VHL-dependent degradation of RNA polymerase II large subunit enhances sensitivity to the novel anticancer agent ecteinascidin 743 Free

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Ecteinascidin 743 (Et-743) is a novel anticancer agent with a unique spectrum of antitumor activity. Et-743 can be given safely to patients and exhibits activity in phase II/III clinical trials in a variety of tumors. Notably, patients with heavily pretreated renal carcinoma are among those that have exhibited objective clinical responses to single-agent Et-743 therapy. Et-743 alkylates DNA in the minor groove forming Et-743-DNA adducts that are targeted for repair by transcription-coupled nucleotide excision repair (TC-NER). The initial cellular signal for TC-NER is the stalling of an actively transcribing RNA polymerase II (RNAPII) large subunit at DNA helix-distorting lesions. Previously, we showed Et-743 exposure results in ubiquitylation of the RNAPII large subunit and degradation by the 26S proteasome. Interestingly, the von Hippel-Lindau (VHL) protein, a critical tumor suppressor gene deleted in 50-80% of sporadic clear cell renal carcinoma cases, was recently established as the ubiquitin-E3-ligase for the RNAPII large subunit. Our ongoing work indicates sensitivity to Et-743 tightly correlates with the ability of cells to degrade the RNAPII large subunit. Accordingly, the goals of the studies presented herein were to study Et-743-induced RNAPII large subunit degradation, drug sensitivity, and apoptosis in isogenic cell lines with a deficiency in the VHL protein. We exposed two isogenic cell lines, 786-0 renal carcinoma cells and stably transfected 786-0 cells expressing VHL protein, to an Et-743 dose response and assessed cytotoxicity by MTT proliferation assay. Both of these cell lines were sensitive to Et-743 at doses less than 1 nM. 786-0 cells expressing functional VHL protein were consistently 2-3 fold more sensitive to Et-743, suggesting that Et-743-mediated cytotoxicity is enhanced by a VHL-dependent mechanism. Additionally, Et-743 induced rapid degradation of the RNAPII large subunit in 786-0 cells expressing functional VHL protein. In contrast, no degradation of RNAPII large subunit was observed in 786-0 cells deficient in VHL protein. Finally, in VHL-deficient 786-0 cells, co-treatment with non-toxic doses of the 26S proteasome inhibitor MG132 had no effect on apoptosis quantified by DNA filter elution. Strikingly, 786-0 cells expressing functional VHL exhibited 30% less apoptosis when MG132 was present during Et-743 exposure as compared to Et-743 alone, indicating prevention of RNAPII degradation protects against apoptotic cell death by a VHL-dependent mechanism. Collectively, these results suggest degradation of the RNAPII large subunit induced by Et-743 exposure is an important molecular event in the cytotoxic response of renal tumors to this novel anticancer agent. In addition, renal tumors that do not harbor VHL mutations and thus have an intact RNAPII large subunit degradation pathway may be more suited to clinical Et-743 therapy.