Breast cancer cells metastasize to bone where they cause bone destruction. Here, cancer cells promote bone loss through the recruitment of osteoclasts, specialised macrophage-like cells that resorb bone. Bone resorption releases bone matrix factors that increase tumor growth, resulting in vicious cycle of bone resorption and tumor progression.

Previously we found 17-AAG, an Hsp90 inhibitor and anti-cancer therapeutic significantly increases MDA-MB-231 breast cancer cell growth in bone following intracardiac inoculation in nude mice. 17-AAG also increases osteoclast formation in vitro and in vivo even though Hsp90 client proteins are important in osteoclast formation. In addition to degradation of Hsp90 client proteins, inhibition by 17-AAG also causes cell stress responses through the activation of Heat Shock Factor 1 (HSF1). Therefore we sought to characterize 17-AAG and new HSP90 inhibitors, to examine the role of HSF1 in their effects and to determine the molecular mechanism of 17-AAG.

17-AAG and new, but structurally unrelated, Hsp90 blockers CCT018159 and NVP-AUY922 dose-dependently enhanced osteoclast formation from mouse bone marrow and RAW264.7 cells. Ablating HSF1 action by shRNA transfection and by pharmacological inhibition using KNK437 inhibited 17-AAG effects upon osteoclast formation. Osteoclast formation from HSF1 knockout mouse bone marrow were unresponsive to 17-AAG, but wild type cells did respond. To determine the mechanism of how 17-AAG is increasing osteoclast formation, the effect of 17-AAG upon critical transcription factors in the osteoclast formation pathway was observed. 17-AAG treatment did not affect transcriptional activity of NFκB, AP-1 or NFATc1. Moreover 17-AAG did not increase NFATc1 or cFos protein expression over time. In contrast, 17-AAG treatment increased protein expression of MITF,in a dose and time dependent manner. RAW264.7 cells were then transiently transfected with a luciferase reporter construct containing the promoter region of a MITF target gene v-ATPase-d2. 17-AAG treatment increased the transcriptional activity of this promoter. RANKL and 17-AAG combination treatment increased reporter activity of the v-ATPase-d2 promoter in an additive manner. The stress induced kinase p38 is known to be important in osteoclast formation. Our results show inhibiting p38 with SB203580 decreases MITF protein expression.

These data indicate that Hsp90 inhibition causes an increase in osteoclast formation in an HSF1-dependent manner. The data provides a mechanism of action, whereby 17-AAG is increasing osteoclast numbers, through the increased protein expression of Mitf.

Citation Format: A. Gabrielle J. van der Kraan, Ryan R. Chai, Michelle M. Kouspou, Ben J. Lang, Preetinder P. Singh, Jiake Xu, Damien Eeles, Matthew T. Gillespie, Julian M. Quinn, John T. Price. HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2013-2933