RAP80 Expression Research Articles

RAP80 Phase Separation at DNA Double-Strand Break Promotes BRCA1 Recruitment and Tumor Radio-Resistance

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 are not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, may be implicated. Recently, we and other groups have reported that liquid-liquid phase separation (LLPS) is a pivotal mechanism underlying DNA repair factors condensation at DSBs and their function. In this study, we aim to disclose whether RAP80 undergoes LLPS at DSBs and whether it is required for BRCA1 recruitment. To verify RAP80 is an LLPS protein and its function in DNA damage response (DDR): (1) candidate-mEGFP fusion protein formed condensates in cells and showed fluorescence recovery after photobleaching (FRAP); (2) candidate protein was expressed in Escherichia coli and purified with GST; (3) intrinsically disordered region (IDR) of RAP80 were predicted and tested in cell and in vitro; (4) lentivirus were used to construct RAP80-Knock out (KO) and RAP80 re-expression cell lines; (5) length gradient K63 poly-ubiquitin chains were chemically synthesized and incubated with RAP80 protein in vitro; (6) BRCA1 and RAP80 location were determined through immunofluorescence; (7) RAP80 protein expression in tissue was determined by IHC staining. Thin layer scanning and 3D reconstruction of the RAP80-mEGFP-expressing cells under a fluorescence microscope showed that RAP80-mEGFP formed spherical condensates with fast FRAP. Observation of purified proteins revealed that GST-RAP80-mEGFP protein formed liquid-like droplets, presenting as a FRAP and the fusion event among adjacent droplets. PEG-8000 and Ficol-400 strengthened the formation of GST-RAP80-mEGFP droplets in vitro. Later, we used a previously developed optoIDR tool to verify that IDR1 (1-254aa) is critical for RAP80 LLPS. To investigate whether the interaction between RAP80 and K63 poly-ubiquitin chains could enhance the condensation of RAP80, we chemically synthesized K63 ubiquitin chains and incubated them with purified GST-RAP80-mCherry proteins. The results showed that supplementation of ubiquitin multipolymer (poly-ubiquitin) significantly induced the LLPS of RAP80, and the ability of RAP80 condensates formation potency was positively correlated with the length of the ubiquitin chain. Consistent with their LLPS capacity, RAP80-WT-mEGFP, RAP80-(IDR1+AIR)-mEGFP groups showed prominent BRCA1 foci, while RAP80-IDR1-mEGFP and RAP80-(SIM+UIM)-mEGFP groups showed delayed BRCA1 recruitment. In rectal cancer tissues, positive staining of the RAP80 protein was mainly observed in the nucleus of cancer cells and high RAP80 expression was correlated with a shorter overall survival time. RAP80 undergoes LLPS to form liquid-like condensates at DSB sites, which is important for BRCA1 recruitment and enhances tumor radio-resistance.

Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?

