BRCA1 Levels Research Articles

Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.

Abstract Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR. Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies. Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P < 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P < 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm. Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC. Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2013-1788

Abstract LB-202: Responses to sequential sapacitabine and seliciclib in patients with BRCA-deficient solid tumors.

Abstract Background: Sapacitabine is an orally administered nucleoside analogue; the active metabolite, CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine), generates single-strand DNA breaks that are converted to double-strand DNA breaks (DSBs) during subsequent replication, resulting in cell death if unrepaired. Repair of CNDAC-induced DSBs is dependent on the homologous recombination (HR) repair pathway. Depletion or inhibition of components of the HR pathway (including ATM, BRCA1/2, Rad 51, and XRCC3) greatly sensitizes tumor cell lines to CNDAC-induced cell death in vitro. Seliciclib is an orally bioavailable inhibitor of cyclin-dependent kinases (CDKs) 2, 7 and 9. CDK2 has been shown to participate in DNA repair and to be a therapeutic target in BRCA-deficient cancers. Seliciclib inhibits DSB repair, and also reduces BRCA1 and BRCA2 mRNA levels in cancer cell lines, sensitizing tumor cells to CNDAC. This phase I study evaluates sequential sapacitabine and seliciclib. Methods: Dose escalation was conducted in patients with incurable solid tumors and adequate organ function with sapacitabine bid x 7 consecutive days (d1-7), seliciclib bid x 3 consecutive days (d8-10) followed by 11 days of rest. At least 3 patients were evaluated per dose level. MTD was the highest dose level at which less than one-third of at least 6 patients experienced cycle 1 DLT. Skin biopsies were obtained to assess DNA damage following sapacitabine (d8 vs pre-treatment) and further augmentation of DNA damage after seliciclib (d11 vs d8). Results: 38 patients were treated. The MTD is sapacitabine 50 mg bid/seliciclib 1200 mg bid. DLTs were reversible transaminase elevations and neutropenia. The most frequent adverse events (all cycles, regardless of causality) included, fatigue, abdominal pain, diarrhea, constipation, decreased appetite, nausea, vomiting, anemia, neutropenia, pyrexia, AST elevation, alkaline phosphatase elevation, creatinine elevation, hyperglycemia, hypophosphatemia, cough, and alopecia, the majority mild to moderate in intensity. Skin biopsies showed a 2.3-fold increase in γ-H2AX staining post-sapacitabine (n=16; p=0.007) and a further 0.58-fold increase post-seliciclib (n=12; p=0.069). Four confirmed PRs occurred in patients with pancreatic, breast (2 pts) and ovarian cancer, all BRCA mutation carriers, lasting 21, 78+, 36+ and 42+ weeks, respectively. SD as best response ≥= 12 weeks was observed in 8 additional patients, including two BRCA mutation carriers with ovarian and breast cancer, lasting 64 and 21 weeks, respectively. Conclusions: Sequential sapacitabine and seliciclib is safe with preliminary antitumor activity. BRCA mutation carrier status may be a potential biomarker for response across multiple tumor types. An alternative schedule with concomitant administration of sapacitabine and seliciclib is currently under evaluation. Citation Format: Geoffrey I. Shapiro, John Hilton, James M. Cleary, Sara M. Tolaney, Leena Ghandi, Eunice L. Kwak, Jeffrey W. Clark, Andrew Wolanski, Tracy Bell, John Schulz, Sheelagh Frame, Chiara Saladino, Morag Hogben, Scott J. Rodig, Judy H. Chiao, David Blake. Responses to sequential sapacitabine and seliciclib in patients with BRCA-deficient solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-202. doi:10.1158/1538-7445.AM2013-LB-202

Abstract 1850: BRCA1-regulating microRNAs, miR-146a and miR-638, are overexpressed in triple negative breast cancer tumors.

