Investigation of BRCAness associated miRNA-gene axes in breast cancer: cell-free miR-182-5p as a potential expression signature of BRCAness.

The concept of the 'BRCAness' phenotype implies the properties that some sporadic breast cancers (BC) share with BRCA1/2-mutation carriers with hereditary BC. Breast tumors with BRCAness have deficiencies in homologous recombination repair (HRR), like BRCA1/2-mutation carriers, and consequently could benefit from poly-(ADP)-ribose polymerase (PARP) inhibitors and DNA-damaging chemotherapy. Triple-negative breast cancers (TNBC) show a higher frequency of BRCAness than the other BC subtypes. Therefore, looking for BRCAness-related biomarkers could improve personalized management of TNBC patients. microRNAs (miRNAs) play a pivotal role in onco-transcriptomic profiles of tumor cells besides their suitable features as molecular biomarkers. The current study aims to evaluate the expression level of some critical miRNAs-mRNA axes in HRR pathway in tumors and plasma samples from BC patients. The expression levels of three multi-target miRNAs, including miR-182-5p, miR-146a-5p, and miR-498, as well as six downstream HRR-related protein-coding genes, have been investigated in the breast tumors and paired adjacent normal tissues by Real-time PCR. In the next step, based on the results derived from the previous step, we examined the level of cell-free miR-182-5p in the blood plasma samples from the patients. Our results highlight the difference between TNBC and non-TNBC tumor subgroups regarding the dysregulation of the key miRNA/mRNA axes involved in the HRR pathway. Also, for the first time, we show that the level of cell-free miR-182-5p in plasma samples from BC patients could be a clue for screening BC patients eligible for receiving PARP inhibitors through a personalized manner. Altogether, some sporadic BC patients, especially sporadic TNBC, have epigenetically dysregulated HRR pathway that could be identified and benefit from BRCAness-specific therapeutic agents.

Clinicopathological Features of BRCA1/2 Mutation-Positive Breast Cancer.

PURPOSE: The BRCA1/2 gene is the most well-known and studied gene associated with hereditary breast cancer. BRCA1/2 genetic testing is widely performed in high-risk patients of hereditary breast cancer in Korea. This study aimed to investigate the clinicopathological characteristics of BRCA1/2 mutation-positive breast cancer patients. METHODS: The clinical data of 188 Korean breast cancer patients who underwent genetic testing of BRCA1/2 mutation between March 2015 and February 2020 at Pusan National University Yangsan Hospital were retrospectively reviewed. The characteristics of breast cancer according to the expression of BRCA1 and BRCA2 mutations were analyzed using the Health Insurance Review and Assessment Service guideline criteria and other clinicopathological factors. RESULTS: The factor associated with BRCA1/2 gene expression was cancer stage, and mutation expression was significantly decreased in stage I compared to stage 0 (p = 0.033; odds ratio [OR], 0.169; 95% confidence interval [CI], 0.033-0.867), and there was a tendency to increase in stage II (p = 0.780; OR, 1.150; 95% CI, 0.432-3.064). BRCA1 was significantly associated with triple-negative breast cancer (TNBC) (p = 0.004; OR, 5.887; 95% CI, 1.778-19.498). Gene expression of BRCA2 was significantly reduced under 40 years of age (p = 0.040; OR, 0.198; 95% CI, 0.042-0.930). There was no difference in disease-free survival (p = 0.900) and overall survival (p = 0.733) between the BRCA1/2 mutation-positive and -negative groups. CONCLUSION: In this study, the clinicopathological characteristics of breast cancer patients with BRCA1/2 gene mutations were identified. BRCA1 gene expression was highly correlated with TNBC. BRCA1/2 mutation did not have a poor prognosis regarding recurrence and death.

Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.

OBJECTIVE: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. METHODS: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. RESULTS: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. CONCLUSION: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Discovery of MTR-106 as a highly potent G-quadruplex stabilizer for treating BRCA-deficient cancers.