Background: Several studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Most recently, KRAS 12c data suggest specific treatment for improving ORR and OS. There is a clear need for therapies specifically developed for these patients. Moreover, data that might be suggestive of a response to specific therapies, such as BRCA1, are needed, and two mutations that were studied in other malignancies show more response to PARP inhibitors. Molecular profiling has the potential to identify other potential targets that may provide better treatment and novel targeted therapy for KRAS-mutated NSCLC. Methods: We purified RNA from archived tissues of patients with stage I and II NSCLC with wild-type (wt) and mutant (mt) KRAS tumors; paired normal tissue adjacent to the tumor from 20 and 17 patients, respectively, and assessed, using real-time reverse transcriptase–polymerase chain reaction (RT-PCR), the expression of four genes involved in DNA synthesis and repair, including thymidylate synthase (TS), BRCA1, ECCR1, RAP80, and the proto-oncogene SRC. Additionally, we assessed the expression of PD-L1 in mt KRAS tumors with immunohistochemistry using an antibody against PD-L1. Results: Our results show that in mt KRAS tumors, the level of expression of ERCC1, TS, and SRC was significantly increased in comparison to paired normal lung tissue (p ≤ 0.04). The expression of BRCA1 and RAP80 was similar in both mt KRAS tumors and paired normal tissue. Furthermore, the expression of BRCA1, TS, and SRC was significantly increased in wt KRAS tumors relative to their expression in the normal lung tissue (p < 0.044). The expression of ERCC1 and RAP80 was similar in wt KRAS tumors and paired normal tissue. Interestingly, SRC expression in mt KRAS tumors was decreased in comparison to wt KRAS tumors. Notably, there was an expression of PD-L1 in the tumor and stromal cells in a few (5 out of 20) mt KRAS tumors. Our results suggest that a greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt KRAS tumor might be suggestive of the sensitivity of taxanes. Our data also suggest that the combination of an SRC inhibitor with a TS inhibitor, such as pemetrexed, might improve the outcome of patients with NSCLC and in particular, patients with wt KRAS tumors. PD-L1 expression in tumors, and especially stromal cells, suggests a better outcome. Conclusion: mt KRAS NSCLC patients might benefit from a treatment strategy that targets KRAS in combination with therapeutic agents based on pharmacogenomic markers, such as SRC and BRCA1. mt KRAS tumors are likely to be platinum-, taxane-, and pemetrexed-resistant, as well as having a low level of PD-L1 expression; thus, they are less likely to receive single-agent immunotherapy, such as pembrolizumab, as the first-line therapy. wt KRAS tumors with BRCA1 positivity tend to be sensitive to taxane therapy and, potentially, platinum. Our results suggest the need to develop targeted therapies for KRAS-mutant NSCLC or combine the targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor.

Competing endogenous RNA network features and prostate tumor cell sensitivity to radiotherapy.

e17008 Background: Radiotherapy (RT) is one of the main treatments for prostate cancer (PC). The effectiveness of RT depends on the initial radioresistance of tumor cells provided by their certain molecular features - the transcriptional activity of genes, miRNAs and long non-coding RNAs (lncRNAs). Together, these indicators form a competing endogenous RNAs network (ceRNAs). The aim of this work was to study the ceRNA features in PC patients with different responses to RT. Methods: The study included 400 patients with PC, 90 of them developed biochemical recurrence after RT (Novalis TX, TFD = 75 Gy). Tumor tissue samples were obtained by biopsy. The RNA fraction was isolated and purified using the RNeasy Plus Universal Kits. Bioinformatic search for miRNAs was performed using a modified TarPmiR algorithm. The miRNA-lncRNA interactions were assessed by bioinformatics. Expression of AKT, ATM, BRIP, BRCA1/2, CDK1, CDKN1B, CCND1/3, EXO1, H2AX, KU70, PTEN, RAD50, RAP80, RIF1, RNF168, TOPB1, TP53, XRCC4, BAX, CASP-8, -3, -9, MDM2, BCL2, RBBP8, EP300 genes, miRNA and lncRNA were determined by RT-qPCR. Differences were assessed using the Mann-Whitney test (Bonferroni correction was used). Results: The expression of CDK1, CDKN1B, RBBP8, XRCC4, BRCA2, RAD50 genes in tumor tissues of patients with biochemical recurrence (group 1) was higher (p &lt; 0.005) and the expression of TP53 and BCL2 was lower than the values in tumor tissues in patients without recurrence (group 2). Bioinformatic search for miRNAs targeting the CDK1, CDKN1B, RBBP8, XRCC4, BRCA2, RAD50, TP53 and BCL2 revealed 1725 miRNAs, 78 of which were validated (miRDB) and had a minimum free energy of miRNA-mRNA interaction. Patients in group 1 showed differential expression of miRNAs targeting genes BCL2 (increased miR-139-5p, miR-4446-5p, miR-211-5p); CDK1 (decreased miR-330-3p, miR-5580-3p, miR-429), CDKN1B (decreased miR-6841-3p); RAD50 (decreased miR-433-3p, miR-3691-5p); RBBP8 (decreased miR-130b-3p); TP53 (increased miR-6884-5p, miR-149-3p, miR-6860, miR-4728-5p, let-7b-5p); XRCC4 (decreased miR-4489) and BRCA2 (decreased miR-7151-3p). The interaction of these miRNAs with 160 lncRNAs was mathematically predicted. Patients in group 1 demonstrated increased expression of lncRNAs LINC00641, SCAMP1, MALAT1, MATN1-AS1 (miR-429) , LINC00657 and GAS5 (miR-433-3p), CCAT1 (miR-130b-3p) and reduced expression of S NHG3, DHRS4-AS1 (miR-139-5p) and MAL2 (let-7b-5p). Conclusions: Our study of the ceRNA network features and RT effectiveness for PC established the mechanisms of radioresistance development and its predictors.

RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells.

BackgroundRAP80 is a member of BRCA1-A complex, which plays an important role in regulating the cell cycle checkpoint and DNA damage repair in the nucleus.MethodWe investigated RAP80 expression in breast cancer and its paired normal breast tissues to further analyze its role in the biological behavior of breast cancer cells.ResultsRAP80 expression in breast cancer (62.3%, 101/162) was significantly lower than that in adjacent normal breast tissues (P<0.05). RAP80 expression was related to tumor size, lymph node metastasis, TNM stage, and molecular subtype (P<0.05). RAP80 mRNA expression was significantly lower in triple-negative breast cancer than other types. The mRNA and protein of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231, so we picked MCF-7 to be transfected with RAP80 siRNA. The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7 according to MTT. RAP80 siRNA transfection upregulated the apoptosis and downregulated invasive or migrating ability of MCF-7. RAP80 siRNA also upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, Bax, and Fas, and downregulated the protein expression of Bcl-2.ConclusionRAP80 expression was related to ER or PR activity. Inhibition of RAP80 expression can induce apoptosis in breast cancer cells and improve chemosensitivity to cisplatin. Tumor cells can activate protective responses to inhibit cell cycle progression, which may be related to RAP80, and repair cisplatin-induced DNA damage. RAP80 is related to BRCA1’s effect, which can be used as an interesting target for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

Association of BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression levels between primary tumors and infiltrated regional lymph nodes in patients with resectable non-small cell lung cancer

Differences in gene expression levels between the primary tumors (PTs) and matched regional lymph nodal metastases (LNs) in patients with totally excised non-small cell lung cancer (NSCLC) were explored. Microdissected formalin-fixed paraffin-embedded (FFPE) samples from (PT) and their matched infiltrated LNs, from 239 patients [183 (with matched PT and LNs samples)-case and 56 PT only samples-control cohorts] were analyzed for BRCA1, ERCC1, RAP80, PKM2, RRM1, RRM2, TS, TSP1, and TXR1 mRNA expression by quantitative real-time polymerase-chain reaction (PCR). Moderately positive correlation between the expression of each gene in the PT and the matched LNs was observed. Concordance rates between the PT and the LNs were: BRCA1 (67.7%), ERCC1 (68.4%), PKM2 (63.4%), RAP80 (68.8%), RRM1 (70.9%), RRM2 (69%), TS (72.9%), TSP1 (69.8%), TXR1 (63.7%). Expression levels and their differences were correlated with Relapse-Free Survival (RFS) and Overall Survival (OS). High BRCA1 PT in patients with squamous histology was associated with increased OS (p = 0.036). High TSP1 PT levels were shown to be the only independent prognostic factor for OS and RFS (p = 0.023 and p = 0.007). PKM2 low levels in both PT and matched LNs were associated with better OS irrespective of the underlying histology (p = 0.031). RRM1 discordant levels between PT and matched LNs were associated with worse OS in squamous tumors (p = 0.019) compared to patients with both low expression in PT and LN.TXR1 high levels in both PT and matched LNs were associated with better OS in patients with squamous tumors (p = 0.007).These findings indicate that there is different gene expression between PT and matched LNs which may affect the outcome in early NSCLC and therefore PT's molecular biology should not be the sole determinant for prognostication.

Low RAP80 mRNA expression correlates with shorter survival in sporadic high-grade serous ovarian carcinoma.

