Excision Repair Cross-complementing 1 Protein Research Articles

Chemoresistance in gastric cancer is attributed to the overexpression of excision repair cross-complementing 1 (ERCC1) caused by microRNA-122 dysregulation.

MicroRNAs are deemed as key regulators of gene expression. In particular, the elevated expression of excision repair cross-complementing 1 (ERCC1) significantly reduced the effectiveness of gastric cancer treatment by cisplatin (CDDP)-based therapies. In this paper, qRT-PCR and western blot were adopted to measure miR-122 and ERCC1 messenger RNA (mRNA) expression in all samples. Luciferase assay was carried out to verify the role of ERCC1 as a target of miR-122. The CCK-8 assay was carried out to study the effect of ERCC1 and miR-122 on cell survival and apoptosis. The results demonstrated that miR-122 expression was reduced in cisplatin-resistant gastric cancer. Using bioinformatic analysis, miR-122 was shown to target the 3'-UTR of human ERCC1. A dual-luciferase assay demonstrated that miR-122 downregulated ERCC1 expression, while the mutations in ERCC1 3'-UTR abolished its interaction with miR-122. Transfection of miR-122 mimics decreased the levels of ERCC1 mRNA and protein expression, while the transfection of miR-122 inhibitors increased the levels of both ERCC1 mRNA and protein expression. Furthermore, we found that overexpressed miR-122 promoted the proliferation of MKN74 cells and reduced their apoptotic by targeting ERCC1. In addition, the levels of miR-122 and ERCC1 were negatively correlated in gastric cancer samples. In summary, the reduced miR-122 expression may play an essential role in the induction of cisplatin-resistance by increasing ERCC1 expression.

Polymorphism in ERCC1 confers susceptibility of coronary artery disease and severity of coronary artery atherosclerosis in a Chinese Han population

Excision repair cross-complementing 1 (ERCC1) gene encodes ERCC1 protein, which is mainly responsible for the repair of DNA damage in different diseases including coronary artery atherosclerosis by acting as a rate-limiting element in nucleotide excision repair (NER). Using a three-stage case-control study with 3037 coronary artery disease (CAD) patients and 3002 controls, we investigated associations of three single nucleotide polymorphisms (SNPs) with CAD risk and severity of coronary artery atherosclerosis in Chinese Han population. In the discovery set, the variant allele T of rs11615 was significantly associated with higher CAD risk (adjusted OR = 1.27, P = 0.006) and severity of coronary artery atherosclerosis (adjusted OR = 1.54, P = 0.003). These associations were more remarkable in the merged set (adjusted OR = 1.23, P = 8 × 10−6 for CAD risk; adjusted OR = 1.36, P = 4.3 × 10−5 for severity of coronary artery atherosclerosis). And the expression level of ERCC1 was significantly higher in CAD cases than controls. Multiplicative interactions among SNP rs11615, alcohol drinking, history of T2DM, and history of hyperlipidemia could increase 5.06-fold risk of CAD (P = 1.59 × 10−9). No significant association of rs2298881 and rs3212986 with CAD risk was identified. Taken together, SNP rs11615 in ERCC1 gene might confer susceptibility to CAD and severity of coronary atherosclerosis in a Chinese Han population.

Prognostic role of ERCC1 protein expression in upper tract urothelial carcinoma following radical nephroureterectomy with curative intent.

Excision repair cross-complementing 1 (ERCC1) has been associated with outcomes of urothelial carcinoma of the bladder, but was not yet studied in upper tract urothelial carcinoma (UTUC). The aim of this study was to assess the prognostic role of ERCC1 expression in a large international cohort of UTUC patients. Immunohistochemical ERCC1 expression was evaluated in 716 UTUC patients who underwent radical nephroureterectomy with curative intent. ERCC1 was considered positive when the H-score was >1.0. Associations with overall survival and cancer-specific survival were assessed using univariable and multivariable Cox models. ERCC1 was expressed in 303 tumors (42.3%) and linked with the presence of tumor necrosis (16.2 vs. 10.4%, p=0.023), but not with any other clinical or pathological variable. ERCC1 status did not predict cancer-specific survival and overall survival on both univariable (p=0.70 and 0.32, respectively) and multivariable analyses (p=0.48 and 0.33, respectively). ERCC1 is expressed in a significant proportion of UTUC and is linked with tumor necrosis, but its expression appears not to be associated with prognosis following radical nephroureterectomy.

