Tumor Suppressor Gene BRCA1 Research Articles

Efficient suppression of premature termination codons with alanine by engineered chimeric pyrrolysine tRNAs.

Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to translate PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that a pyrrolysine (Pyl) tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers a favorable scaffold for developing sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyl tRNAScaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing fluorescent reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent PTC suppression in multiple reporter genes, including pathogenic mutations in the tumor suppressor gene BRCA1 associated with breast and ovarian cancer. Moreover, PTC suppression occurred with high codon specificity and no observed cellular dysregulation. Collectively, these results unveil a new class of sup-tRNAs with encouraging potential for tRNA-based therapeutic applications.

BRCAness, DNA gaps, and gain and loss of PARP inhibitor-induced synthetic lethality.

Mutations in the tumor-suppressor genes BRCA1 and BRCA2 resulting in BRCA1/2 deficiency are frequently identified in breast, ovarian, prostate, pancreatic, and other cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cancer cells by inducing synthetic lethality, providing an effective biomarker-guided strategy for targeted cancer therapy. However, a substantial fraction of cancer patients carrying BRCA1/2 mutations do not respond to PARPis, and most patients develop resistance to PARPis over time, highlighting a major obstacle to PARPi therapy in the clinic. Recent studies have revealed that changes of specific functional defects of BRCA1/2-deficient cells, particularly their defects in suppressing and protecting single-stranded DNA gaps, contribute to the gain or loss of PARPi-induced synthetic lethality. These findings not only shed light on the mechanism of action of PARPis, but also lead to revised models that explain how PARPis selectively kill BRCA-deficient cancer cells. Furthermore, new mechanistic principles of PARPi sensitivity and resistance have emerged from these studies, generating potentially useful guidelines for predicting the PARPi response and design therapies for overcoming PARPi resistance. In this Review, we will discuss these recent studies and put them in context with the classic views of PARPi-induced synthetic lethality, aiming to stimulate the development of new therapeutic strategies to overcome PARPi resistance and improve PARPi therapy.

Bronchial Basaloid Papillary Tumor of Uncertain Malignant Potential: A Case Report.

Bronchial papillomas are benign tumors, which can be divided into different subtypes based on the cellular features. So far, no bronchial papilloma with basaloid cell features has been reported. We report a bronchial basaloid papillary tumor in a 67-year-old woman. Tumor recurrence and malignant transformation were observed after a long-term follow-up. The clinical, histological, immunohistochemical, and genetic features were reappraised. The primary tumor was characterized by papillary growth pattern and basaloid neoplastic cells, only a small amount of neoplastic cells showed mature characteristics. The tumor originated from respiratory epithelium and had a low proliferation index by Ki-67. Keratin (KRT) 5/6 and KRT7 showed patchy or partial positivity. Myoepithelial markers were negative. P63 was diffusely positive, but it was negative in the small amount of tumor cells with mature characteristics. The common genetic alterations (EGFR, KRAS, BRAF V600E, HER2, and ALK) of lung cancers were not detected. However, tumor recurrence was observed in the mediastinum and esophagus 12 years after surgery. The recurrent tumor had a morphology overlapping with that of the primary tumor; however, it displayed significantly malignant characteristics. The recurrent tumor was not related to high-risk HPV. A high variant allele frequency was observed in tumor suppressor gene BRCA1, TP53, oncogene GNA11, and KIT, which were all missense mutations. Considering the bland features of the primary tumor and the fact of tumor recurrence and undisputed malignant transformation, the basaloid papillary tumor was considered a tumor with uncertain malignant potential.

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.

Left ventricular global longitudinal strain is worse in BRCA mutation positive breast cancer patients prior to cancer treatment and premature menopause.

