BRCA1 Expression Research Articles

Downregulation of TGF-\u03b21 suppressed proliferation and increased chemosensitivity of ovarian cancer cells by promoting BRCA1/Smad3 signaling

BackgroundStudies have demonstrated that transforming growth factor beta-1 (TGF-β1) exhibits oncogenic activity in different types of cancer, including ovarian cancer (OC). However, its regulatory mechanism in OC and whether TGF-β1 is involved in chemosensitivity regulation remains unclear. Thus, the aim of this study was to investigate the role of TGF-β1 in OC.MethodsThe OC cell line SKOV3 was employed, and TGF-β1 overexpression or knockdown vectors were constructed. The cell proliferation of SKOV3 was evaluated with the cell counting kit (CCK8) kit after treatment with different concentrations of cis-platinum. Western blot and protein immunoprecipitation were employed to detect changes in BRCA1 and Smad3 expression and their interactions. Tumor growth in nude mice was evaluated.ResultsThe results showed that TGF-β1 knockdown increased chemosensitivity by promoting BRCA1 expression and Smad3 phosphorylation. In vivo studies showed that TGF-β1 knockdown significantly inhibited the growth of tumors, also by upregulating BRCA1 expression and Smad3 phosphorylation.ConclusionTaken together, our results suggest that TGF-β1 knockdown inhibits tumor growth and increases chemosensitivity by promotion of BRCA1/Smad3 signaling.

Analysis of BRCA1 and BRCA2 Mutations and Gene Expression in Hematological Malignancies - Implications for Differential Involvement in Pathogenesis of Lymphoid Neoplasms

Background: BRCA1 and BCRA2 (breast cancer 1 and 2) are tumor suppressor genes involved in repair of DNA double-strand breaks. Loss of BRCA1/2 activity by (germline) mutations (mut) or downregulation (DR) of gene expression (GE) correlates with an increased risk of breast and ovarian cancer. Mut in or reduced GE of BRCA1/2 have also been implicated in some hematological malignancies, although data are so far scarce. Moreover, PARP1 inhibitors, known to induce synthetic lethality in tumors harboring BRCA1/2 mut were shown to affect FLT3(ITD)-positive AML (acute myeloid leukemia) cells.Aims: Investigation of BRCA1/2 mut and GE in 3340 patients with different hematological malignancies by whole genome sequencing (WGS) and RNA sequencing.Patient cohorts and methods: WGS was performed for 3340 patients with different hematological malignancies: 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. All reported p-values are two-sided and were considered significant at p<0.05. For GE analysis, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of GE in each sample.Results: We identified 14/3340 patients diagnosed with a variety of hematological neoplasms with a pathogenic (according to ClinVar) BRCA1/2 mut (BRCA1: n=11; BRCA2: n=3; incidence: 0.42%). Analysis of the mut frequencies of 1067 cancer associated genes revealed RNF213 (6/14, 43%), ARID1B, CREBBP (5/14, 36%, respectively), JAK3, TET2, NUMA1, POLD1, ZFHX3 (4/14, 29%), ATM, KMT2D and POLE (3/14, 21%) as most frequently mutated genes in cases with BRCA1/2 mut. Three of them (ATM, POLD1, POLE) are known to be directly involved in DNA repair. Comparison of mut frequencies of these 11 genes in cases with/without BRCA1/2 mut revealed JAK3 and POLD1 to be significantly more abundant in BRCA1/2 mut (JAK3: 29% vs. 9%, p=0.028; POLD1: 29% vs. 8%, p=0.026). GE analyses of BRCA1/2 showed DR of BRCA1 and/or BRCA2 in several lymphoid malignancies: for BRCA1, the strongest reduction was detected in PPBL (persistent polyclonal B-cell lymphocytosis; normalized BRCA1 expression: 1.5; control: 5.2), CLL (chronic lymphocytic leukemia; 1.6), T-PLL (T-cell prolymphocytic leukemia; 1.7) and MZL (marginal zone lymphoma; 2.2). DR of BRCA2 was less pronounced (control: 6.3; PPBL: 3.5; HCL, hairy cell leukemia: 4.0; T-PLL: 4.1; MZL: 4.6). In myeloid malignancies reduction was less (see Figure). For further analysis, the 50 lymphoid cases with the lowest and highest BRCA1/2 GE (BRCA1/2 high/low) were selected: these included for BRCA1 low (median GE: 0.8) mainly CLL (33/50), T-PLL (4/50) and PPBL (4/50), whereas BRCA1 high (median: 5.4) comprised B-ALL (23/50), T-ALL (15/50) and Burkitt lymphoma (7/50). BRCA2 low (median: 3.4) included only 2/50 CLL cases, as mainly PPBL (14/50), T-PLL (10/50), multiple myeloma (9/50) and HCL (9/50) cases showed a low BRCA2 GE. BRCA2 high (median: 6.6) included T-ALL (21/50), B-ALL (16/50) and Burkitt lymphoma (4/50). Regarding molecular mut, all entities with a strong DR of BRCA1/2 show a high mut frequency of ATM (analyzed cohort: PPBL, 22%; MZL, 24%; CLL, 25%; HCL, 28%, T-PLL: 74%); BRCA1 (p=0.023) and BRCA2 (p<0.001) GE was significantly lower in ATM mut cases. Comparison of BRCA1/2 low/high revealed several significant differences in detected gene mut: for BRCA1 low vs. high, JAK3 was more frequently mutated in low (18% vs. 4%, p=0.05), whereas KAT6B (4% vs. 24%, p=0.008), BCL11B (0% vs. 18%, p=0.003) and PHF6 (0% vs. 18%, p=0.003) were more abundant in high. For BRCA2, results were similar (JAK3: 20% vs. 4%, p=0.028; BCL11B: 2% vs. 18%, p=0.025; PHF6: 0% vs. 24%, p<0.001), moreover BRAF (20% vs. 2%, p=0.008) and STAT5B (16% vs. 0%, p=0.006) were more frequently mutated in low.Conclusions: 1) The incidence of BRCA1/2 mut in patients with hematological malignancies is comparable to the frequency in a normal population. 2) Mut in JAK3 and POLD1 are significantly more frequent in cases with BRCA1/2 mut. 3) GE analyses revealed a strong DR of BRCA1 in CLL, T-PLL and MZL. 4) T-ALL and B-ALL were not found to show a BRCA1/2 DR. 5) DR correlates with a high mut frequency of ATM and JAK3, for BRCA2 also of BRAF and STAT5B. 6) Thus, not molecular mut in, but DR of BRCA1/2 in CLL, T-PLL and MZL could play a more important role in pathogenesis than so far expected. [Display omitted] DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines.