Homologous recombination (HR) is frequently impaired in sporadic high-grade serous ovarian carcinoma (sHGSOC) due to deficiencies in BRCA1/2 genes, a situation associated with hypersensitivity to platinum compounds. Alterations in other genes can also cause HR deficiency. Preclinical data show that RAP80 is an HR-pathway-related gene that influences BRCA1 activity. RAP80 has been reported to affect outcome in some solid neoplasms. This study investigates the role of RAP80 in sHGSOC survival. mRNA expression of RAP80 was analyzed in tumor samples from 35 patients who postoperatively received standard platinum-based chemotherapy. The effects of RAP80 expression on progression-free survival (PFS) and overall survival (OS) were examined by means of Cox regressions. The clinical variables known to have prognostic value (FIGO stage, residual disease at surgery, and debulking surgery) were included as covariates in the analysis. BRCA1 was analyzed given the moderate correlations with RAP80. Median follow-up, PFS and OS were 61.3, 20.2 and 62.8 months, respectively. Low RAP80 expression levels were associated with shorter PFS (HR = 1.449, p = 0.007) and OS (HR = 1.331, p = 0.047). This is the first study to show a potential prognostic role of RAP80 expression in patients with HGSOC. The results suggest that HR deficiency due to low RAP80 expression is not associated with hypersensitivity to platinum compounds in sHGSOC.

A novelly synthesized phenanthroline derivative is a promising DNA-damaging anticancer agent inhibiting G1/S checkpoint transition and inducing cell apoptosis in cancer cells.

The study mainly aimed to determine the biological function of a novelly synthesized phenanthroimidazole derivative, named L233, and to explore its potential mechanisms. Cell survival was examined using the MTT assays, and the DNA-damaging role of L233 was explored using the comet assay. Moreover, the western blotting assays and immunofluorescence assays were used to detect DNA damage biomarkers. Afterward, the flow cytometry was used to assess the effects of L233 on cell cycle distribution. As for the detection of cell apoptosis upon L233 treatment, the Hoechst 33342 staining, flow cytometry, and western blotting assays were all put into practice. We find that L233 inhibits tumor cell growth more efficiently and safely than cisplatin. Moreover, it is a DNA-damaging agent, interrupting the cell cycle G1/S checkpoint transition and inducing cell apoptosis by not only activating ATM/CHK1 signaling pathway, but also targeting CHK1 to reduce the expression of RAP80 and PARP-1 to compromise the DNA damage repair in tumor cells. In summary, L233 is a promising anticancer drug for the development of novel chemotherapies in the future.

PARP1-driven poly-ADP-ribosylation regulates BRCA1 function in homologous recombination-mediated DNA repair.

BRCA1 promotes homologous recombination-mediated DNA repair (HRR). However, HRR must be tightly regulated to prevent illegitimate recombination. We previously found that BRCA1 HRR function is regulated by the RAP80 complex, but the mechanism was unclear. We have now observed that PARP1 interacts with and poly-ADP-ribosylates (aka PARsylates) BRCA1. PARsylation is directed at the BRCA1 DNA binding domain and downmodulates its function. Moreover, RAP80 contains a poly-ADP-ribose-interacting domain that binds PARsylated BRCA1 and helps to maintain the stability of PARP1-BRCA1-RAP80 complexes. BRCA1 PARsylation is a key step in BRCA1 HRR control. When BRCA1 PARsylation is defective, it gives rise to excessive HRR and manifestations of genome instability. BRCA1 PARsylation and/or RAP80 expression is defective in a subset of sporadic breast cancer cell lines and patient-derived tumor xenograft models. These observations are consistent with the possibility that such defects, when chronic, contribute to tumor development in BRCA1+/+ individuals. We propose a model that describes how BRCA1 functions to both support and restrict HRR. BRCA1 PARsylation is a key event in this process, failure of which triggers hyper-recombination and chromosome instability. Thus, hyperfunctioning BRCA1 can elicit genomic abnormalities similar to those observed in the absence of certain BRCA1 functions.

Abstract A01: KRAS molecular profiling in non-squamous non-small cell lung cancer (NSCLC).

