Targeting Hsp90 using EC154, a novel synthetic Hsp90 inhibitor, impairs multiple signaling pathways in human gastric and pancreatic cancer cells and reduces tumor growth in vivo

C170

Background: Heat shock protein 90 (Hsp90) has been identified to be crucial for stability and functionality of oncogenic signaling molecules, including EGFR, Her-2, Akt, Erk, and various transcription factors. We recently demonstrated that gastric and pancreatic cancer harbors multiple such molecular targets, which can effectively be inhibited by using geldanamycin derivates to block Hsp90, thus leading to significant growth inhibition. We now followed-up on this aspect of targeting Hsp90 for therapy of gastrointestinal cancers, and investigated effects of a novel synthetic Hsp90 inhibitor (EC154) in vitro and in vivo. Methods: Human pancreatic cancer cells (L3.6pl, HPAF-II), gastric cancer cells (TMK-1), and endothelial cells (HUVEC) were employed. Cytotoxic effects were assessed by MTT analyses. Effects of Hsp90 blockade on activation of signaling pathways (Erk, Akt, STAT3, EGFR, VEGFR) were investigated by Western blotting. The impact of EC154 on cell migration was evaluated in modified Boyden chambers. Effects of EC154 tumor growth and vascularization were investigated in subcutaneous xenograft models. Mice received either EC154 (60mg/kg; 2x/week) or vehicle by intraperitoneal injections. Tumor diameters were measured every other day and volumes calculated. Tissue was harvested for immunohistochemical analyses and Western blotting. Results: The drug dose-dependently reduced proliferation of gastric and pancreatic cancer cells in vitro and Western blot analyses showed that EC154 (at IC₅₀) effectively blocked STAT3, Akt and Erk activation in cancer cells, in addition to down-regulating EGFR and IGF-IR. Furthermore, EC154 led to marked inhibition of tumor cell migration in vitro (P<0.05). Interestingly, in endothelial cells, EC154 not only significantly reduced cell survival and proliferation in MTT analyses, but also markedly decreased VEGFR2 activation and STAT3 phosphorylation. In addition, EC154 significantly reduced VEGF-A mediated endothelial cell migration in vitro, suggesting a potential antiangiogenic effect of this compound. In vivo, treatment with EC154 significantly reduced tumor growth rates of both pancreatic and gastric cancer cells (P<0.05, for both). Analyses of tumor tissues are pending and will be presented at the meeting (expression of Hsp90-client proteins for monitoring drug efficacy). Conclusion: EC154 is a novel potent inhibitor of Hsp90 that effectively disrupts multiple oncogenic signaling cascades in gastric and pancreatic cancer cells, and significantly reduces tumor growth in vivo. Moreover, direct effects of EC154 on endothelial cells were detectable, suggesting that EC154 could be used for antineoplastic and antiangiogenic therapy concepts for treatment of gastric and pancreatic cancer.