Julie Friedland

α6β4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-κB-dependent resistance to apoptosis in 3D mammary acini

Malignant transformation and multidrug resistance are linked to resistance to apoptosis, yet the molecular mechanisms that mediate tumor survival remain poorly understood. Because the stroma can influence tumor behavior by regulating the tissue phenotype, we explored the role of extracellular matrix signaling and tissue organization in epithelial survival. We report that elevated (α6)β4 integrin-dependent Rac-Pak1 signaling supports resistance to apoptosis in mammary acini by permitting stress-dependent activation of the p65 subunit of NF-κB through Pak1. We found that inhibiting Pak1 through expression of N17Rac or PID compromises NF-κB activation and renders mammary acini sensitive to death, but that resistance to apoptosis could be restored to these structures by overexpressing wild-type NF-κB p65. We also observed that acini expressing elevated levels of Pak1 can activate p65 and survive death treatments, even in the absence of activated Rac, yet will die if activation of NF-κB is simultaneously inhibited through expression of IκBαM. Thus, mammary tissues can resist apoptotic stimuli by activating NF-κB through α6β4 integrin-dependent Rac-Pak1 signaling. Our data emphasize the importance of the extracellular matrix stroma in tissue survival and suggest that α6β4 integrin-dependent Rac stimulation of Pak1 could be an important mechanism mediating apoptosis-resistance in some breast tumors.

Targeted inhibition of Hsp90 by ganetespib is effective across a broad spectrum of breast cancer subtypes

SummaryHeat shock protein 90 (Hsp90) is a molecular chaperone essential for the stability and function of multiple cellular client proteins, a number of which have been implicated in the pathogenesis of breast cancer. Here we undertook a comprehensive evaluation of the activity of ganetespib, a selective Hsp90 inhibitor, in this malignancy. With low nanomolar potency, ganetespib reduced cell viability in a panel of hormone receptor-positive, HER2-overexpressing, triple-negative and inflammatory breast cancer cell lines in vitro. Ganetespib treatment induced a rapid and sustained destabilization of multiple client proteins and oncogenic signaling pathways and even brief exposure was sufficient to induce and maintain suppression of HER2 levels in cells driven by this receptor. Indeed, HER2-overexpressing BT-474 cells were comparatively more sensitive to ganetespib than the dual HER2/EGFR tyrosine kinase inhibitor lapatinib in three-dimensional culture. Ganetespib exposure caused pleiotropic effects in the inflammatory breast cancer line SUM149, including receptor tyrosine kinases, MAPK, AKT and mTOR signaling, transcription factors and proteins involved in cell cycle, stress and apoptotic regulation, as well as providing combinatorial benefit with lapatinib in these cells. This multimodal activity translated to potent antitumor efficacy in vivo, suppressing tumor growth in MCF-7 and MDA-MB-231 xenografts and inducing tumor regression in the BT-474 model. Thus, ganetespib potently inhibits Hsp90 leading to the degradation of multiple clinically-validated oncogenic client proteins in breast cancer cells, encompassing the broad spectrum of molecularly-defined subtypes. This preclinical activity profile suggests that ganetespib may offer considerable promise as a new therapeutic candidate for patients with advanced breast cancers.