Abstract Recent studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Therefore, there is a clear need for therapies specifically developed for these patients. Molecular profiling has the potential to identify potential targets that might provide a better treatment and novel targeted therapy for KRAS-mutated NSCLC. In this study, we purified RNA from archived tumor and normal lung tissue obtained from 20 patients with wild type (wt) and 17 patients with mutant (mt) KRAS tumors undergoing resection for stage I and stage II NSCLC, and assessed by reverse transcriptase-real time polymerase chain reaction (RT-PCR) the expression of four genes involved in DNA synthesis and repair including thymidylate synthase (TS), BRCA1, ECCR1 and RAP80, and the proto-oncogene SRC. Our results show that in mt KRAS tumors, the levels of expression of ERCC1, TS and SRC were significantly increased in comparison to paired-normal lung tissue (p ≤ 0.04). Expression of BRCA1 and RAP80 were similar in both mt KRAS tumors and paired-normal tissue. Furthermore, the expression of BRCA1, TS and SRC were significantly increased in wt KRAS tumors relative to their expression in normal lung (p ≤ 0.044). Expression of ERCC1 and RAP80 was similar in wt KRAS tumors and paired normal tissue. Interestingly, SRC expression in mt KRAS tumors was decreased in comparison to wt KRAS tumors. Our results suggest that greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt KRAS tumors might be suggestive for the sensitivity to taxanes. Our data also suggests that the combination of a SRC inhibitor with a TS inhibitor such as pemetrexed might improve the outcome of patients with wt KRAS tumors. In conclusion, these results suggest that the most effective treatment strategy for KRAS-mutant NSCLC is combined targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor. Citation Format: Nagla Abdel Karim, Sandra Starnes, John Morris, Peter Pathrose, Ashley Perry, Hassana Fathallah. KRAS molecular profiling in non-squamous non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A01.

Predictive markers in metastatic transitional cell carcinoma (mTCC) patients treated with carboplatin plus pemetrexed regimen.

e15558 Background: Treatment with platinum derivatives plus pemetrexed has shown a benefit in metastatic transitional cell bladder carcinoma (mTCC); however, the subset of patients most likely to benefit has not yet been identified. Predictive markers of pemetrexed response can be inferred from its mechanism of action in inhibiting nucleotide synthesis. In the present study we evaluated the predictive role of BRCA1, RAP80, TS, REV3 and AEG1 mRNA levels in a series of mTCC samples from patients treated with carboplatin plus pemetrexed (CP) and correlated with pathological response to chemotherapy and median survival. Methods: We retrospectively analyzed 18 patients with mTCC. Eligible patients received pemetrexed 500 mg/m2 in combination with carboplatin at an area under the concentration curve of 5. Total treatment dose was given on day 1 of each cycle and repeated every 21 days for 6 cycles. Gene expression levels were quantified by real-time quantitative PCR from paraffin-embedded pre-treatment tumor samples obtained by transurethral resection. Results: Patients were male, mean age 71 years (range 51-83). Median overall survival was 3.5 years (95% CI, 0.377-3.623). Non-responders had average survival of 2.2 years (SE=0.436) compared to an average of 5.5 years for responders (SE=0.661) (Pearson X2=4.667, p=0.031). We found an association between non-responders and elevated RAP80 expression (paired t (df)=-2.69, p=0.028) (paired t (df)=2.372, p=0.039). In the multivariate analysis elevated TS level was the variable most predictive of shorter survival (OR=1.158, p=0.047). Conclusions: CP regimen demonstrated moderate activity in patients with mTCC. Our data indicates that RAP80 and TS could be predictors of poor outcome. Also, RAP80 expression may predict efficacy of CP as first-line treatment and could be a useful tool to customize therapy in bladder cancer patients. A prospective study is warranted to validate these observations.

Predictive models for customizing chemotherapy in advanced non-small cell lung cancer (NSCLC).