Breast cancer patients with mutations in human tumor suppressor genes BRCA1 and BRCA2 are at higher risk of cardiovascular disease (CVD) than the general population, as they are frequently exposed to cardiotoxic chemotherapy, anti-estrogen therapy, radiation, and/or oophorectomy for cancer-related treatment and prophylaxis. Animal and cell culture models suggest that BRCA mutations may play an independent role in heart failure. We sought to evaluate cardiac structure and function in female BRCA1 and BRCA2 mutation carriers with breast cancer compared to BRCA wildtype women with breast cancer. We performed a 1:2 age- and hypertension-matched retrospective cohort study comparing BRCA1 and BRCA2 mutation carriers (n = 38) versus BRCA wildtype controls (n = 76) with a new diagnosis of breast cancer. Echocardiographic data were obtained within 6months of breast cancer diagnosis and prior to chemotherapy, anti-estrogen therapy, radiation, or oophorectomy. Left ventricular global longitudinal strain (LV-GLS), a highly sensitive marker of LV function, was measured using QLab 15 (Philips Healthcare). In the total cohort of 114 patients with a new diagnosis of breast cancer, the median age was 45 ± 11years and the prevalence of hypertension was 8%. There were no differences in traditional cardiovascular disease risk factors between cases and controls. BRCA carriers had lower LV-GLS (-18.1% ± 4.7% vs. -20.1% ± 3.8%, p = 0.02) and greater right atrial area (12.9cm2 ± 2.7cm2 vs. 11.8cm2 ± 2.0cm2, p = 0.04) compared to controls; however, both LV-GLS and right atrial area were within the normal range. Compared to controls, BRCA carriers had a trend toward worse LV posterior wall thickness (0.89cm ± 0.15cm vs. 0.83cm ± 0.16cm, p = 0.06) although not statistically significant. In women with newly diagnosed breast cancer and prior to treatment, LV-GLS was worse in BRCA1 and BRCA2 mutation carriers compared to those with BRCA wildtype. These findings suggest that BRCA mutations may be associated with subtle changes in cardiac function. Whether differences in GLS translate to increased cardiovascular risk in women with BRCA mutations needs to be further characterized.

Abstract 6285: Defining androgen receptor function and the effects of PARP inhibition in BRCA1/2-deficient vs. proficient prostate cancer

Abstract Mutation or loss of the tumor suppressor genes BRCA1 and BRCA2 promotes the development of aggressive prostate cancer with poor outcomes and a high risk of metastatic relapse. Approximately 15% of metastatic prostate tumors are BRCA1/2-deficient. These tumors are frequently resistant to standard-of-care treatments that block androgen receptor (AR) function, although the mechanisms underlying this effect remain unclear. PARP inhibitors are synthetically lethal with BRCA1/2-deficiency and are commonly used as a late-line treatment in metastatic prostate cancer; however, both preclinical and clinical data have provided conflicting results on whether PARP inhibitors can be effective in BRCA1/2-proficient prostate tumors. BRCA1, BRCA2, PARP-1, and PARP-2 all have proposed roles in regulating transcription and the chromatin landscape. We therefore hypothesized that loss of BRCA1, BRCA2, and/or inhibition of PARP modulates AR function through changes in global chromatin structure and transcriptional pathways. We used patient-derived xenograft models and next-generation sequencing techniques to profile AR function in BRCA1/2-deficient vs. proficient prostate tumors. We found that loss of BRCA1 and BRCA2 is associated with chromatin accessibility changes, and differentially accessible regions correlate with DNA binding motifs for AR and AR cofactors. ChIP-seq for AR in these tumors demonstrated that BRCA1/2-deficiency is associated with an altered AR cistrome and a global decrease in AR genomic binding. Corresponding RNA-seq data confirmed a downregulated androgen response in BRCA1/2-deficient tumors. In cell line models, inhibition of PARP1 and PARP1/2 inhibited AR-driven transcription. Combining PARP inhibition with either androgen deprivation or the AR antagonist enzalutamide dampened the growth-inhibitory effects of PARP inhibitor treatment. Together, these results indicate that BRCA1/2-deficiency and PARP inhibition modulate AR function in prostate cancer. Citation Format: Nicole A. Traphagen, Esme Wheeler, Rong Li, Xintao Qiu, Myles Brown. Defining androgen receptor function and the effects of PARP inhibition in BRCA1/2-deficient vs. proficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6285.

Modulation of Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene BRCA1 in Breast Cancer Cells by Bazedoxifene Acetate (BZA).