ERCC1 siRNA ameliorates drug resistance to cisplatin in gastric carcinoma cell lines

The present study examined the effects of cisplatin (DDP) on gastric carcinoma cells by inhibiting the expression of excision repair cross-complementing 1 (ERCC1) using RNA interference (RNAi). mRNA and protein expression of ERCC1 were measured in various gastric carcinoma cell lines using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. Cells were treated with different concentrations of DDP and the cell viability was measured using an MTT assay. The correlation between the expression of the ERCC1 gene and the resistance to DDP in the cells was determined. The specific ERCC1 small interfering RNA (siRNA) was synthesized and then transfected into SGC-7901/DDP cells. Alterations in intracellular ERCC1 mRNA expression and protein levels were detected using RT-PCR and western blot analysis, the number of apoptotic cells were measured using flow-cytometry and the cell viability was measured using an MTT assay. The gene expression of ERCC1 correlated with the resistance to DDP of the cells. mRNA expression of ERCC1 was significantly reduced 24 h following transfection of ERCC1 siRNA compared with the mock control group. In addition, the number of apoptotic cells was increased and cell viability was significantly decreased in the ERCC1 siRNA-transfected group compared with the mock control group, suggesting that the sensitivity of SGC-7901/DDP cells to DDP had significantly increased. Cells transfected with siRNA1, siRNA2 and siRNA3 were significantly more sensitive to DPP (161, 381 and 249%, respectively) compared with the mock controls (P<0.05). The results of the present study showed that drug resistance to DDP in gastric carcinoma is correlated with increased expression of ERCC1; therefore, inhibition of ERCC1 by siRNA may ameliorate resistance to DDP in gastric carcinoma.

Association between epidermal growth factor receptor gene copy number and ERCC1, BRCA1 protein expression in Chinese patients with non-small cell lung cancer

Mutation in epidermal growth factor receptor (EGFR) gene may predict response to chemotherapy in non-small cell lung cancer (NSCLC). However, the correlation between EGFR gene copy status and protein levels of drug-resistant genes, such as excision repair cross-complementing 1 (ERCC1) and breast cancer 1 (BRCA1), remains unclear. We retrospectively analyzed formalin-fixed, paraffin-embedded tumor tissues from 109 Chinese patients with NSCLC. EGFR gene copy number was evaluated by fluorescence in situ hybridization (FISH), and protein levels of platinum-resistance-associated genes, including ERCC1 and BRCA1, were determined by immunohistochemical staining. High EGFR gene copy (EGFR FISH-positive) was found in 21.1% of the patients (amplification in 7.3% and high polysomy in 13.8%, respectively). Immunohistochemical analysis revealed that ERCC1 protein expression was not associated with clinicopathological factors, whereas a significantly higher BRCA1 positive rate was found in poorly differentiated tumors (P=0.02). Further association studies demonstrated that EGFR gene copy number status was not correlated with protein levels of ERCC1 or BRCA1; however, expression of ERCC1 was significantly associated with that of BRCA1 in this set of Chinese patients with NSCLC (P<0.001, r=0.484). Our study demonstrated that EGFR gene copy number status was not correlated with ERCC1 or BRCA1 protein expression, but ERCC1 protein levels were significantly correlated to BRCA1 protein expression levels in tumor tissues from Chinese patients with NSCLC.