The backbone of first-line treatment for Epidermal Growth Factor (EGFR) wild-type (wt) advanced Non-small cell lung cancer (NSCLC) patients is the use of a platinum-based chemotherapy combination. The treatment is characterized by great inter-individual variability in outcome. Molecular predictive markers are extremely needed in order to identify patients most likely to benefit from platinum-based treatment and resistant ones, thus optimizing chemotherapy approach in NSCLC. Several components of DNA repair response (DRR) have been investigated as potential predictive markers. Among them, high levels of expression of ERCC1, both at protein and mRNA levels, have been associated with resistance to cisplatin in NSCLC. In addition, low levels of expression of RRM1, a target for gemcitabine, have been associated with improved OS in advanced NSCLC patients treated with cisplatin and gemcitabine. Preclinical data and retrospective analyses showed that BRCA1 is able to induce resistance to cisplatin and sensitivity to antimicrotubule agents. In addition, the mRNA levels of expression of RAP80, encoding for a protein cooperating with BRCA1 in homologous recombination (HR), have demonstrated to further sub-classify low BRCA1 NSCLC tumors, improving the predictive model. On the basis of biological knowledge on DNA repair pathway and recent controversial results from clinical validation of potential molecular markers, integrated analysis of multiple DNA repair components could improve predictive information and pave the way to a new approach to customized chemotherapy clinical trials.

BRCA1, RAP80, and AEG-1 mRNA expression in resectable muscle-invasive bladder cancer (MIBC) patients (p) treated with neoadjuvant cisplatin-based chemotherapy.

4579 Background: Cystectomy remains the standard treatment in MIBC and only a minority of p are treated with neoadjuvant chemotherapy, suggesting that predictive markers of chemotherapy outcome are needed. Low BRCA1 mRNA expression is associated with an improvement in survival in bladder cancer p treated with cisplatin-based chemotherapy. However, BRCA1 function can be modulated by other DNA repair genes. RAP80 is required for the accumulation of BRCA1 to sites of DNA breaks, and cells depleted of RAP80 exhibit hypersensitivity to irradiation. AEG-1 can induce BRCA1 expression and cause chemoresistance. 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%. 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 and intermediate levels (p=0.05). 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). No differences in MS were observed according to RAP80 mRNA levels. Conclusions: BRCA1 can be a useful marker to predict the efficacy of neoadjuvant chemotherapy. Cisplatin-based chemotherapy should be recommended in p with low/intermediate BRCA1 expression. Further studies with larger numbers of p are warranted to elucidate the role of AEG-1 in this setting.

Tubuline, BRCA1, ERCC1, Abraxas, RAP80 mRNA expression, p53/p21 immunohistochemistry and clinical outcome in patients with advanced non small-cell lung cancer receiving first-line platinum–gemcitabine chemotherapy

Background The aim of this study was to assess the predictive value of tumor expression of nine genes on clinical outcome in patients with advanced NSCLC receiving platinum–gemcitabine chemotherapy. Methods Quantitative PCR or immunohistochemistry were used to analyze the expression of β-tubuline IIA (TUBB2A), β-tubuline III (TUBB3), BRCA1, ERCC1, Abraxas (ABRX) and RAP80 in mRNA isolated from paraffin-embedded tumor biopsies of 45 NSCLC patients treated as part of a larger observational trial. All patients received first-line platinum–gemcitabine chemotherapy for stage IIIB or IV NSCLC. Results Median progression-free survival (PFS) was 7 months, overall survival (OS) 12 months. A partial treatment response was found in 14 patients (33%). Patients with low ERCC1 or ABRX expression had a significantly better response to chemotherapy ( R = −0.45, p < 0.01 for ERCC1; R = −0.40, p = 0.016 for ABRX). A significant correlation was found between the individual time for PFS and the expression of both ERCC1 ( R = −0.36, p = 0.015) and ABRX ( R = −0.46, p = 0.001). Patients with low ERCC1 expression had a longer OS as compared to patients with high ERCC1 expression (HR = 0.26, log-rank p = 0.02). Conclusions The study confirms tumor expression of ERCC1 as a predictor for clinical outcome in patients with advanced NSCLC receiving platinum-based chemotherapy, and found ABRX expression to be similarly predictive of clinical outcome. Prospective validation is warranted and – if confirmed – non platinum-containing chemotherapy should be explored as the preferred treatment in patients with high ERCC1 or ABRX expression and no activating mutations of EGFR.