G-quadruplexes (G4s) are DNA or RNA structures formed by guanine-rich repeating sequences. Recently, G4s have become a highly attractive therapeutic target for BRCA-deficient cancers. Here, we show that a substituted quinolone amide compound, MTR-106, stabilizes DNA G-quadruplexes in vitro. MTR-106 displayed significant antiproliferative activity in homologous recombination repair (HR)-deficient and PARP inhibitor (PARPi)-resistant cancer cells. Moreover, MTR-106 increased DNA damage and promoted cell cycle arrest and apoptosis to inhibit cell growth. Importantly, its oral and i.v. administration significantly impaired tumor growth in BRCA-deficient xenograft mouse models. However, MTR-106 showed modest activity against talazoparib-resistant xenograft models. In rats, the drug rapidly distributes to tissues within 5 min, and its average concentrations were 12-fold higher in the tissues than in the plasma. Overall, we identified MTR-106 as a novel G-quadruplex stabilizer with high tissue distribution, and it may serve as a potential anticancer agent.

A Microsimulation Model for Evaluating the Effectiveness of Cancer Risk Management for BRCA Pathogenic Variant Carriers: miBRovaCAre.

OBJECTIVES: To develop a validated model for evaluating the real-world effectiveness of long-term clinical management strategies for women with germline BRCA1 or BRCA2 pathogenic variants. METHODS: A microsimulation model was developed that included a BRCA-specific natural history for breast and ovarian cancer, a clinical framework for carrier follow-up, and cancer risk management strategies (breast screening, risk-reducing mastectomy, and bilateral salpingo-oophorectomy). Adherence rates and outcomes for breast screening and risk-reducing surgery were obtained from BRCA carriers seen through a familial cancer service in Melbourne, Australia. The model was assessed for internal and external validity. The model was used to compare women perfectly adhering to screening recommendations versus actual adherence of the clinical cohort. RESULTS: The model accurately predicted cancer incidence, pathology, and mortality. Using actual adherence for breast screening resulted in additional breast cancer deaths (per 1000 women: BRCA1, 2.7; BRCA2, 1.6) compared with perfect screening adherence. This decreased average life expectancy by 0.30 life-years for BRCA1 and 0.07 life-years for BRCA2. When carriers had access to risk-reducing mastectomy, the benefit from improved screening adherence was not significant. CONCLUSIONS: The developed model is a good descriptor of BRCA carriers' lifetime trajectory and its modification by use of risk management strategies alone or in combination. Evaluations of breast screening in BRCA carriers may overestimate the benefits of screening programs unless adherence is considered. By incorporating real-world clinical practice and patient behavior, this model can assist in developing clinical services and improving clinical outcomes for carriers.

Epigenetic modulation of BRCA-1 and MGMT genes, and histones H4 and H3 are associated with breast tumors.

Aberrant patterns in promoter methylation of tumor-suppressor genes and posttranslational modifications of histone proteins are considered as major features of malignancy. In this study, we aimed to investigate promoter methylation of three tumor-suppressor genes (BRCA-1, MGMT, and P16) and three histone marks (H3K9ac, H3K18ac, and H4K20me3) in patients with breast tumors. This case-control study included 27 patients with malignant breast tumors (MBT) and 31 patients with benign breast tumors (BBT). The methylation-specific PCR was used for determining promoter methylation of BRCA-1, MGMT, and P16 genes. Western blot analysis was performed to detect histone lysine acetylation (H3K9ac and H3K18ac) and lysine methylation (H4K20me3). BRCA-1 promoter methylation was detected in 44.4% of the MBT whereas this alteration was found in 9.7% of BBT (P = 0.005). The Kaplan-Meier analysis indicated that hypermethylation in BRCA-1 promoter was significantly associated with poor overall survival of patients with breast cancer (P = 0.039). MGMT promoter methylation was identified in 18.5% of MBT and 0.0% of the BBT (P = 0.01). The frequency of P16 promoter methylation was 25.8% in BBT and 11.1% in MBT (P = 0.12). As compared with BBT, MBT samples displayed the aberrant patterns of histones marks with hypomethylation of H4K20 and hypoacetylation of H3K18 (P = 0.03 and P = 0.04, respectively). There was a negative significant correlation between H3K9ac levels and tumor size in MBT group (r = -0.672; P = 0.008). The present findings suggest that promoter hypermethylation of MGMT and BRCA-1 genes along with alterations in H3K18ac and H4K20me3 levels may have prognostic values in patients with breast cancer. Moreover, the detection of these epigenetic modifications in breast tumors could be helpful in finding new methods for breast cancer therapy.