Clinical research of individualized therapy in advanced non-small cell lung cancer guiding by detection of ERCC1 protein

Background and purpose: Excision repair cross-complementing 1(ERCC1) has been associated with cisplatin resistance.The study aimed to explore the role and clinical significance of detection of ERCC1 protein in individualized therapy of advanced non-small cell lung cancer(NSCLC) patients.Methods: From Aug.2006 to Jul.2009,222 stage ⅢB/Ⅳ NSCLC patients were enrolled.The expressions of ERCC1 protein in advanced stage NSCLC tissues were qualitatively detected by immunohistochemical methods.Patients were randomly assigned in a 2∶1 ratio to either the individualized treatment group or the standard treatment group before ERCC1 assessment.Patients in the control arm received gemcitabine plus cisplatin or vinorelbine plus cisplatin.In the genotypic arm,patients with low ERCC1 levels received gemcitabine plus cisplatin or vinorelbine plus cisplatin,and those with high levels received gemcitabine plus vinorelbine.Main outcome measures include response rate,overall survival and time to progression.Differences between the groups were statistically analyzed by chi-square test.Survival differences were analyzed by temporal inspection and Kaplan-Meier survival curves.Results: Follow-up data was up to Sep.30,2012.Objective response was obtained by 20 patients(26.6%) in the standard treatment group and 40 patients(27.2%) in the individualized treatment group(P=0.931).One year survival rate was 40.0% in the standard treatment group and 48.3% in the genotypic arm(P=0.24).The median survival time was 10.2 months(95%CI was 8.67 months to 11.73 months) in the standard treatment group and 13.3 months(95%CI was 12.46 months to 14.14 months) in the individualized treatment group(P=0.041).The time to progression was 4.8 months(95%CI was 4.12 months to 5.48 months) in the standard treatment group and 4.7 months(95%CI was 3.88 months to 5.52 months) in the individualized treatment group(P=0.395).Conclusion: The median survival time has extended in the individualized treatment group.But individualized therapy in advanced NSCLC guiding by detection of ERCC1 protein has not reflected advantage in response rate,overall survival and time to progression.Additional studies are warranted to optimize detections of biomarkers in guiding rational clinical chemotherapy regimens.

Impact of ERCC1 expression on treatment outcome in small-cell lung cancer patients treated with platinum-based chemotherapy

IntroductionThe excision repair cross-complementing 1 (ERCC1) protein is an extensively investigated molecular marker because it may decrease sensitivity to platinum-based chemotherapy. Low ERCC1 expression has already been correlated with better treatment efficacy in non-small-cell lung cancer patients treated with platinum-based chemotherapy. However, the data on a prognostic and/or predictive value of ERCC1 in small-cell lung cancer (SCLC) are still very limited. MethodsThis retrospective pilot study evaluated the impact of ERCC1 expression levels on response to first-line platinum-based chemotherapy with or without radiotherapy and survival outcomes of 77 SCLC patients. ERCC1 protein expression was determined immunohistochemically in primary tumour tissue. ResultsERCC1 protein expression was positive in 40/77 (51.9%) of our patients. No significant association was found between ERCC1 protein expression and response rate to first-line platinum-based chemotherapy, progression-free survival (PFS), or overall survival (OS), either in the overall population or in patients stratified by disease stage. ConclusionsIn our limited group of 77 SCLC patients, ERCC1 protein expression was not found to correlate with either response rate to platinum-based chemotherapy or survival outcomes. Multi-centric prospective trials using a validated method of ERCC1 determination are mandatory in order to obtain a definitive answer on the predictive value of ERCC1 in SCLC.

International tailored chemotherapy adjuvant trial: Itaca trial.