BRCA1 and RAP80 mRNA levels and median survival (MS) of advanced gastric cancer patients (p) treated with oxaliplatin plus 5-fluorouracil (FOLFOX).

e14602 Background: In a previous study (Wei et al. BJC 2008), advanced gastric cancer p with high ERCC1 mRNA expr ession had significantly shorter MS when treated with FOLFOX. To further identify predictive biomarkers in gastric cancer, we examined the expression of genes involved in the repair of chromatin damage through the homologous recombination pathway. Low levels of BRCA1 mRNA confer hypersensitivity to radiotherapy and cisplatin. RAP80 is a linker of BRCA1 and can cause DNA repair regardless of BRCA1 status. Methods: We have examined the expr ession of BRCA1 and RAP80 by RT-QPCR in a new series of 200 advanced gastric cancer p treated with FOLFOX. Expression levels by terciles were correlated with MS. In ancillary analyses, we examined the expression of PIAS1 and PIAS4 as a surrogate of BRCA1 SUMOylation and the expression of SIAH2 and ERCC1. Results: Interim data is available on 135 p: median age, 61 years (range, 34- 92); 106 males; prevalent histology, adenocarcinoma (118 p); 49 stage IIIA, 45 stage IIIB, 41 stage IV. Median expression levels: BRCA1, 6.72 (range, 0.58-39.4); RAP80, 2.85 (range, 0.34-12.13). MS was 13.5 months (m) overall, compared to 7.5 m for p with high RAP80 levels and intermediate or high BRCA1 levels. The influence of second-line docetaxel is being examined. Conclusions: While the role of BRCA1 and RAP80 expression levels is not conclusive in the interim analysis, the effect could have been influenced by second-line treatment. The role of BRCA1 SUMOylation could further clarify the potential relevance of BRCA1 as a predictive biomarker. No significant financial relationships to disclose.

Abstract PL2-2: The genomic landscape in lung cancer

Abstract PL2-2: The genomic landscape in lung cancer

A Regulatory Loop Composed of RAP80-HDM2-p53 Provides RAP80-enhanced p53 Degradation by HDM2 in Response to DNA Damage

The ubiquitin interaction motif-containing protein RAP80 plays a key role in DNA damage response signaling. Using genomic and functional analysis, we established that the expression of the RAP80 gene is regulated in a DNA damage-responsive manner by the master regulator p53. This regulation occurs at the transcriptional level through a noncanonical p53 response element in the RAP80 promoter. Although it is inducible by p53, RAP80 is also able to regulate p53 through an association with both p53 and the E3 ubiquitin ligase HDM2, providing HDM2-dependent enhancement of p53 polyubiquitination. Depletion of RAP80 by small interfering RNA stabilizes p53, which, following DNA damage, results in an increased transactivation of several p53 target genes as well as greater apoptosis. Consistent with these observations, exogenous expression of RAP80 selectively inhibits p53-dependent transactivation of target genes in an mdm2-dependent manner in MEF cells. Thus, we identify a new DNA damage-associated role for RAP80. It can function in an autoregulatory loop consisting of RAP80, HDM2, and the p53 master regulatory network, implying an important role for this loop in genome stability and oncogenesis.

Customized Treatment in Non-Small-Cell Lung Cancer Based on EGFR Mutations and BRCA1 mRNA Expression

BackgroundMedian survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC) treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels.Methodology/Principal FindingsWe treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1 levels (hazard ratio, 1.3 [95% CI, 1–1.7]; P = 0.05).Conclusions/SignificanceChemotherapy customized according to BRCA1 expression levels is associated with excellent median and 2-year survival for some subsets of NSCLC patients , and RAP80 could play a crucial modulating effect on this model of customized chemotherapy.Trial RegistrationClinicalTrials.gov NCT00883480

Spanish Lung Adenocarcinoma Trial (SLAT) of customized treatment based on EGFR mutations (mut) and BRCA1 mRNA expression: Ancillary analyses of Abraxas and RAP80 expression

8037 Background: We carried out a study in metastatic NSCLC where treatment was customized according to EGFR mut status and BRCA1 mRNA levels. Loss of BRCA1 function increases sensitivity to cispla...