docking scores and had optimum binding interactions with key residues were selected for synthesis and in vitro assay. The compounds NY1 and NY2 exhibited significant effects on suppressing MDAMB- 231 cells in the concentration of 24 μg/ml and 44 μg/ml, respectively.

INHERITED MUTATION IN BRCA1 AND BRCA2 IN BREAST CANCER

BRCA1 and BRCA2 are unrelated proteins, but both are normally expressed in the cells of breast and other tissue, where they help repair damaged DNA, or destroy cells if DNA cannot be repaired. They are involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double-strand breaks. If BRCA1 or BRCA2 itself is damaged by a BRCA mutation, damaged DNA is not repaired properly, and this increases the risk for breast cancer. BRCA1 and BRCA2 have been described as "breast cancer susceptibility genes" and "breast cancer susceptibility proteins". The predominant allele has a normal, tumor suppressive function whereas high penetrance mutations in these genes cause a loss of tumor suppressive function which correlates with an increased risk of breast cancer.
 Keywords: BRCA1; BRCA2; Breast cancer; Mutation; Gene.