TPS7109 Background: This is an ongoing phase III multicenter randomized trial comparing adjuvant pharmacogenomic-driven chemotherapy, based on thymidilate synthase (TS) and excision-repair cross-complementing -1 (ERCC1) gene expression versus standard adjuvant chemotherapy in completely resected stage II-IIIA non-small cell lung cancer (EudraCT #: 2008-001764-36). Methods: In all registered patients, before randomization, expression of ERCC1 and TS is assessed by qRT-PCR on paraffin-embedded tumor specimens in a central laboratory. Randomization is stratified by stage and smoking status. Trial was emended on Feb, 2011 with the 7th staging system. Primary end point is overall survival; secondary end points include recurrence-free survival, therapeutic compliance, toxicity profile and comparative evaluation of ERCC1 and TS mRNA versus protein. It is assumed that the 5-year survival in the control arm is 45% and the hazard reduction associated to the experimental treatment is 30%. With a power of 90% to detect the estimated effect with log-rank test, a significant level of 5% (2 tails), 336 events have to be observed; the expected total number of patients is 700. The final statistical analysis will group together all control arms and all tailored chemotherapies groups. Efficacy analysis will be done on an intent-to-treat basis. Cox proportional hazard model will be used for estimating hazard ratios after adjusting for relevant variables. Within 45 days post-surgery, patients in each genetic profile are randomized to receive either a standard chemotherapy selected by the investigator (cisplatin/vinorelbine, cisplatin/docetaxel or cisplatin/gemcitabine) or an experimental treatment (tailored arms) selected as follows: 1) high ERCC1 and high TS 4 cycles of single agent paclitaxel 2) high ERCC1 and low TS 4 cycles of single agent pemetrexed 3) low ERCC1 and high TS 4 cycles of cisplatin/gemcitabine 4) low ERCC1 and low TS) 4 cycles of cisplatin/pemetrexed. All chemotherapy regimens are administered for a total of 4 cycles on a 3-weekly basis. Currently, 312 patients have been randomized from 26 institutions mainly located in Italy and Germany (average enrolment: 13 patients/month).

Genotoxic stress and activation of novel DNA repair enzymes in human endothelial cells and in the retinas and kidneys of streptozotocin diabetic rats

Mammalian excision repair cross-complementing 1 (ERCC1) and ERCC4 (a.k.a xeroderma pigmentosum complementation group F) are nucleotide excision repair enzymes, which excise the 5' end of damaged DNA. ERCC1 and ERCC4 have an interactive relationship with poly (adenosine diphosphate ribose) polymerase (PARP). We studied the role of ERCC1 and ERCC4 in glucose-induced extracellular matrix protein production in human endothelial cells and in the retinas and kidneys of streptozotocin diabetic rats. Human umbilical vein endothelial cells were grown with low (5 mM) and high glucose (25 mM). The cells were subjected to ERCC1 and ERCC4 small interfering RNA transfections, PARP blocker (3-aminobenzamide, ABA) and p300 blocker (curcumin). Retinas and kidneys from 1-month-old streptozotocin diabetic rats with or without treatment with curcumin and ABA were examined. Cells and tissues were studied for oxidative stress markers, fibronectin, ERCC1 and ERCC4, PARP and p300 mRNA. Western blot of nuclear proteins was performed. ERCC1 and ERCC4 messenger RNA and protein levels were higher in high glucose than in low glucose, along with increasing oxidative stress and augmented p300 and fibronectin production. ABA, curcumin, ERCC1 and ERCC4 silencing reduced such upregulations and oxidative stress. Similar changes were seen in the kidneys and retinas of diabetic rats. ABA and curcumin treatment significantly reduced such changes. These data indicate that glucose-induced ERCC1 and ERCC4 upregulation leads to increased fibronectin production via a p300-dependent pathway in umbilical endothelial cells, as well as in the retina and in the kidneys of streptozotocin diabetic rats. ERCC1 and ERCC4 may play important roles in the development of diabetic retinopathy and nephropathy.

