Mutant Rad50-mediated cisplatin chemosensitization for the treatment of human head and neck cancer

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 3314 In 2006, there were 40,000 new cases of head and neck squamous cell carcinoma (HNSCC) and 11,000 deaths in the US. Survival from HNSCC has not improved over many decades. Cisplatin is the mainstay of adjuvant treatment. Escalating cisplatin dose to overcome drug resistance leads to severe systemic toxicity. Repair of DNA double-strand breaks (DSBs) by the Mre11, Rad50, and Nbs1 (MRN) complex contributes to resistance. With the goal of developing molecular therapies to enhance the cytotoxicity of cisplatin and, thus, improve therapeutic outcome in the treatment of HNSCC, we have created a novel, recombinant adenoviral vector containing a mutant Rad50 gene (Ad-Rad50). Ad-Rad50 interrupts the function of the MRN DNA repair complex, thereby sensitizing HNSCC cells to cisplatin-induced damage. Cell proliferation assay showed that Ad-Rad50, with cisplatin, produced significantly slower cancer cell growth than any other treatment group. Neutral comet assay demonstrated a higher number of DNA DSBs in the Ad-Rad50 with cisplatin group, versus all other groups (P<0.01). Ad-Rad50, through impairment of MRN-mediated DNA repair, permits lethal cell damage to accumulate in cisplatin-treated cells. It has been demonstrated that 90% of HNSCC tumor cells achieve immortality through telomerase activation. MRN is required for telomerase function. Telomere shortening beyond a critical threshold causes cellular senescence. PNA-FISH telomere length analysis showed significantly shorter telomeres in the Ad-Rad50 with cisplatin group, relative to all other groups (P<0.01), contributing to enhanced cisplatin cytotoxicity. Cisplatin induces dose-dependent G2/M cell cycle arrest. Flow cytometry revealed that, at tested doses, cisplatin did not increase the proportion of cells in G2/M arrest. The addition of Ad-Rad50 produced G2/M arrest in the majority of HNSCC cells (P<0.005). Abrogation of MRN function by Ad-Rad50 may allow cisplatin-induced damage to accumulate, producing arrest. An animal model of human HNSCC was used to further investigate the feasibility of this novel treatment strategy. Mice, treated with Ad-Rad50 and cisplatin in combination, had significantly smaller tumors than untreated controls, cisplatin alone, or Rad50 alone groups. Preliminary data suggests that low-dose cisplatin, with Ad-Rad50, results in smaller tumors than a higher cisplatin dose, used as the standard in clinical applications. Our study demonstrates that Ad-Rad50 enhances the cytotoxicity of cisplatin in HNSCC by increasing DNA DSBs, telomere shortening, and enhancing G2/M cell cycle arrest. This translates to tumor volume reduction in vivo. The implications are considerable as sensitizing HNSCC to cisplatin with Ad-Rad50 may allow for the use of lower cisplatin doses, clinically. This work is supported by FAMRI.