Abstract microRNAs are non-coding RNA sequences of about 22 nucleotides long that participate in post transcriptional regulation of mRNAs. Its deregulation has been associated to breast cancer development and treatment resistance. Triple negative breast tumors are characterized by its difficult treatment due to lack of estrogen and progesterone receptors and ERBB2. In addition, a high proportion do not express BRCA1; a breast cancer high susceptibility gene. BRCA1 expression can be regulated by different mechanisms as we have confirmed in our laboratory, such as methylation of its gene promoter, mutations and deletions due genomic rearrangements. Although there are tumors that do not present any of these abnormalities, they express low nuclear BRCA1 levels or they do not express the protein at all. This is why analyzing levels of microRNAs that are shown to regulate BRCA1 mRNA expression is an interesting additional mechanism to be evaluated. There is experimental evidence that miR-146a and mir-638 regulate BRCA1 expression. We therefore analyzed miR-146a and miR-638 in triple negative breast cancer tumors (n=41). Briefly, total RNA was extracted from formalin-fixed tumors. Then, both microRNAs were evaluated using qPCR TaqMan assay and compared with BRCA1 expression previously determined by immunohistochemistry. Our results showed overexpression of miR-146a in 68% of the analyzed tumors and miR-638 in 82%. Among those tumors presenting abnormal BRCA1 expression (n=24), 50% showed an increase of miR-146a and 75% presented miR-638 overexpression. Both microRNAs showed a discrete overexpression in tumors with normal levels of BRCA1 (n=16). Finally, this data suggest that microRNAs might regulate BRCA1 expression in triple negative tumors. Citation Format: Valentina Zavala, Wanda Fernandez, Adolfo Cruz, Laura Segovia, Mauricio Camus, Pilar Carvallo. BRCA1-regulating microRNAs, miR-146a and miR-638, are overexpressed in triple negative breast cancer tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2013-1850

A three-gene signature as potential predictive biomarker for irinotecan sensitivity in gastric cancer

ObjectivePersonalized chemotherapy based on molecular biomarkers can maximize anticancer efficiency. We aim to investigate predictive biomarkers capable of predicting response to irinotecan-based treatment in gastric cancer.MethodsWe examined gene expression of APTX, BRCA1, ERCC1, ISG15, Topo1 and methylation of SULF2 in formalin-fixed paraffin-embedded gastric cancer tissues from 175 patients and evaluated the association between gene expression levels or methylation status and in vitro sensitivity to irinotecan. We used multiple linear regression analysis to develop a gene-expression model to predict irinotecan sensitivity in gastric cancer and validated this model in vitro and vivo.ResultsGene expression levels of APTX, BRCA1 and ERCC1 were significantly lower in irinotecan-sensitive gastric cancer samples than those irinotecan-resistant samples (P < 0.001 for all genes), while ISG15 (P = 0.047) and Topo1 (P = 0.002) were significantly higher. Based on those genes, a three-gene signature were established, which was calculated as follows: Index =0.488 - 0.020× expression level of APTX + 0.015× expression level of Topo1 - 0.011 × expression level of BRCA1. The three-gene signature was significantly associated with irinotecan sensitivity (rho = 0.71, P < 0.001). The sensitivity and specificity for the prediction of irinotecan sensitivity based on the three-gene signature reached 73% and 86%, respectively. In another independent testing set, the irinotecan inhibition rates in gastric samples with sensitive-signature were much higher than those with resistant-signature (65% vs. 22%, P < 0.001). Irinotecan therapy with 20 mg/kg per week to immunodeficient mice carrying xenografts with sensitive-signature dramatically arrested the growth of tumors (P < 0.001), but had no effect on mice carrying xenografts with resistant-signature.ConclusionsThe three-gene signature established herein is a potential predictive biomarker for irinotecan sensitivity in gastric cancer.

Customized chemotherapy in metastatic non-small cell lung cancer (NSCLC).

Metastatic non-small cell lung cancer (NSCLC) unfortunately remains a lethal disease, despite recent genetic characterization of subclasses of NSCLC, mainly adenocarcinoma, which has led to the development of targeted therapies that improve progression-free survival (PFS). Ultimately, however, patients fatally relapse. In this review we will focus on the search to improve survival for NSCLC patients deemed to be pan-negative for the common driver alterations susceptible to targeted therapy, above all those with EGFR mutations or ALK, ROS or RET translocations. Other uncommon driver mutations such as HER2 and BRAF mutations should be tested in order to rule out targeted treatment before assigning patients to chemotherapy. Chemotherapy yields short lived response with median survival still less than one year. Customized chemotherapy represents one way to attempt to prolong survival, although to date no prospective randomized customized studies have reported sufficient evidence to support this. In one attempt to demonstrate the role of tailoring chemotherapy, the Spanish Lung Cancer Group (SLCG) phase II customized chemotherapy trial (NCT00883480) showed that RAP80, a component of the BRCA1-A complex, influenced outcome in patients with low BRCA1 expression treated with cisplatin/gemcitabine, and in patients with intermediate/high BRCA1 levels receiving cisplatin/docetaxel or docetaxel alone. We are currently performing a prospective, randomized phase III trial comparing non-customized cisplatin/docetaxel with customized therapy in metastatic NSCLC patients (NCT00617656/GECP-BREC) and a parallel phase II study (ChiCTR-TRC-12001860) is being carried out in China (BREC-China) under the auspices of the SLCG.

SiRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/− with susceptibility versus C57BL/6-Trp53+/− mice with resistance to mammary tumors

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/− female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/− females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/− mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/− versus C57BL/6J-Trp53+/− mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/−, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/− by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/− mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Role of Conventional Chemosensitivity Test and Tissue Biomarker Expression in Predicting Response to Treatment of Peritoneal Carcinomatosis From Colon Cancer

Background5-Fluorouracil- or oxaliplatin-based regimens are the treatments of choice in patients with PC from colon cancer. There are currently no useful preclinical evaluations to guide the decision-making process for tailored therapy. The aim of the present study was to compare the advantages and limits of a conventional in vitro chemosensitivity test with those of a panel of biomolecular markers in predicting clinical response to different drugs used to treat colon cancer-derived PC. Patients and MethodsFresh surgical biopsy specimens were obtained from 28 patients with peritoneal carcinomatosis from colon cancer. TS, TP, DPD, MDR1, MRP-1, MGMT, BRCA1, ERCC1, GSTP1, and XPD gene expression levels were determined by real-time reverse transcription polymerase chain reaction. An in vitro chemosensitivity test was used to define a sensitivity or resistance profile to the drugs used to treat each patient. ResultsExpression levels of the genes analyzed were generally poorly related to each other. TS and ERCC1 expression was inversely related to response to 5-FU-and/or oxaliplatin-containing regimens. Significant predictivity in terms of sensitivity but poor predictivity of resistance (56.2%) (P = .037) were observed for ERCC1 expression (90%), and high predictivity of resistance (100%) but very low predictivity of sensitivity (40%) (P = .014) were registered for TS. The best overall and significant predictivity was observed for chemosensitivity test results (62.5% sensitivity and 89% resistance; P = .005). ConclusionsSensitivity and resistance to drugs used in vivo was better defined by the chemosensitivity test than by biomarker expression.

Association between clinical pathology and multiple genes mRNA expression in Chinese patients with NSCLC

The aim of this study was to evaluate whether there was an association between pathology type and ERCC1, BRCA1, RRM1, TUBB3, STMN1, TOP2A and epidermal growth factor receptor (EGFR) messenger ribonucleic acid (mRNA) expression level in Chinese patients with non-small cell lung carcinoma (NSCLC). mRNA expression level of these genes was analyzed in 181 cancer tissues by using xTAG-step liquid-chip array. The mRNA expression level of the seven genes was evaluated in association with the clinical pathology type. The average mRNA expression level of the seven genes were ERCC1 (1.02 ± 0.03), BRCA1 (0.15 ± 0.04), RRM1 (0.19 ± 0.05), TUBB3 (0.31 ± 0.06), STMN1 (2.78 ± 0.42), TOP2A (3.04 ± 0.42) and EGFR (0.58 ± 0.09), respectively in Chinese patients with NSCLC. The mRNA expression level of ERCC1, STMN1 and TOP2A genes were statistical different with different pathology type (p(a) < 0.05); STMN1 and TOP2A genes mRNA expression were much higher in squamous cell lung carcinoma than that in non-squamous cell lung carcinoma (p(a) < 0.05). And ERCC1 gene expression was much lower in squamous cell carcinoma than that in non-squamous cell carcinoma (p(a) < 0.05). mRNA expression level of STMN1, TOP2A and ERCC1 were correlated with the clinical pathology type.

Loss of BRCA1 expression leads to worse survival in patients with gastric carcinoma

To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival. Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (I/II vs III, 10.7% vs 20.5; I/II vs IV, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (I/II vs III, 12.9% vs 16.9%; I/II vs IV, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (I/II vs III, 0% vs 12%; I/II vs IV, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and MDC1 may serve as predictive factors in tumor development or progression in GC patients.