Abstract 1038: Inhibition of mTOR enhances the activity of HSP90 inhibitors in part through cessation of heat shock protein synthesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of hundreds of client proteins, many of which promote tumor growth. While HSP90 inactivation abrogates multiple oncogenic pathways, it subsequently triggers a heat shock response that may limit its full cytotoxic potential. To overcome this limitation, we sought to identify a clinically feasible method to block heat shock protein synthesis induced by the HSP90 inhibitor ganetespib. Results: An immunoassay was developed to screen a library of >300 late stage or approved drugs for their ability to block the upregulation of HSP70 subsequent to HSP90 inhibition. A number of effective agents were identified, with the most prevalent and effective being those that target PI3K/mTOR signaling. Validating this result, mTOR inhibitors diminished ganetespib induced HSP70 and HSP90 protein levels in multiple cancer cell lines. To determine the mechanism of HSP regulation by mTOR inhibitors the expression of >80 heat shock related genes was analyzed. Inhibitors of mTOR had no effect on HSP RNA levels but suppressed the upregulation of HSP genes induced by ganetespib. Heat shock factor 1 (HSF1) is the major transcriptional regulator of HSPs. Under conditions of stress or HSP90 inhibition, HSF1 is released from repressive cytoplasmic chaperone complexes and translocates into the nucleus to initiate heat shock gene transcription. Nuclear HSF1 levels increased within 1 h of ganetespib exposure but were dramatically reduced in the presence of an mTOR inhibitor. A major function of mTOR is regulating protein synthesis through activation of the cap-binding protein EIF4E, therefore we investigated whether inhibition of protein synthesis would affect HSF1 nuclear accumulation. Cycloheximide or an EIF4E inhibitor reduced ganetespib-mediated HSF1 entry into the nucleus suggesting that inhibition of mTOR can prevent HSP induction by blocking protein synthesis. Combinations of ganetespib with mTOR or dual mTOR/PI3K inhibitors resulted in a significant increase in anticancer activity compared to monotherapy in multiple in vitro and in vivo cancer models. Conclusions: Inhibition of mTOR counteracts ganetespib induced upregulation of HSPs by blocking translocation of HSF1 into the nucleus, an effect potentially mediated by a decrease in mTOR driven protein synthesis. Attenuation of heat shock response may contribute to the synergy observed for ganetespib and PI3K/mTOR inhibitors in preclinical cancer models. Targeting mTOR signaling thus represents a promising, clinically feasible approach to maximize the therapeutic potential of ganetespib. Citation Format: Suqin He, Jaime Acquaviva, Julie C. Friedland, Jim Sang, Donald L. Smith, Manuel Sequeira, Chaohua Zhang, David A. Proia. Inhibition of mTOR enhances the activity of HSP90 inhibitors in part through cessation of heat shock protein synthesis. [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 1038. doi:10.1158/1538-7445.AM2013-1038

P3-17-05: Beyond HER2 and Hormonal Agents: The Heat Shock Protein 90 Inhibitor Ganetespib as a Potential New Breast Cancer Therapy.

Background: Ganetespib is a fully synthetic and selective inhibitor of heat shock protein 90 (HSP90), a molecular chaperone recognized as a key facilitator of breast cancer initiation, progression and metastasis. Methods: Preclinical activity of ganetespib across the four major breast cancer subtypes and inflammatory breast cancer was assessed in vitro and in vivo. Modulation of cell proliferation and viability was determined both in monolayer and three-dimensional cultures. HSP90 client protein expression and activity was monitored by Western blot and protein array. To recapitulate clinical dosing, kinetics of client protein destabilization were measured following short exposures to drug in vitro. Anticancer activity of ganetespib was further investigated in vivo using breast cancer xenografts. Results: Ganetespib displayed potent, low nanomolar activity in luminal (A and B), basal (A and B) and inflammatory breast cancer cell lines grown as monolayers in vitro. BT-474 (HER2 amplified) luminal cells grown as mammospheres in 3D were equally as sensitive to ganetespib as those grown in monolayer. In luminal cells, ganetespib simultaneously disrupted multiple signaling components including the estrogen and progesterone receptor, several receptor and non-receptor tyrosine kinases, as well as the MAPK pathway. Further, ganetespib effectively inhibited AKT, PDK1 and SGK3 activity in PIK3CA mutant cells suggesting that HSP90 is essential for both AKT-dependent and AKT-independent signaling. Clinically relevant exposure times to ganetespib in vitro resulted in potent, long term destabilization of HER2. In the basal-like breast cancer cell line MDA-MB-231, enriched in CD44+CD24- stem like cells that commonly display chemotherapeutic resistance and activated JAK2/STAT3 signaling, ganetespib (50 nM) induced significant degradation of JAK2 concordant with loss of both tyrosine and serine phosphorylation of STAT3, followed by cell death. The potent anticancer activity in vitro translated in vivo , where ganetespib was effective in modulating breast cancer xenograft growth as a single agent in both luminal and basal-like breast cancer models. Finally, ganetespib has demonstrated encouraging signs of clinical activity in breast cancer patients, including confirmed partial responses in both a triple negative breast cancer patient and a HER2 positive breast cancer patient. Conclusions: Ganetespib is a highly potent HSP90 inhibitor that displays preclinical activity in breast cancer due to its ability to simultaneously perturb multiple oncogenic signaling pathways. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-05.