High UDG and BRCA1 expression is associated with adverse outcome in patients with pemetrexed treated non-small cell lung Cancer.

OBJECTIVE: The antifolate chemotherapy agent pemetrexed has been widely used to treat non-small-cell-lung-cancer (NSCLC), but there is no clinically validated biomarker to select patients likely to respond. The aim of this study was to assess two proteins involved in DNA repair mechanisms, uracil DNA glycosylase (UDG) and BRCA1 as potential prognostic biomarkers in NSCLC patients treated with pemetrexed-based chemotherapy. MATERIAL AND METHODS: Formalin-fixed-paraffin-embedded tumor specimens from 119 patients with advanced NSCLC treated with pemetrexed between 2004 and 2011 were retrospectively analyzed. Expression of UDG, BRCA1, and known prognostic factors ALK, TTF-1, thymidylate synthase and folylpolyglutamate synthase was assessed by immunohistochemistry using H-SCORE (product of percent stained cells and intensity of expression). Progression-free (PFS) and overall survival (OS) served as reference endpoint. RESULTS: Most NSCLC tumor samples had UDG positivity in at least 5% of tumor cells and 34% samples had more than 50% positive tumor cells. Using the median expression value as threshold, high UDG expression (H-SCORE≥75) was significantly associated with shorter median PFS (3-year PFS 7% vs. 37%, p = 0.045) and a trend for shorter OS (3-year OS 15% vs 42%, p = 0.066) compared to patients with low UDG. In multivariable Cox analysis, the association between high UDG and shorter PFS was close to statistically significant (p = 0.08) at a significance level of 0.05 after controlling for age, gender, ALK- and TTF1-status with hazard ratio of 2.1. Grouping patients according to combined UDG and BRCA1 expression, patients with a profile of UDGhigh/BRCA1high had the shortest PFS and OS compared to all other patient groups (p = 0.007 and 0.02, respectively). CONCLUSION: Our results demonstrate an important prognostic role for high UDG expression in pemetrexed-treated NSCLC patients, in addition to its previously reported role in pemetrexed cytotoxicity. High UDG expression was predictive of shorter PFS and OS, and patients with a combined profile of UDGhigh/BRCA1high had the poorest outcome following pemetrexed treatment.

19q12 amplified and non-amplified subsets of high grade serous ovarian cancer with overexpression of cyclin E1 differ in their molecular drivers and clinical outcomes.

OBJECTIVES: Readily apparent cyclin E1 expression occurs in 50% of HGSOC, but only half are linked to 19q12 locus amplification. The amplified/cyclin E1hi subset has intact BRCA1/2, unfavorable outcome, and is potentially therapeutically targetable. We studied whether non-amplified/cyclin E1hi HGSOC has similar characteristics. We also assessed the expression of cyclin E1 degradation-associated proteins, FBXW7 and USP28, as potential drivers of high cyclin E1 expression in both subsets. METHODS: 262 HGSOC cases were analyzed by in situ hybridization for 19q12 locus amplification and immunohistochemistry for cyclin E1, URI1 (another protein encoded by the 19q12 locus), FBXW7 and USP28 expression. Tumors were classified by 19q12 amplification status and correlated to cyclin E1 and URI1 expression, BRCA1/2 germline mutation, FBXW7 and USP28 expression, and clinical outcomes. Additionally, we assessed the relative genomic instability of amplified/cyclin E1hi and non-amplified/cyclin E1hi groups of HGSOC datasets from The Cancer Genome Atlas. RESULTS: Of the 82 cyclin E1hi cases, 43 (52%) were amplified and 39 (48%) were non-amplified. Unlike amplified tumors, non-amplified/cyclin E1hi tumor status was not mutually exclusive with gBRCA1/2 mutation. The non-amplified/cyclin E1hi group had significantly increased USP28, while the amplified/cyclin E1hi cancers had significantly lower FBXW7 expression consistent with a role for both in stabilizing cyclin E1. Notably, only the amplified/cyclin E1hi subset was associated with genomic instability and had a worse outcome than non-amplified/cyclin E1hi group. CONCLUSIONS: Amplified/cyclin E1hi and non-amplified/cyclin E1hi tumors have different pathological and biological characteristics and clinical outcomes indicating that they are separate subsets of cyclin E1hi HGSOC.