SLUG silencing increases radiosensitivity of melanoma cells in vitro

Melanoma radioresistance has been attributed to the presence of tumor cells with highly efficient DNA damage repair mechanisms. We examined the expression of genes involved in DNA damage repair and DNA damage sensing, and assessed their modulation by SLUG silencing, which is potentially capable of increasing radiosensitivity. Two melanoma cell lines (M14 and M79) were used to evaluate in vitro radiation-induced cytotoxicity before and after SLUG silencing. mRNA expression levels of BRCA1, ERCC1, DNA-PK, PARP, MGMT, ATM and TGM2 were determined by real-time RT-PCR, and protein expression levels of SLUG, caspase 3, p21, PUMA and pMAPK by Western blotting. The cytotoxic effect of radiation was high in M14 and low in M79 cells. SLUG silencing increased the interference of radiation on cell cycle distribution and cell killing by 60 % and 80 % in M79 cells after a 2.4 Gy and 5 Gy radiation dose, respectively. It also led to a significant inhibition of expression of genes involved in DNA damage repair and DNA damage sensing in all cell lines maintained after radiation. An almost total inhibition was observed for TGM2, which is expressed at a high basal level in the most radioresistant cell line (M79). Protein expression of PUMA was induced by radiation and was enhanced after SLUG silencing. Our results reveal a pivotal role of SLUG in regulating a cellular network involved in the response to DNA damage, and highlight the importance of TGM2 in radiosensitivity modulation. SLUG silencing appears to increase radiation sensitivity of the melanoma cells tested.

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines

Five-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance.

A Tissue-specific Long Noncoding RNA Controls Homologous Recombination in Prostate Cancer

Our group has recently identified 121 novel long non-coding RNAs (lncRNAs) whose dysregulated expression stratifies prostate cancer progression. One of these, PCAT1, was upregulated in aggressive disease, and PCAT1 expression inversely correlated with expression levels of BRCA2, a homologous recombination (HR) repair protein. The purpose of this study was to determine the role of PCAT1 in DNA repair and response to genotoxic stress.

807P - Outcome to Neoadjuvant Cisplatin-Based Chemotherapy in Resectable Muscle-Invasive Bladder Cancer (MIBC) Patients (P) According to Expression Levels of BRCA1, RAP80, and AEG-1

ABSTRACT Background Response to neoadjuvant chemotherapy has been associated with an improvement in survival in MIBC. The analysis of genetic markers involved in DNA repair pathways could be useful to predict chemosensitivity to cisplatin-based regimens. BRCA1 plays a central role in DNA repair and cell-cycle checkpoint control. However, BRCA1 function can be modulated by other DNA repair genes. RAP80 acts upstream of BRCA1 and regulates BRCA1 function after DNA damage. AEG-1 can induce BRCA1 expression and cause chemoresistance by activating PI3K/Akt and NF-KB pathways. Methods Paraffin-embedded pre-treatment tumor samples were collected by transurethral resection from 65 p with resectable MIBC stage T2-4N0M0 treated with neoadjuvant cisplatin-based chemotherapy. Gene expression levels of BRCA1, RAP80 and AEG-1 were quantified by real-time quantitative PCR. Expression levels were divided into terciles and correlated with median survival (MS). Results 33 p were treated with cisplatin, methotrexate and vinblastine (CMV) and 32 p with cisplatin and gemcitabine. Chemotherapy was followed by cystectomy in 60 p. Overall MS was not reached and 5-year survival was 51%. 16 p (38%) with low/intermediate BRCA1 levels had a complete pathological response (pR), while only 1 p (5%) with high BRCA1 levels had a complete pR (P = 0.006). MS was 45 months (m) and 5-year survival was 27% in 21 p with high BRCA1 mRNA levels vs 168 m and 59% in 44 p with low/intermediate levels (P = 0.05). No differences in MS were observed according to RAP80 mRNA levels. MS was 50 m in 15 p with high AEG-1 levels, 45 m in 15 p with intermediate levels, and was not reached in 18 p with low levels, although these differences were not statistically significant (P = 0.3). Conclusions Low/intermediate BRCA1 mRNA expression is associated with better pR and longer MS and 5-year survival to cisplatin-based neoadjuvant chemotherapy in p with MIBC. BRCA1 levels can be useful in customizing chemotherapy in these p. Further studies with larger numbers of p are warranted to elucidate the role of other molecular markers, including AEG-1, in this setting. Disclosure All authors have declared no conflicts of interest.