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER+ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER + cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.

Alteration of BRCA-1 tumor suppressor gene expression in serous and mucinous ovarian neoplasms in the benign-borderline-malignant pathway

Alteration of expression of the tumor suppressor gene BRCA-1 has been widely studied in breast and ovarian carcinoma. However, pattern of this alteration in the benign-borderline-carcinoma sequence in serous and mucinous ovarian neoplasms have not yet fully described. Tissue sections from 214 formalin-fixed paraffin-embedded ovarian specimens were stained immunohistochemically with BRCA-1 antibody. Specimens were 10 normal ovarian surface epithelium, 10 fallopian tube epithelium, 70 benign adenoma (50 serous and 20 mucinous), 28 borderline (13 serous and 15 mucinous), 78 carcinoma (58 serous and 20 mucinous), and 18 metastatic deposit (13 serous and 5 mucinous). Expression was evaluated into 0, +1, +2, and +3. Score +3 staining similar to normal tissues was considered normal and other scores were considered altered expression. Strong expression was seen in all normal epithelium specimens. Altered expression was seen in 34 serous neoplasms; 17 of 50 (34%) of benign cystadenomas, 6 of 13 (46%) of borderline tumors, 43 of 58 (74%) of primary carcinoma, and in 8 of 13 (62%) of metastatic carcinoma. This alteration was significantly associated with higher histopathologic grade (P = 0.049), presence of necrosis (P = 0.0001), and higher proliferation rate (P = 0.001). In mucinous neoplasms; altered BRCA-1 was detected in 25 specimens; 7 of 20 (41%) of benign cystadenomas, 5 of 15 (33%) of borderline neoplasms, 9 of 20 (45%) of primary carcinoma, and 4 of 5 (80%) of the metastatic deposits. This alteration was not associated with any of the clinicopathologic tumor characteristics. In conclusion, alteration of BRCA-1 expression is more frequent in serous than in mucinous carcinomas and is associated with tumors of higher grades and high proliferation rate.

