bioRxiv preprint doi: https://doi.org/10.1101/521989; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. C-terminal HSP90 Inhibitors Block the HSP90:HIF-1α Interaction and Inhibit the Cellular Hypoxic Response Nalin Katariaa, Bernadette Kerra, Samantha S. Zaiterb, Shelli McAlpineb and Kristina M Cooka† a. University of Sydney, Faculty of Medicine and Health, Charles Perkins Centre, Sydney, Australia. b. School of Chemistry, University of New South Wales, Sydney, Australia. † To whom correspondence should be addressed: Kristina M Cook, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia 2006; [email protected]; Tel: +61 286274858. Hypoxia Inducible Factor (HIF) is a transcription factor cancer cells known as a heat shock response (HSR)12. The activated by low oxygen, which is common in solid compounds also have poor selectivity for HSP9013,14. tumours. HIF controls the expression of genes involved in C-terminus inhibitors of HSP90 (SM molecules)11 act in a angiogenesis, chemotherapy resistance and metastasis. The selective manner, and in contrast to the N-terminus chaperone HSP90 (Heat Shock Protein 90) stabilizes the inhibitors, they do not induce a heat shock response13,15. subunit HIF-1α and prevents degradation. Previously Although the SM molecules block all co-chaperones that identified HSP90 inhibitors bind to the N-terminal pocket bind to the C-terminus of HSP90 and inhibit HSP90 of HSP90 which blocks binding to HIF-1α, and produces function16,17, there is no data discussing whether C- HIF-1α degradation. N-terminal inhibitors have failed in terminal inhibitors will impact the binding event between the clinic as single therapy treatments due in part because HSP90 and HIF-1α or the subsequent hypoxic response. they induce a heat shock response, which increases This is the first study examining the effect of C-terminal chemotherapy resistance. SM molecules are HSP90 HSP90 inhibitors on the activity of HIF-1α in hypoxia and inhibitors that bind to the C-terminus and do not activate downstream gene expression. the heat shock response. The effects of C-terminal HSP90 Herein we report that the SM compounds prevent inhibitors on HIF-1α are unreported. Herein we show that accumulation of HIF-1α in HCT116 colorectal cells in SM compounds block binding between HSP90 and HIF-1α, hypoxia (0.5% O2) (Fig 1). HIF-1α substantially increases leading to HIF-1α degradation through the proteasome in HCT116 cells under hypoxia. When hypoxic HCT116 using the PHD/pVHL pathway in hypoxic conditions. The cells are incubated in the presence of SM122, HIF-1α SM compounds decrease HIF-1α target gene expression at protein levels decreased to normoxia levels. SM253 and the mRNA and protein level under hypoxia in colorectal SM258 also decreased HIF-1α in hypoxia (Fig 1a and 1b). cancer cells, leading to cell death, without inducing a heat The HSP90 inhibitors were also compared to chetomin, a shock response. Our results suggest that targeting the C- potent HIF-1 inhibitor that blocks the interaction between terminus of HSP90 blocks the hypoxic response and may HIF-1α and p300 by a zinc ejection mechanism2. SM122 be an effective anti-cancer strategy. reduces HIF-1α levels similar to that seen with chetomin (Fig. 1a and b). Hypoxia is common in tumors and activates a transcription Cells treated with 17-AAG (10 µM, 100 fold over the factor known as Hypoxia Inducible Factor-1 (HIF-1). HIF- GI50), an N-terminal HSP90 inhibitor16,17 show similar 1 controls the expression of genes involved in glycolysis, degradation of HIF-1α when treated with SM122 (10 µM), angiogenesis and cell survival, and is associated with a or SM252 (25µM) or SM258 (25µM). The C-terminal poor cancer prognosis1. Given that HIF-1 is upregulated in majority of solid tumors, there are ongoing drug development efforts to inhibit its activity. Previous work has focused on inhibiting the HIF-1α subunit from binding to partners such as p3002-5 or HIF-1β6, or binding to the DNA itself7. HIF-1 is a heterodimer composed of α and β subunits. Activity of the HIF-1 transcriptional complex is regulated post-translationally through degradation of HIF-1α in an oxygen-dependent process1,8. HIF-1α stability and Fig. 1 SM compounds inhibit HIF-1α stabilization under hypoxia. a) Nuclear HIF-1α levels in HCT116 cells after 6 h of hypoxia and SM degradation is further regulated by the chaperone Heat treatment. b) Densitometry of HIF-1α western blots. mean± SEM of 4-5 Shock Protein 90 (HSP90)9,10. N-terminal HSP90 experiments (except 25 µM SM122 which is n = 2). **P < 0.01, ***P < inhibitors such as geldanamycin and related analogues 0.001, as compared