Selective estrogen receptor modulators (SERMs) are steroid analogs with dual functionality, acting as partial estrogen receptor agonists to preserve postmenopausal bone density and as estrogen receptor antagonists in breast tissue. Bazedoxifene acetate (BZA) is an FDA-approved, third-generation SERM used in the treatment of osteoporosis in women. It demonstrates potential as a therapeutic option for breast cancer patients undergoing endocrine therapy. Our study aimed to assess BZA's effects on Estrogen Receptor Alpha (ERα) and tumor suppressor gene BRCA1 in T-47D and MCF-7 breast cancer cells, using Western blots, cellular viability, apoptosis assays, and RT-qPCR. Cells were cultured in 5% charcoal-stripped fetal bovine serum for six days to deplete endogenous steroids. Following a 24 h exposure to 2 µM BZA (optimal concentration determined from 1 nM-2 µM studies), Western blot analyses revealed reduced ERα and BRCA1 protein levels in both cell lines. ERα decreased by 48-63% and BRCA1 by 61-64%, indicating sensitivity to antiestrogens. Cytolocalization of ERα and BRCA1 remained unchanged after BZA and 17-β-estradiol (E2) treatment. ESR1 mRNA expression correlated with Western blot findings. Image cytometric analysis using the stain, propidium iodide, detected decreased cellular proliferation in T-47D and MCF-7 cells following a 6-day treatment ranging from 1 nM to 2 µM BZA. BZA treatment alone led to a tenfold reduction in cellular proliferation compared to estrogen-treated cells, suggesting antiproliferative effects. Understanding BZA's modulation of BRCA1 and ERα, along with their mechanistic interactions, is vital for comprehending its impact on breast cancer tumor suppressors and hormone receptors.

PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via spindle assembly checkpoint reactivation.

Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in Caenorhabditis elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.

Procena rizika u korelaciji sa gojaznošću i laboratorijskim monitoringom najčešćih tipova karcinoma dojke kod žena u Kantonu Sarajevo

Abstract: Introduction: Breast cancer (BC) is a malignant disease that predominantly affects women, with known genetic components such as mutations in tumor suppressor genes BRCA1 and BRCA2. Other risk factors include unhealthy lifestyles, lack of physical activity, and consumption of alcohol and cigarettes. Aging also plays a role in BC development, with hormonal influences such as estrogen and progesterone promoting cancer growth. Material and Methods: Research was conducted using data collection tools for risk factors and tumor markers from primary healthcare unit records. The sample comprised 200 women, divided into two groups based on BC diagnosis, with complete medical documentation. Male BC cases were excluded. Results: Statistical significance was found between genetic components, family history, aging, obesity, alcohol and cigarette consumption, longer hormone exposure, and female BC development using the Chi-Square test, confirmed by Fisher's Exact test. Tumor markers CA 15-3, CEA, CA 19-9, and CA 125 were useful for BC screening and metastasis detection, as determined by the One Sample T-test. In Sarajevo Canton, invasive ductal BC was the most common type among women, while lobular carcinoma in situ was the least common. Conclusion: Correlations between risk factors, including aging, unhealthy lifestyles, and hormone exposure, and increased BC risk were confirmed. Tumor markers CA 15-3, CEA, CA 19-9, and CA 125 were effective in diagnosis, screening, and metastasis detection in females, with sensitivity for regression detection at 81.8% and specificity at 100%.

Role of Single Nucleotide Polymorphisms in BRCA1 and BRCA2 Genes Relative to Previous Studies in Pakistan in the Prognosis of Breast Cancer

Breast cancer is a complex disease characterized by a myriad of genetic alterations. Single nucleotide polymorphisms are particularly relevant due to small allelic variations. BRCA1 and BRCA2 genes are notably associated with inherited breast and ovarian cancers. Alterations in other genes were also identified. Mutations in BRCA1 and BRCA2 tumor suppressor genes play a significant role in development. Studies in the Pakistani population revealed significant differences in gene mutations. In 31 families, 8 had male and 23 had female breast cancer, substantial findings were observed. The analysis encompassed full coding sites of these genetic variations. Among eight families with male BC, changes were detected in 2 BRCA1 and 4 BRCA2 genes. Additionally, eight alterations were noted in 23 females from site-specific families, with 4 in BRCA1 and 4 in BRCA2. These findings underscore the importance of genetic variations, especially in the BRCA1 and BRCA2 genes, in the context of breast cancer susceptibility within diverse populations.

Comparison of BRCA1 Gene Expression and CA15‐3 Tumor Marker Level in Different Stages of Breast Cancer

Breast cancer (BC), a globally prevalent malignancy, shows significant variability in incidence across different geographical regions. In this study, we examined the expression of the tumor suppressor gene BRCA1 and the tumor marker CA15‐3 in women diagnosed with BC, focusing on different cancer grades. Our research, conducted at the Baqiyat Elah Hospital in Tehran in 2021, involved collecting blood and serum samples from BC patients. These samples underwent BRCA1 gene expression analysis and CA15‐3 tumor marker assessment. Using the AJCC grading system, we categorized BC patients into various grades. Our findings revealed that BRCA1 gene expression was present in 28.57% of patients, while 71.43% showed negative expression. Both BRCA1 expression and CA15‐3 levels significantly increased with advanced cancer stages (P < 0.001). These results suggest the potential utility of BRCA1 gene expression and CA15‐3 tumor marker assessment in BC prognosis and management, particularly concerning staging and disease progression. This study provides valuable insights into the biology of BC and the development of prognostic markers for improved patient outcomes.

