Abstract 3895: Functional characterization of ERCC1 isoforms in NSCLC

Abstract

Abstract Background: ERCC1 is a DNA repair protein whose expression may influence platinum-based chemotherapy efficiency in solid tumors and is used as a predictive biomarker of cisplatin sensitivity. 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 (8-12 residues) 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 with frozen matched tumor/normal specimens by qRT-PCR. We established a functional DNA repair assay by monitoring the accumulation of cisplatin intrastrand Pt-[GG] adducts over time with a specific antibody RC-18 (courtesy of Juergen Thomale, Germany). To investigate thoroughly isoforms functionality, we have knocked-out ERCC1 gene with Zinc Finger Nucleases technology (Sigma) to establish stable cell lines overexpressing each isoforms. Results: The 2 monoclonal antibodies, clone 8F1 (used for clinical characterization of patients) and 3H11 recognize a conserved region shared by all isoforms of ERCC1 protein, whereas the polyclonal antibody FL297 binds to 7 epitopes along the protein. Therefore, these antibodies 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. Other isoforms were homogenously expressed in normal and tumor tissues. We are currently exploring isoform function by analyzing DNA repair efficiency in the different stable cell lines by monitoring the kinetic of the decrease of cisplatin DNA adducts. Preliminary results suggested that isoform 201 is unable to remove cisplatin adducts. We compared DNA repair efficiency and cisplatin sensitivity among 20 lung cancer cell lines. Interestingly, we observed a significant negative-correlation between DNA repair efficiency and cisplatin sensitivity. Conclusions: Our data (a) demonstrate that DNA adduct repair efficiency is directly correlated with chemosensitivity, (b) suggest possible alternative roles for ERCC1 isoforms. 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 NSCLC tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3895. doi:10.1158/1538-7445.AM2011-3895