Abstract 3120: Functional characterization of ERCC1 isoforms in NSCLC

Background: ERCC1 is a DNA repair protein whose expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. Five alternative transcripts of the ERCC1 gene have been described or hypothesized to exist (isoforms 201 to 205). Nevertheless, little is known about the function and relative expression of these isoforms. We explored 3 main questions: a- Which ERCC1 isoforms are detected by clinically validated antibodies? b- Which isoforms are expressed in lung cancer cell lines and patient samples? c- Which isoforms are functional for removal of cisplatin DNA-adducts and potentially relevant as predictors of cisplatin resistance? Methods: To determine the specificity of anti-ERCC1 antibodies, a screening of isoform specific peptide sequences was achieved using a peptide array (Epitope Mapping CelluSpots™). The expression of ERCC1 isoforms was evaluated in 20 lung cancer cell lines and in 123 NSCLC patients by qRT-PCR. We established a functional DNA repair assay by monitoring the accumulation of cisplatin adducts over time with a specific antibody RC-18 (courtesy of Juergen Thomale, Germany). To investigate isoforms functionality, we created stable ERCC1 attenuated A549 cells with Zinc Finger Nucleases technology (Sigma). Using lentiviral constructs we rescued ERCC1 isoforms expression. Results: The 2 monoclonal antibodies, clone 8F1 and 3H11 recognize a conserved region shared by all isoforms of ERCC1 protein, and so, are unable to distinguish the different isoforms. The 5 isoforms were all detected at mRNA level, both in cancer cell lines and in patient tissues. Interestingly, a significant increase in isoform 201 expression was observed in all tumor tissues compared to normal counterparts. We obtained 7 A549 clones harboring a 85-95% decrease in ERCC1 expression. In these cells we observed a 7-fold increase in IC50 of cisplatin accompanied by a significant decrease in the rate of nucleotide excision repair (NER). We are currently exploring isoform function by analyzing DNA repair efficiency in the different stable cell lines. Conclusions: Alternative roles for ERCC1 beyond NER are currently emerging. Our 7 A549 clones, with stable and strong ERCC1 attenuation, provide a very promising tool to analyse all functions of ERCC1. Since most antibodies used to test ERCC1 positivity in patients do not distinguish the different isoforms, it is urgent to characterize their function further, particularly the role of isoform 201 overexpression in