Breast Cancer and Genetic BRCA1/2 Testing in Routine Clinical Practice: Why, When and For Whom?

Pathogenic variants of the tumor suppressor genes BRCA1 and BRCA2 are responsible for the majority of hereditary breast cancers; they are also becoming increasingly important to identify whether patients are suitable for targeted therapy with poly ADP-ribose polymerase inhibitors (PARPi). Patients with HER2-negative breast cancer and BRCA1/2 germline mutations can benefit significantly from PARPi therapy, and the findings of the OlympiAD and the EMBRACA phase III clinical trials for regulatory approval were recently expanded by the addition of the most recent OlympiA data on the treatment of patients with early disease and a high risk of recurrence. This means that BRCA1/2 germline testing to plan patient therapy is now also relevant for patients with early breast cancer and therefore has a direct impact on survival. Healthcare research data shows, however, that BRCA1/2 testing rates are strongly affected by familial history, cancer subtype (particularly triple-negative subtypes), and patient age at onset of disease (especially with regards to younger patients with breast cancer), despite the existing clear recommendations for BRCA1/2 germline testing to identify whether PARPi therapy is indicated. This article presents the clinical implications of identifying BRCA1/2 germline mutations in patients with breast cancer, the current recommendations on molecular diagnostics, and their implementation in practice. The treatment of patients with breast cancer has progressed greatly in recent years and now offers individual treatment concepts which can only be implemented after the targeted identification of individual parameters. As detection of a BRCA1/2 germline mutation is essential for planning individual therapy, where indicated, testing should be arranged as early as possible. It is the only way of identifying patients suitable for PARPi therapy and ensuring they receive the best possible treatment. This also applies to patients with a negative familial history, HR-positive disease, or who are older at onset of disease.

Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

Gene augmentation therapy entails replacement of the abnormal tumor suppressor genes in cancer cells. In this study, we performed gene augmentation for BRCA1/2 tumor suppressors in order to retard tumor development in breast cancer mouse model. We formulated inorganic carbonate apatite (CA) nanoparticles (NPs) to carry and deliver the purified BRCA1/2 gene- bearing plasmid DNA both in vitro and in vivo. The outcome of BRCA1/2 plasmid-loaded NPs delivery on cellular viability of three breast cancer cell lines such as MCF-7, MDA-MB-231 and 4T1 were evaluated by MTT assay. The result in MCF-7 cell line exhibited that transfection of BRCA 1/2 plasmids with CA NPs significantly reduced cancer cell growth in comparison to control group. Moreover, we noticed a likely pattern of cellular cytotoxicity in 4T1 murine cancer cell line. Following transfection with BRCA1 plasmid-loaded NPs, and Western blot analysis, a notable reduction in the phospho-MAPK protein of MAPK signaling pathway was detected, revealing reduced growth signal. Furthermore, in vivo study in 4T1 induced breast cancer mouse model showed that the tumor growth rate and final volume were decreased significantly in the mouse group treated intravenously with BRCA1 + NPs and BRCA2 + NPs formulations. Our results established that BRCA1/2 plasmids incorporated into CA NPs mitigated breast tumor growth, signifying their application in the therapy for breast cancer.

Fine-tuning the cytotoxicity of ruthenium(II) arene compounds to enhance selectivity against breast cancers.

Ruthenium-based complexes have been suggested as promising anticancer drugs exhibiting reduced general toxicity compared to platinum-based drugs. In particular, Ru(η6-arene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), or RAPTA, complexes have demonstrated efficacy against breast cancer by suppressing metastasis, tumorigenicity, and inhibiting the replication of the human tumor suppressor gene BRCA1. However, RAPTA compounds have limited cytotoxicity, and therefore comparatively high doses are required. This study explores the activity of a series of RAPTA-like ruthenium(II) arene compounds against MCF-7 and MDA-MB-231 breast cancer cell lines and [Ru(η6-toluene)(PPh3)2Cl]+ was identified as a promising candidate. Notably, [Ru(η6-toluene)(PPh3)2Cl]Cl was found to be remarkably stable and highly cytotoxic, and selective to breast cancer cells. The minor groove of DNA was identified as a relevant target.