International tailored chemotherapy adjuvant trial: ITACA trial.

e17514 Background: This is an ongoing phase III multicenter randomized trial comparing adjuvant pharmacogenomic-driven chemotherapy, based on thymidilate synthase (TS) and excision-repair cross-complementing -1 (ERCC1) gene expression versus standard adjuvant chemotherapy in completely resected stage II-IIIA non-small cell lung cancer (EudraCT #: 2008-001764-36). In all registered patients, before randomization, expression of ERCC1 and TS is assessed by qRT-PCR on paraffin-embedded tumor specimens in a central laboratory. Methods: Randomization is stratified by stage and smoking status. Primary end point is overall survival; secondary end points include recurrence-free survival, therapeutic compliance, toxicity profile and comparative evaluation of ERCC1 and TS mRNA versus protein. It is assumed that the 5-year survival in the control arm is 45% and the hazard reduction associated to the experimental treatment is 30%. With a power of 90% to detect the estimated effect with log-rank test, a significant level of 5% (2 tails), 336 events have to be observed; the expected total number of patients is 700. The final statistical analysis will group together all control arms and all tailored chemotherapies groups. Efficacy analysis will be done on an intent-to-treat basis. Cox proportional hazard model will be used for estimating hazard ratios after adjusting for relevant variables. Results: Within 45 days post-surgery, patients in each genetic profile are randomized to receive either a standard chemotherapy selected by the investigator (cisplatin/vinorelbine, cisplatin/docetaxel or cisplatin/gemcitabine) or an experimental treatment (tailored arms) selected as follows: 1) high ERCC1 and high TS 4 cycles of single agent paclitaxel 2) high ERCC1 and low TS 4 cycles of single agent pemetrexed 3) low ERCC1 and high TS 4 cycles of cisplatin/gemcitabine 4) low ERCC1 and low TS) 4 cycles of cisplatin/pemetrexed. All chemotherapy regimens are administered for a total of 4 cycles on a 3-weekly basis. Conclusions: Currently 180 patients have been enrolled from 24 institutions mainly located in Italy and Germany (average enrolment: 20 patients/month).

Correlation between expression of excision repair cross-complementing 1 and cisplatin sensitivity in non-small cell lung cancer

To investigate the correlation between excision repair cross-complementing 1 (ERCC1) expression and cisplatin sensitivity in non-small cell lung cancer (NSCLC). A total of 168 NSCLC patients were selected from our hospital from January 2005 to December 2007. The expression of ERCC1 protein was analyzed by immunohistochemistry and ERCC1 mRNA tested by RT-PCR. Analyses were performed to determine the correlation between expression of ERCC1 and chemotherapeutic sensitivity in NSCLC. A positive expression of ERCC1 protein was found in 99 (58.93%) patients. The expression of ERCC1 had no correlation with gender, age, stage and pathological types (P > 0.05). Among all patients, 133 were followed up for about 3-5 years and 91 cases belonged to the responding group. Three cases with stage Ia-Ib underwent only operation without chemotherapy. And 76 (58.46%) patients were positive for ERCC1 expression in 130 cases. In the responding group, 38 (43.18%) cases had a positive expression of ERCC1 and 50 (56.82%)cases a negative expression of ERCC1. In the non-responding group, 37 (88.10%)cases had a positive expression of ERCC1 and 5 (11.90%) cases a negative expression of ERCC1. The expression of ERCC1 decreased in the responding group versus the non-responding group (χ(2) = 23.50, P < 0.01). The expressions of ERCC1 mRNA were (0.624 ± 0.275) and (2.758 ± 0.771) in the responding and non-responding groups respectively (t = 11.54, P = 0.013). An elevated expression of ERCC1 is an important factor for cisplatin insensitivity in NSCLC. It may provide an useful reference for designing individualized chemotherapeutic regimens for NSCLC patients.

Excision repair cross-complementing 1 (ERCC1) and survival in advanced bladder cancer: Confirmatory results using immunohistochemistry