1661P - The Predictive Role of the 53BP1 Pathway in Advanced Non-Small-Cell Lung Cancer (NSCLC) Patients (P) Treated with First-Line Platinum-Based Chemotherapy

ABSTRACT Background Preclinical and clinical data have shown that low BRCA1 expression increases sensitivity to platinum. However, the complexity of DNA repair pathways suggests that the BRCA1 function could be modulated by several proteins. MDC1, the protein initiating the response to DNA double strand breaks, activates two parallel pathways: 53BP1 and BRCA1. We hypothesized that the mRNA expression of components involved in the 53BP1 pathway could influence the predictive value of BRCA1. Methods mRNA expression levels of BRCA1, MDC1, UBC13, RNF8, 53BP1, PIAS4, MMSET and UBC9 were assessed by real-time PCR analysis in 115 paraffin-embedded tumor samples collected from advanced NSCLC p treated with first-line platinum-based chemotherapy without taxanes. Results Expression levels of BRCA1 were correlated with MDC1 (r = 0.47, P Conclusions Advanced NSCLC p with low levels of both BRCA1 and 53BP1 showed significantly improved outcome to first-line platinum-based chemotherapy. The assessment of BRCA1 and 53BP1 can be useful for customizing chemotherapy. Disclosure All authors have declared no conflicts of interest.

1655P - High mRNA Expression of LMO4, A BRCA1 Downregulator, Correlates with Better Prognosis in Erlotinib-Treated Non-Small-Cell Lung Cancer (NSCLC) Patients (P) with EGFR Mutations

ABSTRACT Background High BRCA1 mRNA levels affected progression-free survival (PFS) to erlotinib in EGFR-mutant NSCLC p (Rosell et al. CCR 2011). LMO4 is a negative regulator of BRCA1 function, and CtIP can bind to BRCA1 and LMO4. We have assessed the expression of CtIP, LMO4 and BRCA1 and examined the impact of CtIP and LMO4 levels on outcome. Methods mRNA expression of BRCA1, LMO4 and CtIP was examined by RT-PCR in the original pretreatment tumor biopsies of 72 erlotinb-treated NSCLC p with sensitive EGFR mutations. Results p characteristics: median age, 68; 61.8% female; 98.2% Caucasian; 63.6% never-smokers; 81.8% ECOG PS Conclusions Low BRCA1 and high LMO4 levels were associated with longer PFS in erlotinib-treated advanced NSCLC p with EGFR mutations. We recommend baseline assessment of BRCA1 and LMO4 mRNA expression to predict outcome and provide alternative individualized treatment to patients when indicated. HR 95%CI P BRCA1 ≤4.92 1 ref. 4.92-10.7 5.32 1.45-19.52 0.03 >10.7 5.13 1.25-20.99 0.02 CtIP ≤1.21 1 ref. 1.21-2.1 0.69 0.24-2.02 0.50 >2.1 1.10 0.42-2.89 0.84 LMO4 ≤1.29 6.02 1.51-23.94 0.01 1.29-1.86 2.81 0.85-9.21 0.09 >1.86 1 ref. Disclosure All authors have declared no conflicts of interest.

PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells

Two cellular proteins encoded by the breast and ovarian cancer type 1 susceptibility (BRCA1 and BRCA2) tumor suppressor genes are essential for DNA integrity and the maintenance of genomic stability. Approximately 5–10% of breast and ovarian cancers result from inherited alterations or mutations in these genes.Remarkably, BRCA1/BRCA2-deficient cells are hypersensitive to selective inhibition of poly(ADP-ribose)polymerase 1 (PARP-1), whose primary functions are related to DNA base excision repair; PARP-1 inhibition significantly potentiates the cytotoxicity of various anti-cancer drugs, including inhibitors of topoisomerase I and II.In the present study, we examined the anti-proliferative and pro-apoptotic effects of C-1305, a selective inhibitor of topoisomerase II, on human breast cancer cell lines with different BRCA1 and p53 statuses. BRCA1-competent breast cancer cell lines exhibited different responses to topoisomerase II inhibition. BT-20 cells that express high levels of BRCA1 levels were most resistant to C-1305 than other tested cells. Surprisingly, pharmacological interference with PARP-1 activity strongly inhibited their proliferation and potentiated the efficacy of C-1305 treatment. In contrast, PARP-1 inhibition only weakly affected the proliferation of BRCA1-deficient SKBr-3 cells and was not synergistic with the effects of C-1305. Further experiments revealed that the inhibition of PARP-1 in BT-20 cells caused the accumulation of DNA strand breaks and induced caspase-3 dependent apoptosis. These results seem to indicate that PARP-1 inhibition can potentiate the cytotoxicity of anti-cancer drugs in cancer cells with functional BRCA1 and suggest that mutations in other DNA repair proteins may render cancer cells more sensitive to interference with PARP-1 activity.