Bile acids upregulate BRCA1 and downregulate estrogen receptor 1 gene expression in ovarian cancer cells.

Two major risk factors for ovarian cancer include loss-of-function mutations in the BRCA1 (breast cancer 1, early onset) gene and aspects of estrogen metabolism. Modulation of the levels of the normal BRCA1 allele and estrogen receptor expression may therefore be a preventive strategy. Consensus binding motifs for the bile acid-responsive transcription factor farnesoid X receptor were identified in the BRCA1 and estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) genes, supported by chromatin immunoprecipitation sequencing data. Two major bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), resulted in a greater than four-fold induction of BRCA1 transcript levels at 10 μmol/l and a greater than six-fold induction at 50 μmol/l relative to untreated control OVCAR3 ovarian cancer cells. Conversely, CDCA and DCA at 10 μmol/l resulted in about a 75% decrease in ESR1 expression in response to 10 μmol/l CDCA and DCA and close to 90% reduction with 50 μmol/l CDCA and DCA. Bile acids had no effects on ESR2 gene transcript levels. The inverse regulation of BRCA1 and ESR1 gene expression in response to physiological levels of bile acids could have important implications for disease penetrance and chemoprevention strategies in carriers of BRCA1 mutations.

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

Effect of lamin-A expression on migration and nuclear stability of ovarian cancer cells

ObjectiveNuclear lamina plays important roles in nuclear shape and mechanical stability. Many studies demonstrated that defects of lamin-A were associated with several diseases, but little research was found on its potential roles in ovarian cancer. MethodsGEPIA and GEO database were used to analyze lamin-A in ovarian tissues, followed by assessing lamin-A and prognosis of ovarian cancer patients with Kaplan-Meier plotter. Then, transient transfected HO-8910 cells with shRNA to knockdown lamin-A. Knockdown efficiency was determined by western blot, qRT-PCR and immunofluorescence. Meanwhile, lamin-A was overexpressed in HO-8910 PM cells. Then, 2D migration, 3D migration through 3 μm and 8 μm pores were carried out, followed by immunofluorescence and TEM observation. ResultsLamin-A tended to be lower in ovarian cancer, and higher expression of lamin-A was associated with better survival. After lamin-A knockdown, 2D and 3D migration (3 μm, 8 μm) abilities of HO-8910 cells were significantly increased (p < 0.001), while overexpression of lamin-A in HO-8910PM impeded migration. Meanwhile, when HO-8910 cells migrated through 3 μm pores, nuclei became strikingly elongated, and down-regulation of lamin-A promoted nuclear plasticity, making the circularity of nucleus increased. Besides, further knockdown group had the highest proportion of γ-H2AX, with micronuclei forming. Furthermore, western blot showed that the expression of BRCA1, Ku80 and Rad50 decreased significantly after further knockdown, suggesting impairment of DNA damage repair. ConclusionsLamin-A was down-regulated in ovarian cancer, and higher lamin-A was associated with better prognosis. Nuclei with high lamin-A were severely deformed through constricted pores. Moderate lamin-A enhanced nuclear plasticity, so as to strengthen migration ability. When lamin-A was further knockdown, ovarian cancer cells that migrated through restricted pores decreased, with DNA damage, genomic instability and impairment of DNA damage repair.

High expression of the breast cancer susceptibility gene BRCA1 in long-lived termite kings.