to hypoxic control. bind in the ATP binding pocket on the amino terminus (N- terminus) of HSP9011, disassociating it from HIF-1α and inhibitors, SM122, SM253, and SM258, all have a GI50 = 16,17 inducing pVHL-independent degradation of HIF-1α9,10. ~5 µM , yet they are highly effective at inducing the While N-terminal HSP90 inhibitors effectively block the degradation of HIF-1α when cells are treated with 2-5 fold due to their ability to induce a survival mechanism in over their GI50 (Fig. 1a and b), compared to 17-AAG, bioRxiv preprint doi: https://doi.org/10.1101/521989; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. which is treated at 100 fold over its GI50. The considerable indicate that SM molecules functionally reduce the amount of HIF-1α degradation in the presence of SM glucose transporter and are more potent than the N- compounds indicates that targeting the C-terminus of terminal inhibitor 17-AAG. HSP90 is a more effective strategy for decreasing HIF-1α SM253 and SM258 reduced LDHA (lactate than targeting the ATP binding pocket in the N-terminus. dehydrogenase A) mRNA expression under hypoxia (Fig HCT116 cells treated with SM compounds showed 2a). SM122 had little effect on LDHA mRNA. SM253 and varying degrees of inhibition of HIF-1 target gene SM258 both decreased LDHA protein to similar levels as expression in hypoxia based on both mRNA (Fig. 2a) and chetomin (~33%) (Fig 2b and c). Protein levels of protein levels (Fig 2b). The expression of carbonic hexokinase 2 (HK2), another enzyme involved in anhydrase IX (CA9) is regulated by HIF-1 and increases glycolysis, were significantly reduced by SM122 and under hypoxia (Fig. 2a). Both the N-terminal (17-AAG) SM253 (~40%) (Fig. 2b and c). SM258 decreased HK2 by and C-terminal compounds (SM) significantly decreased ~60%. Thus, SM122 was highly effective at reducing HIF- the expression of CA9 mRNA (Fig. 2a), indicating that 1 target protein expression for Glut1, LDHA and HK2 HIF-1 target gene expression is inhibited by both series. compared to N-terminal HSP90 inhibitor 17-AAG, whilst We also examined the mRNA and protein expression of SM253 and SM258 were slightly more effective than 17- multiple glycolytic genes under HIF-1 control upon AAG at reducing Glut1. This indicates that C-terminal SM exposure to hypoxia and HSP90 inhibitors. At the mRNA compounds, and particularly SM122, are effective at level, 25 µM SM258 and 25 µM and 10µM 17-AAG reducing glycolytic enzymes that are commonly significantly inhibited glucose transporter SLC2A1 upregulated in cancers. (Glut1) mRNA expression (Fig. 2a). To test the cytotoxic effect of SM compounds, we Comparing the compounds’ impact on Glut1 protein measured HCT116 cell viability at 18 and 72 h and expression, SM122 (25 and 10 µM), produced a compared to 17-AAG. The majority of cells were still significant reduction in the amount of Glut1, similar to viable at concentrations up to 25 µM at 18 h (see levels seen in normoxia (Fig. 2b and c). In contrast, 17- supplementary), where HIF-1 inhibition was documented AAG, slightly increased Glut-1 protein levels in hypoxia (Fig. 2). However, after 72 h, cell viability significantly decreased under normoxia and hypoxia for all drugs indicating HSP90 is essential for cell survival. A pitfall of N-terminal HSP90 inhibitors is that they induce a heat shock response (HSR). The HSR activates heat shock factor-1, a transcription factor that controls expression of HSP90, 70, 40 and 2718. The HSR therefore increases levels of HSP90, the protein targeted by N- terminal drugs, and activates mechanisms that block apoptosis and increase chemo-resistance18. SM HSP90 inhibitors do not induce the HSR in normoxia thus greatly increasing their cellular efficacy13. The HSR under hypoxic conditions is unknown, as is the impact of SM compounds on HSP protein levels. To test if a heat shock response is activated by N- and C- terminal HSP90 inhibitors in hypoxia, we measured HSP related proteins following an 18 h drug treatment. Treatments using 10 µM 17-AAG produced high levels of HSP70, and a slight increase in HSP90, while the SM compounds did not (Fig. 3a and b). We also compared the heat shock response from the HSP90 inhibitors to chetomin (Fig. 3a and b). At 1 µM chetomin, HSP70 significantly increases. This is not entirely unexpected given that the heat shock response can be induced by cell Fig 2 HIF-1 target gene expression is decreased by SM compounds in hypoxia. a) HIF-1 target mRNA expression. HCT116 cells were incubated in the presence or absence of compounds in hypoxia for 18 h. Total RNA was isolated, converted to cDNA and used for qPCR.