5025 Background: DNA damaging agents are the backbone of combination chemotherapy regimens for the treatment of advanced bladder cancer. We hypothesized that levels of DNA repair genes such as ERCC1 could predict survival in patients receiving platinum based therapy. Our previous work (Bellmunt J et al, Ann Oncol. 2007) showed that survival was significantly higher in advanced bladder cancer patients with low gene expression levels of ERCC1 measured by RT-qPCR (25.4 versus 15.4 months; p = 0.03). We aimed to confirm these findings using immunohistochemistry (IHC) in an independent cohort of advanced bladder cancer patients treated with cisplatin-based chemotherapy at our institution. Methods: Formalin-fixed paraffin-embedded tumor tissue was available from 51 patients. IHC stains for ERCC1 protein levels were scored as percentage and intensity of positive cells. ERCC1 staining was considered positive if detected in ≥1% tumor cells. The Kaplan-Meier method was used to calculate survival and the Cox proportional hazards model was used to examine the prognostic value of protein expression levels. Results: ERCC1 staining was negative in 60% of patients, weakly positive (1–10% positive cells) in 21% and strongly positive (&gt;10% positive cells) in 19%. Median overall survival for all patients was 14.4m (95% CI 6.7–16.1m). Median disease-specific survival was significantly higher in patients negative for ERCC1 by IHC (12.6m versus 8.6m; p = 0.032). Conclusions: Our results using the more simple IHC technique confirm the prognostic value of ERCC1 expression in advanced bladder cancer. Patients with high expression of ERCC1 by IHC have a worse disease-specific survival than patients with no expression. A correlative study of IHC and RT-qPCR in both sets of samples is ongoing. No significant financial relationships to disclose.

Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant known to cause DNA damage, which may be repaired through nucleotide excision repair (NER). However, little is known about dynamic changes in levels of DNA damage and their correlations with levels of NER proteins in cells exposed to BaP. In a series of experiments using the human bronchial epithelia cells (16HBE) exposed to different concentrations of BaP for different times, we measured dynamic changes in levels of DNA damage and expression of NER subunit xeroderma pigmentosum (XP) groups A, C, F, G (XPA, XPC, XPF, XPG) and excision repair cross-complementing 1 (ERCC1), and analyzed their possible correlations. We found that in vitro exposure to BaP reduced cell viability in a dose-dependent manner ranging from 2 to 64 μM and increased DNA damage in a dose- and time-dependent manner. Our results showed that levels of these NER proteins significantly increased and peaked at 12th or 24th, 8th or 12th and 4th or 8th hours in cells exposed to 2, 8 and 16 μM BaP, respectively. ERCC1 expression increased by 2.4-, 4.2- and 19.3-fold for exposure to 2, 8 and 16 μM BaP, respectively, compared with control group. Moreover, levels of ERCC1 in cells exposed 16 μM BaP significantly higher than those in 2 and 8 μM BaP from 2nd to 48th hours. In addition, there was a significant positive correlation between Olive tail moments and relative ratios of ERCC1 in cells exposed to BaP. Our results suggested ERCC1 may be an important limiting factor for NER, but the mechanisms underlying this observation needs further investigation.

Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin

The level of excision repair cross-complementing 1 (ERCC1) gene expression, which is important in the repair of the cisplatin–DNA adducts, is reported to be related to the level of cisplatin resistance in tumor cells. Therefore, ERCC1 is an attractive target to confer increased cellular sensitivity to cisplatin-based chemotherapy. We designed, synthesized, and utilized small interfering RNAs (siRNAs) that were selective for ERCC1 and investigated their effectiveness in altering the repair capacity of the cells to cisplatin–DNA damage as well as the resistance of the cells to cisplatin. Twenty-four and 48 h after transfecting ERCC1 siRNA1 and siRNA2 targeting the two different regions of the ERCC1 transcript, both the ERCC1 mRNA and protein expression were significantly inhibited, whereas the mock or control siRNA had no effect. The suppression of ERCC1 expression in the HeLa S3 cells led to a decrease in the repair activity of cisplatin-induced DNA damage along with a decrease in the cell viability against platinum-based drugs, such as cisplatin, carboplatin, and oxaliplatin. A similar increased sensitivity to cisplatin and decreased repair activity were also observed for siRNA-mediated ERCC1 silencing in the MCF-7 and HCT116 cells. This study is the first to demonstrate the feasibility of utilizing ERCC1 siRNAs to specifically reduce the ERCC1 expression level in human cancer cells and provides direct evidence for the potential use of ERCC1 siRNA as a chemotherapy-sensitizing agent.