Abstract CN4-3: Factors that influence the regulation of ERCC1 in drug-resistant cells

Abstract

Abstract ERCC1 is an excellent marker for nucleotide excision (NER) repair activity. ERCC1 expression in tumor tissues has been closely correlated with clinical resistance to platinum coordination complexes in lung cancer, ovarian cancer, gastric cancer, and other malignancies. We have previously demonstrated that up-regulation of ERCC1 is coordinated with up-regulation of other genes in the NER excision repair protein complex, such as XPA, XPD, XPB, etc (Reed and colleagues; Mut Res 1993, J Clin Invest 1994, Cancer Res 1995, Biochem Pharm 2000). The positive transcriptional regulator for ERCC1 is activator protein 1, or AP1, a heterodimer of c-jun and c-fos (Reed, JBC 1998). The negative transcriptional regulator for ERCC1 is the myeloid zinc finger gene MZF1 (Reed, Biochem Pharm 2006). Cancer stem cells are presumed to be highly resistant to chemotherapy. Activation of the Hedgehog pathway has been reported as necessary, for the cancer stem cell state. A key gene in the Hedgehog pathway is Gli1. Resistance to platinum compounds is due in part, to up-regulated nucleotide excision repair, NER. Upon treatment with cisplatin, cisplatin-resistant A2780-CP70 cells normally up-regulate c-jun and c-fos, which then up-regulate ERCC1 and other NER genes. More specifically, the c-jun Ser63/73 cascade is up-regulated, with up-regulation of activator protein 1 (Reed, JBC 1998). We therefore studied the relationships between Gli1 expression, c-jun expression, ERCC1 expression, and cisplatin resistance in the human ovarian cancer cell lines A2780 and A2780-CP70. A2780-CP70 cells are 10–15 fold more resistant to cisplatin than A2780 cells. We used two different probes to modulate the Hedgehog pathway: a) cyclopamine as a pharmacologic inhibitor of the Smoothened; and, b) an anti-Gli1 shRNA to specifically inhibit Gli1. At baseline, cisplatin-resistant cells, expressed >10-fold more Gli1 protein than cisplatin-sensitive cells. In cisplatin-resistant cells, Gli1 protein was down-regulated in response to cyclopamine, and to anti-Gli1 shRNA. When A2780-CP70 cisplatin-resistant cells are pretreated with cyclopamine, or anti-Gli1 shRNA, the following events occur. The c-jun Thr91/93 cascade is upregulated, which is associated with pro-apoptotic processes. The c-jun Ser63/73 cascade is suppressed. This cascade is associated with pro-growth processes. If cisplatin is given after cyclopamine or anti-Gli1 pretreatment; Gli1 protein is down-regulated; the c-jun Thr91/93 cascade is upregulated; the c-jun Ser63/73 cascade is suppressed; and cells do not up-regulate ERCC1, XPD, or XRCC1. Thus, the normal cisplatin-induced up-regulation of these three genes involved in NER (ERCC1, XPD) and in base excision repair (XRCC1), is suppressed by suppression of the Hedgehog pathway. We conclude that Gli1 and/or other genes involved in the Hedgehog pathway (which is critical for cancer stem cells), may play key roles in the up-regulation of DNA repair genes, specifically in response to DNA damaging agents such as cisplatin. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN4-3