Aging is associated with the accumulation of DNA damage. High expression of DNA repair genes has been suggested to contribute to prolonged lifespan in several organisms. However, the crucial DNA repair genes contributing to longevity remain unknown. Termite kings have an extraordinary long lifespan compared with that of non-reproductive individuals such as workers despite being derived from the same genome, thus providing a singular model for identifying longevity-related genes. In this study, we demonstrated that termite kings express higher levels of the breast cancer susceptibility gene BRCA1 than other castes. Using RNA sequencing, we identified 21 king-specific genes among 127 newly annotated DNA repair genes in the termite Reticulitermes speratus. Using quantitative PCR, we revealed that some of the highly expressed king-specific genes were significantly upregulated in reproductive tissue (testis) compared to their expression in somatic tissue (fat body). Notably, BRCA1 gene expression in the fat body was more than 4-fold higher in kings than in workers. These results suggest that BRCA1 partly contributes to DNA repair in somatic and reproductive tissues in termite kings. These findings provide important insights into the linkage between BRCA1 gene expression and the extraordinary lifespan of termite kings.

Studies on selected molecular factors in endometrial cancers.

Endometrial carcinomas (EC) differ in etiology, clinical course and prognosis. This multi-center study aimed at a closer recognition of molecular factors linked to heterogeneity of EC by evaluating estrogen and progesterone receptors, proteins dependent on MMR genes, proteins linked to poor prognosis and metastases, and mutations in BRCA1. Using sections of paraffin-embedded preparations, in 115 patients with EC type I and 31 with EC type II, expression of ERα, ERβ1, PR, MLH1, and MSH2 proteins, as well as ARID1A, c-MET and BRCA1, was estimated by immunohistochemistry using specific antibodies. Expression of ERβ1 was augmented in EC type II, in poorly differentiated cancers and with growing clinical advancement. An augmented expression of ERα was noted in well-differentiated EC and at lower clinical stage. An increased expression of PR and decreased of MLH1 were detected in type I EC. The expression of ARID1A and c-MET proteins showed no differences between the types of EC, stages of clinical advancement or grading. In 51.6% patients with type II EC, a loss of BRCA1 expression was disclosed; in this group of cancers a decreased expression of ERα was noted. An augmented expression of ERβ1 was linked to type II EC. A higher expression of ERα in EC cancers was associated with a lower histopathological grade. A decreased expression of MLH1 protein was estimated in EC type I. Type II EC may be connected to BRCA1 mutation.

Elucidating the novel BRCA1 function as a non-genomic metabolic restraint in ER-positive breast cancer cell lines.

Within populations carrying the same genetic predisposition, the penetrance of BRCA1 mutations has increased over time. Although linked to changes in lifestyle factors associated with energy metabolism, these observations cannot be explained by the established role of BRCA1 in DNA repair alone.We manipulated BRCA1 expression using tetracycline in the UBR60-bcl2 cell line (which has an inducible, tetracycline-regulated BRCA1 expression) and siRNA in oestrogen receptor(ER)-positive MCF7 and T47D breast cancer cells. Cellular responses to BRCA1 silencing and IGF-I actions were investigated using western blotting, 3-H Thymidine incorporation assay, cell fractionation and co-immunoprecipitation.We demonstrated that the loss of BRCA1 resulted in downregulation of a phosphorylated and inactive form of acetyl CoA Carboxylase-α (ACCA), with a concomitant increase in fatty acid synthase (FASN) abundance. BRCA1 was predominantly cytoplasmic in ER-positive breast cancer cells, compatible with the observation that BRCA1 physically associates with phosphorylated ACCA, which is a cytoplasmic protein. We also found that IGF-I induced de-phosphorylation of ACCA by reducing the interaction between BRCA1 and phosphorylated ACCA. BRCA1 deficiency enhanced the non-genomic effects of IGF-I, as well as the proliferative responses of cells to IGF-I.We characterized a novel, non-genomic role for BRCA1 in restraining metabolic activity and IGF-I anabolic actions.

Antibody-drug conjugates in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a heterogeneous disease that comprises 15-20% of all breast cancers and is more frequently seen in younger women, African-Americans, and BRCA1 expression. Advanced TNBC carries aggressive features and is associated with overall poor outcomes. Unfortunately, there are no targeted therapies available for non-BRCA associated TNBC, which remains a high unmet therapeutic need. One emerging treatment modality includes antibody-drug conjugates which are highly selective monoclonal antibodies conjugated to cytotoxic agents, designed to deliver cytotoxic drugs to antigen-expressing tumor cells. This review will highlight three antibody-drug conjugates currently being evaluated in TNBC (CDX-011, SGN-LIV1a, IMMU-132), including one that has been given Breakthrough Therapy designation from the US FDA.

