Abstract 834: Inhibition of Gli1 results in altered c-jun activation, inhibition of cisplatin-induced up-regulation of ERCC1, XPD, and XRCC1, and inhibition of platinum-DNA adduct repair

Abstract

Abstract The transcription of ERCC1 and other nucleotide excision repair genes is strongly influenced by c-jun. C-jun is transcriptionally regulated by Gli proteins of the Hedgehog pathway. We therefore studied the possible relationships between Gli1, c-jun, and the upregulation of ERCC1, XPD, and XRCC1 in cisplatin-resistant human ovarian cancer cells. We studied the paired human ovarian cancer cell lines A2780 and A2780-CP70. We used a shRNA construct that specifically degrades Gli1 message; which has been previously published by one of the co-authors (Shevde-Samant et al., 2009). Genes we assessed for mRNA and/or protein levels included: c-jun, ERCC1, XPD, XRCC1, Gli1, Gli2, SHH, IHH, GAPDH, and β-tubulin. Platinum-DNA adduct repair was assessed by atomic absorbance spectrometry with Zeeman background correction. Use of the anti-Gli1 shRNA in cisplatin-resistant cells, resulted in a block of the cells’ ability to up- regulate genes in response to cisplatin treatment, including: c-jun, ERCC1, XPD, and XRCC1. This block in upregulation of c-jun was concurrent with a change in the phosphorylation pattern of the c-jun protein, shifting that pattern from a Ser63/73 dominant pattern, to a Thr91/93 dominant pattern. A2780-CP70 cells were treated at their cisplatin IC50, and DNA repair was assessed after pretreatment with anti-Gli1 shRNA or scrambled shRNA control. Control cells repaired 78% of platinum-DNA adducts at 12 hours; compared to 33% repair in cells pretreated with anti-Gli1 shRNA; a 2.4 fold difference. Pretreatment of A2780-CP70 cells with anti-Gli1 shRNA resulted in supra-additive cell killing with cisplatin; shifting the cisplatin IC50 from 30 µM to 5 µM. Pretreatment of these cells with cyclopamine, did not shift the cisplatin IC50. We conclude that the transcriptional protein Gli1 is important in the upregulation of these three DNA repair genes in human ovarian cancer cells. And that Gli1 thereby strongly influences platinum-DNA adduct repair, and cellular sensitivity to cisplatin. This Gli1 role has c-jun as an intermediate in the pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 834. doi:1538-7445.AM2012-834