Abstract INTRODUCTION: Advanced head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat. In HNSCC, hypoxia is a negative prognostic marker with altered metabolism, increased invasiveness, and resistance to therapy. Natural withanolides exhibit potent, highly selective anticancer effects in HNSCCs in vitro and in vivo through a novel anticancer mechanism. These drugs [withaferin A and the novel compound, withalongolide A-triacetate(WGA-TA)] selectively induce metabolic oxidative stress in cancer cells while inhibiting HSP90 chaperone function via disruption of CDC37 (co-chaperone) binding. We hypothesize that WGA-TA is a potent, novel therapeutic drug for HNSCC that will decrease tumor migration and cancer stem cell endothelial-mesenchymal transition (EMT) even in the setting of hypoxia. METHODS: Human HNSCC cell lines MDA-1986, UMSCC11A, 11B and 12 were grown either under normoxic or hypoxic conditions. CM-H2DCFDA level defined accumulation of intracellular reactive oxygen species. Effects on proliferation and apoptosis were evaluated by flow cytometry (FC), Western blotting (WB), and RT PCR. Viability was evaluated by MTS and clonigenic assay. Scratch wound-healing and Boyden chamber assays were used to follow cell migration. RESULTS: WGA-TA treatment of both hypoxic and normoxic cells showed dose dependent induction of oxidation (p<0.01 vs. controls). This effect was completely blocked by N-acetyl cysteine(NAC) under both conditions (p<0.001). Dose dependent changes in biomarkers of oxidative stress (200-400% increase in HSP32 and HSP70; 80-90% decrease in HSF-1) with functional inhibition of Akt, mTOR, the EMT marker vimentin (80-90%), and cancer stem cell marker (Bmi-1, CD44 & ALDH each 50-90% inhibition (p<0.01 vs. control) and modulation of glycolytic proteins (20-80%; p<0.01) was observed under both conditions after WGA-TA treatment. Oxidative stress effect was further enhanced by hypoxia or pretreatment with L-Buthione-[S,R]-sulfoxime (BSO), whereas NAC treatment completely abrogated this effect even at 500nM WGA-TA. Treatment of hypoxic cells with WGA-TA did not alter the IC50 values (50-100 nM) and NAC pretreatment did not inhibit proliferation, whereas BSO decreased IC50 values by 10-40% indicating the role of ROS in reducing cell proliferation and viability. Dose-dependent induction of apoptosis (starting at 500nM WGA-TA) on FC was confirmed by caspases 3 and PARP cleavage on WB and accelerated by BSO but blocked by NAC treatment. Finally 500-1000 nM WGA-TA concentrations prevented HNSCC cell migration by 80-90% (p<0.01 vs. control). CONCLUSION: WGA-TA potently induces glycolytic oxidative stress in HNSCC leading to decrease in CSC EMT as well as,induction of apoptosis and loss of tumor cell migration even in hypoxic conditions. These exciting findings warrant further evaluation in translational in vivo models. Citation Format: Chitra Subramanian, Qing Zhu, Eileen Brandes, Peter T. White, Barbara N. Timmermann, Mark S. Cohen. A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2015-1739

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