The BRCA1 Methylation and PD-L1 Expression in Sporadic Ovarian Cancer.

OBJECTIVE: This study aimed to assess the relationship between BRCA1 gene methylation, PD-L1 protein expression, and the clinicopathologic features of sporadic ovarian cancer (OC). METHODS: Bisulfite pyrosequencing and immunohistochemistry were used to detect BRCA1 gene methylation and PD-L1 protein expression, respectively, in tumor tissues from 112 patients with sporadic OC. Their levels were analyzed against clinicopathologic characteristics and prognosis using standard statistical methods. RESULTS: Twenty percent (22/112) of the OC cases exhibited BRCA1 gene hypermethylation. The frequency of BRCA1 hypermethylation was significantly higher in serous OC (25%) than in nonserous OC (8%; P < 0.05). No significant correlations were discovered between BRCA1 hypermethylation and age, menstrual status, tumor location, stage, lymph node metastasis, and prognosis (P > 0.05). Among the 112 OC cases, 59% (66/112) cases were positive for PD-L1 protein expression. No significant difference existed between PD-L1 expression and age, menstrual status, histological type, tumor location, stage, lymph node metastasis, and prognosis (P > 0.05). Moreover, no correlation existed between BRCA1 methylation and PD-L1 expression (P > 0.05, r = 0.002). CONCLUSIONS: This is the first study linking BRCA1 hypermethylation variability to PD-L1 protein expression and the clinicopathologic features of OC. The data demonstrated that an epigenetic alteration of BRCA1 was closely associated with serous OC. The expression of PD-L1 was unrelated to the clinicopathologic features or BRCA1 hypermethylation in sporadic OC.

Association of prenatal organochlorine pesticide-dichlorodiphenyltrichloroethane exposure with fetal genome-wide DNA methylation.

AIMS: To investigate whether intrauterine organochlorine pesticide (OCP)-dichlorodiphenyltrichloroethane (DDT) exposure could lead to epigenetic alterations by DNA methylation with possible important lifetime health consequences for offspring. MAIN METHODS: We used Illumina Infinium HumanMethylation 450 K BeadChip to explore the pattern of genome-wide DNA methylation containing >485,000 gene sites in cord blood of 24 subjects in a 12 mother-newborn pairs birth cohort. Based on the genome-wide DNA methylation data, we chose one potential gene, BRCA1, to verify the results in another group comprising 126 subjects. KEY FINDINGS: We identified 1,131 significantly different CpG sites which included 690 hypermethylation sites and 441 hypomethylation sites in the DNA methylation level between case and control group. The identified sites were located in 598 unique genes. In subsequent validation studies, we found that the DNA methylation level of the identified CpGs of BRCA1 increased with increased exposure to dichlorodiphenyltrichloroethane (DDT) and the level of gene expression in the identified CpGs of BRCA1 decreased with increased exposure to dichlorodiphenyltrichloroethane (DDT). SIGNIFICANCE: The results indicated that epigenetic processes played a possible role in the development of fetuses affected by maternal OCP-DDT exposure. Early prenatal exposure to DDT may affect fetal BRCA1 gene methylation, and increased exposure leads to a higher DNA methylation level and lower gene expression level.

BRCA1 and MAD2 Are Coexpressed and Are Prognostic Indicators in Tubo-ovarian High-Grade Serous Carcinoma.

OBJECTIVES: The aim of this study was to investigate the relationship between BRCA1 and mitotic arrest deficiency protein 2 (MAD2) protein expression, as determined by immunohistochemistry, and clinical outcomes in epithelial ovarian carcinoma (EOC). METHODS: A tissue microarray consisting of 94 formalin-fixed paraffin-embedded EOC with fully matched clinicopathological data were immunohistochemically stained with anti-BRCA1 and anti-MAD2 antibodies. The cores were scored in a semiquantitative manner evaluating nuclear staining intensity and extent. Coexpression of BRCA1 and MAD2 was evaluated, and patient survival analyses were undertaken. RESULTS: Coexpression of BRCA1 and MAD2 was assessed in 94 EOC samples, and survival analysis was performed on 65 high-grade serous carcinomas (HGSCs). There was a significant positive correlation between BRCA1 and MAD2 expression in this patient cohort (P < 0.0001). Both low BRCA1 and low MAD2 are independently associated with overall survival because of HGSC. Low coexpression of BRCA1 and MAD2 was also significantly associated with overall survival and was driven by BRCA1 expression. CONCLUSION: BRCA1 and MAD2 expressions are strongly correlated in EOC, but BRCA1 expression remains the stronger prognostic factor in HGSC.