BRCA1 promoter methylation status does not predict response to tamoxifen in sporadic breast cancer patients

The purpose of this study is to investigate whether BRCA1 promoter methylation is associated with poorer outcome in sporadic breast cancer cases treated with tamoxifen. BRCA1 promoter methylation was determined by bisulfite pyrosequencing in two groups of sporadic breast cancer patients, systemically untreated (N = 497) and treated with adjuvant tamoxifen (N = 497). Associations of BRCA1 promoter methylation with clinopathological characteristics and the effect of BRCA1 promoter methylation on time to first recurrence (TTR) and overall survival (OS) were examined. No significant differences were observed between BRCA1 promoter methylation and clinopathological characteristics in untreated and tamoxifen-treated groups. Cut point analysis did not find any promising cut point for BRCA1 promoter methylation that would differentially influence TTR and OS in untreated and tamoxifen-treated group. Using the median (2.53 %) and an arbitrary value of 10 % as a cut point for methylation, we still found no significant effect of BRCA1 promoter methylation on TTR and OS in untreated and tamoxifen-treated group. Despite data suggesting that BRCA1 levels impact estrogen receptor response to tamoxifen, our results indicate that BRCA1 promoter methylation is not associated with poorer outcome in sporadic breast cancer cases treated with tamoxifen.

DNA repair components modulating the function of breast cancer susceptibility gene 1 (BRCA1) in advanced non-small cell lung cancer (NSCLC) patients (p) treated with platinum-based chemotherapy.

e18130 Background: The standard first-line treatment for advanced NSCLC p with wild-type epidermal growth factor receptor (EGFR) is platinum-based chemotherapy, but median overall survival (OS) is about 12 months, with great heterogeneity in terms of efficacy and tolerability. Low BRCA1 mRNA expression has been correlated with sensitivity to cisplatin, but BRCA1 function is modulated by other DNA repair components. For example, AEG1 can induce BRCA1 expression, and CASPASE 3 cleaves the mediator of DNA damage checkpoint 1 (MDC1) preventing the activation of the DDR pathway Methods: Paraffin-embedded samples were collected from 115 advanced NSCLC p treated with first-line platinum-based chemotherapy (not including taxanes and vinca alkaloids). Samples were screened for EGFR, KRAS and p53 mutations, and gene expression levels of BRCA1, RAP80, AEG1, MDC1, CASPASE 3, 53BP1, MMSET, PIAS4, UBC9, UBC13, NPM1 and HERC2 were quantified by real-time PCR. Results: Gene expression levels were significantly higher in squamous tumors, and the correlation among genes confirmed the biological model. Median OS was 11 months and median progression free survival (PFS) was 7.1 months. In p with low levels of both BRCA1 and RAP80, PFS was 12 months and OS was not reached, while in p with high level of both genes, PFS was 5.1 months and OS was 6.2 months and in p with other combinations, PFS was 7.4 months and OS was 10.9 months (P=0.02). Increased levels of AEG1 increased the risk of death (HR: 1.3; P=0.01) and of progression (HR: 1.2; P=0.04). When CASPASE 3 levels were divided into tertiles, intermediate levels were associated with improved PFS (P=0.005) and OS (P=0.05). The multivariate analysis confirmed the role of BRCA1 (P=0.02) and CASPASE 3 (P=0.008). Conclusions: The study showed the predictive value of combined BRCA1 and RAP80 mRNA levels. In addition, AEG1 and CASPASE 3 have emerged as potential predictive markers.