High expression of lncRNA MEG3 participates in non-small cell lung cancer by regulating microRNA-7-5p.

This study was made to investigate whether long noncoding RNA (lncRNA) MEG3 could participate in the occurrence and development of non-small cell lung cancer (NSCLC) by regulating the expression of BRCA1 through competitive binding to microRNA-7-5p. We used quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) to explore the expression of lncRNA MEG3 and BRCA1 in NSCLC tissues and adjacent normal tissues, as well as NSCLC cell lines. The dual luciferase reporter gene assay was used to detect the binding of microRNA-7-5p to lncRNA MEG3 and BRCA1. Meanwhile, the expression of BRCA1, B-cell lymphoma-2 (Bcl-2) and BCL2-associated X (Bax) was detected by Western blot after the cells were overexpressed or knocked down of lncRNA MEG3. All these experiments were designed to investigate whether lncRNA MEG3 participated in the pathogenesis of NSCLC through inhibiting the expression of BRCA1 and Bcl-2 and promoting Bax expression. The expressions of lncRNA MEG3 and BRCA1 in NSCLC tissues and A549 and HCC823 cell lines were significantly lower than those in the normal group. Overexpression of lncRNA MEG3 and BRCA1 in A549 and HCC823 cell lines resulted in increased apoptosis of lung cancer cells. Dual luciferase reporter assay demonstrated that lncRNA MEG3 can regulate the expression of BRCA1 through competitively binding to microRNA-7-5p to form the lncRNA MEG3/microRNA-7-5p/BRCA1 regulatory network. Besides, lncRNA MEG3 could inhibit the apoptosis inhibitory protein Bcl-2 and promote the expression of apoptosis-promoting factor Bax. LncRNA MEG3 was significantly downregulated in NSCLC, and it could regulate the BRCA1 expression by competitive binding to microRNA-7-5p.

SPOP promotes transcriptional expression of DNA repair and replication factors to prevent replication stress and genomic instability.

Mutations in SPOP, the gene most frequently point-mutated in primary prostate cancer, are associated with a high degree of genomic instability and deficiency in homologous recombination repair of DNA but the underlying mechanisms behind this defect are currently unknown. Here we demonstrate that SPOP knockdown leads to spontaneous replication stress and impaired recovery from replication fork stalling. We show that this is associated with reduced expression of several key DNA repair and replication factors including BRCA2, ATR, CHK1 and RAD51. Consequently, SPOP knockdown impairs RAD51 foci formation and activation of CHK1 in response to replication stress and compromises recovery from replication fork stalling. An SPOP interactome analysis shows that wild type (WT) SPOP but not mutant SPOP associates with multiple proteins involved in transcription, mRNA splicing and export. Consistent with the association of SPOP with transcription, splicing and RNA export complexes, the decreased expression of BRCA2, ATR, CHK1 and RAD51 occurs at the level of transcription.

SPOP promotes transcriptional expression of DNA repair and replication factors to prevent replication stress and genomic instability.

Mutations in SPOP, the gene most frequently point-mutated in primary prostate cancer, are associated with a high degree of genomic instability and deficiency in homologous recombination repair of DNA but the underlying mechanisms behind this defect are currently unknown. Here we demonstrate that SPOP knockdown leads to spontaneous replication stress and impaired recovery from replication fork stalling. We show that this is associated with reduced expression of several key DNA repair and replication factors including BRCA2, ATR, CHK1 and RAD51. Consequently, SPOP knockdown impairs RAD51 foci formation and activation of CHK1 in response to replication stress and compromises recovery from replication fork stalling. An SPOP interactome analysis shows that wild type (WT) SPOP but not mutant SPOP associates with multiple proteins involved in transcription, mRNA splicing and export. Consistent with the association of SPOP with transcription, splicing and RNA export complexes, the decreased expression of BRCA2, ATR, CHK1 and RAD51 occurs at the level of transcription.

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

ObjectiveThe 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 methodsFormalin-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. ResultsMost 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). ConclusionOur 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.