BRCA1, Ki67, and ß-Catenin Immunoexpression Is Not Related to Differentiation, Platinum Response, or Prognosis in Women With Low- and High-Grade Serous Ovarian Carcinoma.

OBJECTIVE: The purpose of this study was to compare the immunohistochemical expression of BRCA1, Ki67, and ß-catenin in women with low-grade (LGSOC) and high-grade serous ovarian carcinomas (HGSOC) and their relationship with clinicopathological features, response to platinum-based chemotherapy, and survival. METHODS: For this study, 21 LGSOC and 85 HGSOC stage I to IV cases, diagnosed and treated from 1996 to 2013 and followed-up until December 2016, were included. BRCA1, Ki67, and ß-catenin expression was assessed using tissue microarray-based immunohistochemistry. RESULTS: Women with HGSOC were significantly more likely to have advanced-stage disease (P < 0.001), higher CA125 levels (P < 0.001), postsurgery residual disease (P < 0.01), and higher rates of disease progression and recurrence (P = 0.001). The percentage of women with HGSOC whose tumors expressed Ki67 was significantly higher compared with women with LGSOC (P < 0.001). The expression of BRCA1 and ß-catenin did not differ between LGSOC and HGSOC (P = 0.12 and P = 1.00, respectively). The clinicopathological features and the response to platinum-based chemotherapy did not differ according to the BRCA1, Ki67, and ß-catenin expression in either group. In HGSOC, only International Federation of Gynecology and Obstetrics stage was independently associated with poor survival (PFS and OS). CONCLUSIONS: Ki67 expression was significantly higher in HGSOC. BRCA1 and ß-catenin expression did not differ between LGSOC and HGSOC samples. BRCA1, Ki67, and ß-catenin expression was neither related to clinicopathological features, response to platinum-based chemotherapy, nor survival. Only International Federation of Gynecology and Obstetrics stage remained associated with poor survival in women with HGSOC.

BRCA1 deficiency and synthetic lethality in leukemias; not only gene mutation matters.

BRCA1 (breast cancer 1 susceptibility protein) is one of main regulators of cellular genomic stability. It is responsible for proper segregation of chromatides to daughter cells during mitosis as well as DNA double strand breaks repair by homologous recombination (HR). Genetic alterations of BRCA1 gene are cancer predisposition markers. Mutations or epigenetic alterations have been noticed in breast, ovarian and prostate cancers, significantly increasing risk of cancer development. Such gene alterations are not connected with leukemias. Importantly, BRCA1 deficiency is a factor which makes patients susceptible for personalized therapy with PARP1 inhibitors, which is based on the phenomenon called synthetic lethality. In this review we present our discoveries of novel mechanism leading to BRCA1 deficiency in leukemia, which is not connected with BRCA1 gene mutations or epigenetic alterations, but with attenuated translation of BRCA1 protein linked to the cellular stress response and controlled by RNA binding proteins. Moreover, we found that some treatments or genetic alterations in leukemias might also result in BRCA1 deficits. Our studies provide evidence that PARP1 inhibitors should be considered as efficient treatment in BRCA1-deficient leukemias, leading to elimination of cancer cells, including stem and progenitor cells. Finally we propose a strategy to select leukemia patients which might be sensitive to therapy with PARP1 inhibitors.

BRCA1 and BRCA2 expression patterns and prognostic significance in digestive system cancers.