Brca1 and Tp53 co-deficiency causes a PARP-inhibitor sensitive erythroproliferative neoplasm.

Mutations in the BRCA1 tumor suppressor gene, such as 5382insC (BRCA15382insC), give carriers an increased risk for breast, ovarian, prostate and pancreatic cancers. We have previously reported that, in mice, Brca1 deficiency in the hematopoietic system leads to pancytopenia and, as a result, early lethality. Here we explore the cellular consequences of Brca1 null and BRCA1 5382insC alleles in combination with Tp53 deficiency in the murine hematopoietic system. We find that Brca1 and Tp53 co-deficiency leads to a highly penetrant erythroproliferative disorder that is characterized by hepatosplenomegaly and expanded megakaryocyte erythroid progenitor (MEP) and immature erythroid blast populations. The expanded erythroid progenitor populations in both bone marrow and spleen have the capacity to transmit the disease into secondary mouse recipients, suggesting Brca1 and Tp53 co-deficiency provides a new murine model of hematopoietic neoplasia. This Brca1/Tp53 model replicates Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib sensitivity seen in existing Brca1/Tp53 breast cancer models and has the benefits of monitoring disease progression and drug responses via peripheral blood analyses without sacrificing experimental animals. In addition, this erythroid neoplasia develops much faster than murine breast cancer, allowing for increased efficiency of future preclinical studies.

ODP275 The Effects of Tris(2-Chloroethyl) phosphate (TCEP) on the Expression of Estrogen Receptor Alpha and Tumor Suppressor Gene BRCA-1 in Breast Cancer Cell Lines

Abstract TCEP is an organophosphorus flame-retardant (OPFR) that is used widely in polyurethane foams, furniture, and household products. From these products, TCEP has significant emission into its surrounding environment. Inhalation and dermal absorption are the most common routes of human exposure to OPFRs, which have been measured in human hair, breast milk, and urine. Previous studies of OPFRs have shown their endocrine disrupting actions that have influence on diseases such as cancer. Breast cancer is the second most common cancer among women in the United States, and because of this prevalence, it is imperative to investigate possible causes and treatments for this disease. Due to the endocrine disrupting nature of OPFRs, we are investigating the effects of TCEP on hormone dependent breast cancer cell lines MCF-7 and T-47D. Our study examines the effects of TCEP, alone and in combination with hormones and anti-hormones, on ERα and BRCA1 expression in MCF-7 and T-47D breast cancer cells by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assays and RT-qPCR analyses. In order to deplete any endogenous steroids or effectors, breast cancer cells were cultured in a medium containing 5% charcoal-stripped fetal bovine serum for six days. Western blot analysis revealed alterations in the expression of ER-alpha after 24 hours of treatment with varying concentrations of TCEP (1µM-2mM). A concentration-dependent decrease of ERα protein levels was noted in the T-47D cell line when compared to the control. BRCA1 protein levels also displayed an altered expression compared to the control through the various concentrations of TCEP. Through our concentration studies, optimum concentrations of TCEP were found to be 100 µM for T-47D and 2mM for MCF-7. For our hormone studies, cell lines were treated with their respective optimum concentration of TCEP as well with combinations of hormones and anti-hormones. After 24-hour treatment of E 2, TCEP, and a combination of E2 with TCEP, a decrease in ERα expression was observed when compared to the control in both MCF-7 and T-47D cell lines. A combination of TCEP with ICI treatment revealed a significant down regulation of ERα expression level compared to the control. The same treatment conditions exhibited an increase with BRCA1 expression compared to the control and these effects were sensitive to the presence of antiestrogens in both cell lines. For cell viability studies, cells were treated for 6 days with TCEP concentrations ranging from 10nm-2µM which displayed an increase (40-50%) in cell proliferation compared to the control in both cell lines. Cytolocalization of ERα remained unaltered with the above treatment conditions. Our studies provide interesting findings about the molecular mechanisms of TECP as a potential endocrine disrupting compound on the steroid receptors and tumor suppressor genes in breast cancer cells. Presentation: No date and time listed

PMON13 The Effects of Perfluorooctanesulfonic Acid (PFOS) on the Expression of Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene BRCA1 in Breast Cancer Cells