Abstract A12: Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells

Abstract The precursor of the human microRNA hsa-miR-6855-3p is a 5-tailed mitronic pre-miRNA that is coded by intron #13 of the deubiquitinase USP20 gene. Our preliminary data suggest that the biosynthesis of this miRNA is upregulated by oxidative stress in the SLUG-high metastatic breast cancer cells, promoting DNA double-strand break repair (DSBR) through the indirect enhancement of BRCA2 gene expression. We found that miR-6855-3p favors BRCA2 biosynthesis through the enhancement of the level of an alternately translated form of the BRCA2 gene enhancer protein SPRDX5. We hypothesize that BRCA2 gene silencer acts as a scaffolding to co-recruit SLUG and SPRDX5 and promote SPRDX5-mediated oxidative inactivation of the BRCA2 negative regulator protein SLUG. Here, we present evidence that (a) the expression of miR-6855-3p is essential for the expression of BRCA2 in the SLUG-high metastatic breast cancer cells; (b) knockdown of miR-6855-3p affects DSBR in these cells; and (c) the miR-6855-3p knocked-down cells are susceptible to the synthetic lethality by the PARP inhibitors. Our study reveals a novel mechanism of regulation of BRCA2 gene expression in the aggressive breast cancer cells, offering an alternative strategy to chemically intervene in the progression of hard-to-cure, highly aggressive SLUG-high breast cancer. We propose that the inhibition of miR-6855-3p will induce BRCA2 deficiency in the BLBC cells, thus increasing their susceptibility to standard chemotherapy as well as to PARP inhibitors as has been described for other BRCA2-deficient cells. Supported in part by DOD-CDMRP IDEA Expansion Grant #BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM. Note: This abstract was not presented at the conference. Citation Format: Marshall JD Ellison, Smita Misra, Gautam Chaudhuri. Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A12.

A Dominantly Inherited 5′ UTR Variant Causing Methylation-Associated Silencing of BRCA1 as a Cause of Breast and Ovarian Cancer

Pathogenic variants in BRCA1 or BRCA2 are identified in ∼20% of families with multiple individuals affected by early-onset breast and/or ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1 or BRCA2 have not explained the missing heritability. Here, we report a dominantly inherited 5′ UTR variant associated with epigenetic BRCA1 silencing due to promoter hypermethylation in two families affected by breast and ovarian cancer. BRCA1 promoter methylation of ten CpG dinucleotides in families who are affected by breast and/or ovarian cancer but do not have germline BRCA1 or BRCA2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants. BRCA1 promoter hypermethylation was identified in 2 of 49 families in which multiple women are affected by grade 3 breast cancer or high-grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa, and hair follicles. Pyrosequencing showed that DNA was ∼50% methylated, consistent with the silencing of one allele, which was confirmed by clonal bisulfite sequencing. RNA sequencing revealed the allelic loss of BRCA1 expression in both families and that this loss of expression segregated with the heterozygous variant c.−107A>T in the BRCA1 5′ UTR. Our results establish a mechanism whereby familial breast and ovarian cancer is caused by an in cis 5′ UTR variant associated with epigenetic silencing of the BRCA1 promoter in two independent families. We propose that methylation analyses be undertaken to establish the frequency of this mechanism in families affected by early-onset breast and/or ovarian cancer without a BRCA1 or BRCA2 pathogenic variant.

Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

Cathepsin S (CTSS) is a cysteine protease that is thought to play a role in many physiological and pathological processes including tumor growth, angiogenesis, and metastasis; it has been identified as a radiation response gene. Here, we examined the role of CTSS in regulating the DNA damage response in breast cancer cells. Activating CTSS (producing the cleavage form of the protein) by radiation induced proteolytic degradation of BRCA1, which ultimately suppressed DNA double-strand break repair activity. Depletion of CTSS by RNAi or expression of a mutant type of CTSS enhanced the protein stability of BRCA1 by inhibiting its ubiquitination. CTSS interacted with the BRCT domain of BRCA1 and facilitated ubiquitin-mediated proteolytic degradation of BRCA1, which was tightly associated with decreased BRCA1-mediated DNA repair activity. Treatment with a pharmacological CTSS inhibitor inhibited proteolytic degradation of BRCA1 and restored BRCA1 function. Depletion of CTSS by shRNA delayed tumor growth in a xenograft mouse model, only in the presence of functional BRCA1. Spontaneously uced rat mammary tumors and human breast cancer tissues with high levels of CTSS expression showed low BRCA1 expression. From these data, we suggest that CTSS inhibition is a good strategy for functional restoration of BRCA1 in breast cancers with reduced BRCA1 protein stability.

