Abstract 5401: The mTOR inhibitor everolimus cooperates with the Toll-like receptor 9 (TLR9) agonist IMO in renal cell carcinoma by interfering with both tumor cells and microenvironment
Authors: Tortora Giampaolo , Rosa Roberta , Gelardi Teresa , Damiano Vincenzo , Marciano Roberta , Nappi Lucia , Formisano Luigi , Bianco Roberto 
Published: February 2011

Abstract
Background. The mammalian target of rapamycin (mTOR) kinase plays a key role in renal cell carcinoma by integrating proliferation, survival and angiogenic pathways. Therefore, mTOR targeting by rapamycin analogues including everolimus has become a successful approach for renal cell carcinoma therapy. We previously demonstrated that the novel Toll-like receptor 9 (TLR9) agonist IMO, currently under clinical development, exhibits direct antitumor and antiangiogenic activity and cooperates with both Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor (VEGF) inhibitors.
Methods. Since successful therapeutic interventions in renal cell carcinoma are currently based on a multitargeting approach, in this study we tested the combination of everolimus plus IMO on models of human renal cell carcinoma with different VHL gene status, HIF1α levels and VEGF production capability. We evaluated the activity of these agents on in vitro cancer cell growth, survival, and signal transduction. Moreover, we investigated their effects on in vivo growth and signal transduction of renal cell tumors xenografted in nude mice. Finally, we studied their direct antiangiogenic effects by using the human umbilical vein endothelial cells (HUVEC) model.
Results. We demonstrated that both, everolimus and IMO inhibit in vitro growth and survival of renal cell carcinoma cell lines, and that their combination produces a synergistic inhibitory effect. Since IMO prevents EGFR phosphorylation, while everolimus acts on the downstream transducer mTOR, everolimus plus IMO in combination efficiently interferes with EGFR-dependent signalling. Moreover, the combined treatment reduces VEGF secretion levels in both VHL wild-type and VHL mutant cells, thus interfering with induction of angiogenesis. In renal cell tumors xenografted in nude mice, everolimus plus IMO causes a potent and long-lasting cooperative antitumor activity, with strong reduction of tumor growth, significant prolongation of mice survival and potent inhibition of signal transduction. Immunohistochemistry revealed that the combined treatment is also able to inhibit tumor angiogenesis and to alter the composition of tumor stroma, by reducing fibroblastic response. Moreover, everolimus and IMO, both as single agents or in combination, are able to interfere with the main endothelial cell functions, such as adhesion, migration and capillary-like tubes and network formation.
Conclusion. Our results demonstrated that a combined treatment with everolimus and IMO is effective in different models of renal cell carcinoma by interfering with both cancer cells and microenvironment. Therefore, everolimus plus IMO could represent a potentially effective, rationale-based combination to be translated in the clinical setting.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5401.