Abstract Perfluoroctanesulfonic Acid (PFOS) is a perfluorinated chemical (PFC) and endocrine-disrupting compound (EDC) found in fire-extinguishing foams, food packing, and commonly used household products such as nonstick cookware and cleaning products. PFOS is also an environmental toxicant that has polluted air, soil, and water in past years. Few studies have suggested PFOS may cause endocrine-disrupting effects, therefore it is critical to explore the effects of PFOS on breast cancer cells. This study examined the estrogenic-like effects of PFOS, alone and in combination with hormones and antihormones, on the expression of ERα and BRCA1 in T-47D and MCF-7 breast cancer cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, apoptosis assay and RT-qPCR analyses. The cells were cultured with 5% charcoal-stripped fetal bovine serum (FBS) for six days to deplete endogenous steroids or growth factors. In the concentration-dependency study, cells were treated with various concentrations of PFOS (300µM-800µM) for 24 hours. Western blot analysis revealed alterations in the expression of ERα and BRCA1 related with these varying concentrations of PFOS (300 µM-800µM). In comparison to the control, a concentration-dependent decrease of ERα expression levels were seen in both cell lines. A concentration-dependent increase of BRCA1 was seen in both cells. Western blot analysis revealed the optimal concentration of PFOS to be 700 µM in cells, which exhibited the maximal estrogenic-like effect. To further examine the effects of PFOS on breast cancer cells, the optimal concentration (700 µM PFOS) was then used alone and in combination with hormones and anti-hormones in T-47D cells. Treatment with PFOS, E2and the combination of PFOS and E2 for 24 hours showed a significant down-regulation of ERα in T-47D cells when compared to the control. Treatment of PFOS with antiestrogen ICI revealed a significant down-regulation of ERα (approximately 50%) in T-47D cells. Image cytometric analysis with propidium iodide staining was utilized to examine the effects of PFOS on cellular viability in T-47D cells. PFOS alone and in combination with E2 revealed an increase in cellular proliferation compared to the control in T-47D cells and these effects were sensitive to the presence of antiestrogens. RT-qPCR studies revealed a transcriptional expression of ESR1 and BRCA-1 mRNA levels that correlate with the translational data obtained via western blot analyses. Cytolocalization experiments and effects of PFOS with hormones and antihormones on MCF-7 cells studies are in progress. Our studies provide exciting leads to clearly delineate the molecular mechanisms of PFOS as an EDC on the steroid receptors and tumor suppressor gene in breast cancer cells. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ANALYSIS OF THE mTOR PATHWAY IN OVARIES OF WOMEN WITH BRCA MUTATIONS

Mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose women to cancer and are associated with diminished ovarian reserve through a known impairment in DNA repair function. The PI3K/AKT/mTOR pathway regulates primordial follicle activation, and its dysregulation is implicated in human cancers including BRCA-related ovarian cancers. We sought to investigate whether mTOR activity is upregulated in follicles of women with BRCA mutations, suggesting a potential factor contributing to diminished ovarian reserve.

Evaluation of promoter hypermethylation of tumor suppressor gene BRCA1 in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a serious gynecological issue worldwide and its late detection is the major encumbrance in treatment procedures. Hypermethylation-mediated BRCA1 gene silencing results in failure of the repair system of damaged DNA playing an important role in ovarian carcinogenesis. BRCA1 gene hypermethylation can serve as a safe and highly specific clinical marker for EOC. The present study was conducted to evaluate the promoter hypermethylation of BRCA1 gene in EOC patients. This hospital-based case-control study carried out in the tertiary care hospital in New Delhi. Subjects and Methods: Promoter hypermethylation of BRCA1 gene was examined in 30 EOC diagnosed untreated cases confirmed by histopathological examinations and compared with 30 normal healthy controls matched for age using methylation specific-polymerase chain reaction. We found significantly higher BRCA1 promoter hypermethylation in the serum of EOC cases as compared to controls with P < 0.0001. BRCA1 gene methylation was found to have 70% sensitivity for the diagnosis of EOC with 100% specificity. A significant difference was observed in the range of CA125 levels, B12 and Folate levels between EOC cases and controls. We conclude that BRCA1 gene is significantly hypermethylated in EOC patients and thus can prove to be a noninvasive diagnostic tool. Our results provide prefatory evidence that epithelial ovarian epigenome can be influenced by dietary nutrients.

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair.

Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic; however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele, whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.