CD44v9 as a poor prognostic factor of triple-negative breast cancer treated with neoadjuvant chemotherapy

BackgroundNeoadjuvant chemotherapy (NAC) is the standard therapeutic strategy for triple-negative breast cancer (TNBC). TNBC patients with residual disease after NAC have a significantly worse survival than those with pathological complete response (pCR); however, there is no apparent prognostic factor for non-pCR patients. Cancer stemness or epithelial–mesenchymal transition (EMT) might influence the sensitivity to chemotherapy.Patients and methodsForty-eight patients with TNBC who were treated with NAC were available were included in this study. The expressions of stemness marker CD44v9, EMT marker vimentin and BRCA1, and basal phenotype were evaluated with immunohistochemistry. The relationships between the expression of these proteins and the pCR rate and the prognosis, especially in the patients with residual tumors, were investigated.ResultsAmong the 48 patients, pCR was achieved in 14 cases. High nuclear grade and basal phenotype in the pre-NAC samples were significantly correlated with pCR (p = 0.0458 and 0.0343). There were no significant relationships between the pCR rate and the expression of CD44v9, vimentin, or BRCA1. Achieving pCR was significantly correlated with longer distant metastasis-free survival (DMFS) (p = 0.0206). High CD44v9 expression was significantly associated with shorter DMFS (p = 0.0291). Among the patients in whom pCR was not achieved, high grade in the residual tumor cells, poor pathological response and high CD44v9 expression in the pre-treatment CNB samples were significantly correlated with a poor DMFS (p = 0.0433, 0.0406 and p = 0.0333). In addition, high grade in the residual tumor cells was significantly associated with high CD44v9 expression in the pre-treatment CNB (p = 0.0389).ConclusionsHigh CD44v9 expression in pre-NAC samples was associated with poor prognosis in TNBC patients treated with NAC, especially for those in whom pCR was not achieved.

Abstract 483: BRCA1 protein expression is downregulated by miR-185, miR-93 and miR-107 in breast cancer tumors

Abstract BRCA1 is a tumor-suppressor gene whose mutations confer a high risk to breast cancer. Fifty percent of hereditary breast cancer tumors without germline mutation have impaired BRCA1 expression, as well as 30% of sporadic cases. Somatic events that silence BRCA1 expression have been described in breast cancer tumors, such as promoter hypermethylation and BRCA1 gene deletion; however, these mechanisms do not completely explain the loss of BRCA1 expression in breast tumors. Our aim is to identify miRNAs differentially expressed in tumors with negative or moderate BRCA1 protein, which could regulate BRCA1 expression. Total RNA from 50 fresh-frozen breast cancer tumors was isolated and miRNA profiling was performed using miRNA microarrays. Sixteen miRNAs were found exclusively upregulated in BRCA1-negative, and ten in BRCA1-moderate tumors. Among them, in silico analyses predicted eleven miRNAs that could regulate BRCA1 expression. The effect of five of these microRNAs was assessed using a luciferase reporter assay in HEK293T cells, where the complete BRCA1 3'UTR, or a segment of its coding region, was cloned within the 3' end of the Luciferase gene. Cells were co-transfected with each of the selected microRNAs. MiR-185, miR-93 and miR-107 significantly downregulated luciferase activity by binding to the 3'UTR. Also, we transfected these microRNAs into a nontumorigenic breast cell line (MCF10A), where miR-93 diminished endogenous BRCA1 expression in 48%, miR-185 in 39% and miR-107 in 38%, in relation to non-transfected cells. These results strongly support that these microRNAs are potential regulators of BRCA1 expression in breast cancer tumors. FONDECYT1120200, CONICYT21120269. Citation Format: Valentina A. Zavala, Patricia Gajardo, Paola Faundez, Carolina Alvarez, Pilar Carvallo. BRCA1 protein expression is downregulated by miR-185, miR-93 and miR-107 in breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 483.