Simultaneous inhibition of Hsp90 and mTOR synergistically reduces tumor growth and angiogenesis of hepatocellular cancer in an experimental model

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Hepatocellular carcinoma (HCC) is a disease with minimal response to conventional chemotherapeutic regimens. Hence, novel targeted therapy concepts need to be developed to improve outcome of this highly resistant cancer. Since heat-shock protein 90 (Hsp90) is required for activation of multiple oncogenic signaling pathways that are also involved in HCC progression, we hypothesized that targeting Hsp90 could reduce growth and angiogenesis of HCC in vivo. Secondly, we sought to investigate whether addition of the mTOR inhibitor rapamycin would potentiate antiangiogenic effects. Furthermore, as mTOR inhibitors themselves may lead to activation of signaling pathways in tumor cells, we postulated that Hsp90 inhibition will counteract such pathway activation, thus improving efficacy of rapamycin. Human HCC cell lines (HepG2, Huh-7) were used for experiments with the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) and the mTOR inhibitor rapamycin. By MTT analyses, combination of 17-DMAG with rapamycin substantially improved cytotoxic effects of single agents. In addition, therapy with 17-DMAG lowered constitutive and inducible activation of Erk, Akt and STAT3 in HCC cells, as determined by Western blotting. Furthermore, a hypoxic response in terms of HIF-1α was blunted. In contrast, rapamycin itself induced Akt activation in HCC cells in vitro. However, both agents were able to significantly reduce cancer cell migration in Boyden chambers. The effects of 17-DMAG ± rapamycin on tumor growth were determined in a subcutaneous tumor model with Huh-7 cells. Mice received 17-DMAG (10mg/kg/day) and/or rapamycin (0.1mg/kg/day), or vehicle, by intraperitoneal injections. Results showed that either substance, 17-DMAG or rapamycin, significantly reduced tumor growth (P<0.05). In addition, numbers of proliferating tumor cells (BrdU) and tumor vascularization (CD31) were significantly reduced by monotherapy (P<0.05, for both). Surprisingly, combination of 17-DMAG with the mTOR inhibitor rapamycin synergistically enhanced growth inhibitory and antiangiogenic effects compared to monotherapy, as reflected by final tumor weights, CD31-vessel area, and BrdU-positive cells (P<0.05 vs. 17-DMAG or rapamycin). Interestingly, concurrent Hsp90 inhibition counteracted a rapamycin-mediated increase in Akt phosphorylation in tumor tissues in vivo, as determined by Western blotting. In conclusion, combination of 17-DMAG with the mTOR inhibitor rapamycin led to synergistic growth inhibitory and antiangiogenic effects in an experimental model of HCC in vivo. This finding could, in part, relate to a 17-DMAG mediated prevention of a rapamycin-induced paradoxical activation of Akt in tumors. Hence, inhibition of Hsp90 plus mTOR appears to be a promising strategy for therapy of HCC.