The role of BRCA1 and BRCA2 genes is mainly to maintain genome integrity in response to DNA damage through different mechanisms. Deregulation of BRCA1 and BRCA2 is associated with the development of tumor and altered sensitivity to chemotherapeutic agents. In this study, we determined protein expression of BRCA1 and BRCA2 in 4 digestive system cancers (gastric cancer, colorectal cancer, hepatocellular carcinoma, and pancreatic cancer) by immunohistochemistry on tissue microarrays. A total of 1546 samples of 4 types of cancer tissues, their matched adjacent nontumor tissues, and corresponding benign tissues were studied, respectively. Immunohistochemistry expression patterns of the 2 proteins and their correlation with patients' clinical parameters and overall survival were analyzed. The results showed that low expression of cytoplasmic BRCA1 and BRCA2 was commonly associated with advanced tumor-lymph node-metastasis stage, whereas high expression of nuclear BRCA1 was generally correlated with advanced tumor stages in these cancers. High expression of cytoplasmic BRCA1 and BRCA2 had significantly favorable overall survival in digestive system cancers; in contrast, BRCA1 nuclear expression usually predicted poor outcomes. We conclude that BRCA1 and BRCA2 could be used as clinicopathological biomarkers to evaluate the prognosis of digestive system cancers.

Impact of Etoposide on BRCA1 Expression in Various Breast Cancer Cell Lines.

Breast cancer 1 (BRCA1), as a tumor suppressor, exerts an effective influence on protecting DNA integrity to suppress the development of breast cancer (BC). BRCA1 expression is induced in response to DNA-damaging agents such as etoposide. Germline BRCA1 gene mutations are associated with development of hereditary BC. However, besides BRCA-mutated BCs, some sporadic cancers may also exhibit a BRCA-like phenotype, displaying so-called 'BRCAness'. This common phenotype may respond to similar therapeutic approaches as BRCA-mutated tumors and may thus have important implications for the clinical management of these cancers. In order to determine whether and how etoposide regulates the protein levels of BRCA1 in BC cells, we exposed a panel of five selected cell lines to etoposide, compared the results to untreated control cells, and then stained the cells with the specific, reliable, and reproducible MS110 antibody directed against phosphorylated Ser1423 BRCA1. By evaluating cytoplasmic BRCA1 protein levels, we were able to distinguish three aggressive BC subtypes with BRCAness characteristics. In addition, determination of early and late apoptosis helped to complete the analysis of BRCA1 functions in the DNA damage pathway of aggressive BC. In conclusion, our study suggested that high cytoplasmic BRCA1 protein levels could be considered as a potential predictive marker for response to chemotherapy in both sporadic and hereditary BC. Tumors with either BRCAness phenotype or germline BRCA1 mutation are both aggressive BCs associated with poor prognosis and could both be subjected to targeted therapies against BRCA1-mutated BC in future clinical management strategies.

The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.

The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double-strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

The role of BRCA1-IRIS in the development and progression of triple negative breast cancers in Egypt: possible link to disease early lesion.

BACKGROUND: Breast cancer is the most globally diagnosed female cancer, with the triple negative breast cancer (TNBC) being the most aggressive subtype of the disease. In this study we aimed at comparing the effect of BRCA1-IRIS overexpression on the clinico-pathological characteristics in breast cancer patients with TNBC or non-TNBC in the largest comprehensive cancer center in Egypt. METHODS: To reach this goal, we conducted an observational study at the National Cancer Institute (NCI), Cairo University (Cairo, Egypt). The data on all diagnosed breast cancer patients, between 2009 and 2012, were reviewed. BRCA1-IRIS expression measured using real time RT/PCR in these patients' tumor samples was correlated to tumor characteristics, such as to clinico-pathological features, therapeutic responses, and survival outcomes. RESULTS: 96 patients were enrolled and of these 45% were TNBC, and 55% were of other subtypes (hereafter, non-TNBC). All patients presented with invasive ductal carcinomas. No significant difference was observed for risk factors, such as age and menopausal status between the TNBC and the non-TNBC groups except after BRCA1-IRIS expression was factored in. The majority of the tumors in both groups were ≤5 cm at surgery (p = 0.013). However, in the TNBC group, ≤5 cm tumors were BRCA1-IRIS-overexpressing, whereas in the non-TNBC group they were BRCA1-IRIS-negative (p = 0.00007). Most of the TNBC patients diagnosed with grade 1 or 2 were BRCA1-IRIS-overexpressing, whereas non-TNBCs were IRIS-negative (p = 0.00035). No statistical significance was measured in patients diagnosed with grade 3 tumors. Statistically significant difference between TNBCs and non-TNBCs and tumor stage with regard to BRCA1-IRIS-overexpression was observed. Presence of axillary lymph node metastases was positively associated with BRCA1-IRIS overexpression in TNBC group, and with BRCA1-IRIS-negative status in the non-TNBC group (p = 0.00009). Relapse after chemotherapy (p < 0.00001), and local recurrence/distant metastasis after surgery (p = 0.0028) were more pronounced in TNBC patients with BRCA1-IRIS-overexpressing tumors compared to non-TNBC patients. Finally, decreased disease-free survival in TNBC/BRCA1-IRIS-overexpressing patients compared to TNBC/BRCA1-IRIS-negative patients, and decreased overall survival in TNBC as well as non-TNBC patients was driven by BRCA1-IRIS overexpression. CONCLUSIONS: TNBC/BRCA1-IRIS-overexpressing tumors are more aggressive than TNBC/BRCA1-IRIS-negative or non-TNBC/BRCA1-IRIS-overexpressing or both negative tumors. Further studies are warranted to define whether BRCA1-IRIS drives the early TNBC lesions growth and dissemination and whether it could be used as a diagnostic biomarker and/or therapeutic target for these lesions at an early stage setting.

Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.

The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (P<0.05) during TTFields treatment. DNA double-strand break (DSB) repair foci increased when cells were exposed to TTFields as did the appearance of chromatid-type aberrations, suggesting an interphase mechanism responsible for cell death involving DNA repair. Exposing cells to TTFields immediately following ionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents.

Expresión de BRCA1 en lesiones benignas y malignas de la mama / BRCA1 expression in benign and malignant breast lesions

La mayor parte de los carcinomas de mama son cánceres esporádicos; sin embargo, existe una proporción, estimada entre el 5 y el 10%, en la cual aparece una predisposición hereditaria al cáncer, asociado principalmente a mutaciones germinales en los genes BRCA1 y BRCA2; tales mutaciones incrementan la predisposición para el desarrollo de la enfermedad durante el transcurso de la vida. El objetivo general de este trabajo fue valorar la expresión del gen BRCA1 por inmunohistoquímica (IHQ). El estudio se realizó en mujeres diagnosticadas con lesiones benignas o con carcinoma ductal infiltrante de mama en seguimiento en el Instituto de Oncología “Dr. Miguel Pérez Carreño” de Valencia, Venezuela. Se analizó la expresión de la proteína BRCA1 y los resultados obtenidos se compararon con la clasificación de las lesiones benignas propuesta por Dupont y Page y los subtipos moleculares intrínsecos definidos por IHQ. De este análisis se obtuvo que tanto en las lesiones no infiltrantes (proliferativas y carcinoma in situ), así como en los carcinomas infiltrantes, predominaron los casos con un marcaje nuclear de BRCA1 por IHQ ≤10%. Además, la relación de la expresión de BRCA1 con la media de la supervivencia global, obtuvo valor pronóstico desfavorable, cuando la expresión nuclear y citoplasmática de BRCA1 fue ≤10%, con p<0,05. Finalmente, en base a los resultados, se sugiere que en el algoritmo de abordaje de mujeres con riesgo de padecer cáncer de mama, se incluya la valoración de la expresión de BRCA1 por IHQ.

The majority of breast cancers are sporadic cancers; however, there is an estima¬ted proportion of 5% to 10%, where a hereditary predisposition appears, mainly associated with germline mutations in the BRCA1 and BRCA2 genes. Such mutations increase the predis-position to develop the disease during the course of life. The overall objective of this work was to evaluate the expression of the BRCA1 gene by immunohistochemistry (IHC). The study was conducted in women diagnosed with benign lesions or invasive breast ductal carcinoma in follow-up care at the Institute of Oncology “Dr. Miguel Perez Carreño” in Valencia, Venezuela. Expression of the BRCA1 protein was analyzed and the results were compared with the benign lesions classification given by DuPont and Page and the intrinsic molecular subtypes defined by IHC. From this analysis it was found that in both, non-infiltrative lesions (proliferative and carcinoma in situ), as well as in infiltrating carcinomas, predominated the cases with BRCA1 nuclear labeling by IHC (≤10 %). Furthermore, the relationship of expression of BRCA1 with the average overall survival, showed a poor prognostic value obtained when the nuclear and cytoplasmic expression of BRCA1 was ≤10%, with p<0.05. Finally, based on the results, it is suggested that the assessment of BRCA1 expression by IHC should be included in the approach algorithm of women at risk of developing breast cancer.

The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer.

Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC.