Accepted Manuscript

Hsp90 inhibition in adrenocortical carcinoma: Limited drug synergism with mitotane

Silviu Sbiera, Sabine Kendl, Isabel Weigand, Iuliu Sbiera, Martin Fassnacht, Matthias Kroiss

19SN 0000-7209

Molecular and Cellular Endocrinology

PII:	S0303-7207(18)30290-9
DOI:	10.1016/j.mce.2018.10.009
Reference:	MCE 10311
To appear in:	Molecular and Cellular Endocrinology
Received Date:	22 December 2017
Revised Date:	14 August 2018
Accepted Date:	7 October 2018

Please cite this article as: Sbiera, S., Kendl, S., Weigand, I., Sbiera, I., Fassnacht, M., Kroiss, M., Hsp90 inhibition in adrenocortical carcinoma: Limited drug synergism with mitotane, Molecular and Cellular Endocrinology (2018), doi: https://doi.org/10.1016/j.mce.2018.10.009.

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1 Hsp90 inhibition in adrenocortical carcinoma: limited drug synergism

2 with mitotane

3 Silviu Sbiera1, Sabine Kendi1, Isabel Weigand1, luliu Sbiera1, Martin Fassnacht1,

4 Matthias Kroiss1

5 6 1University Hospital Würzburg; Department of Internal Medicine I, Division of

7 Endocrinology and Diabetes, Würzburg, Germany

9 Corresponding author:

10 Matthias Kroiss, MD, PhD

11 University Hospital Würzburg

12 Division of Endocrinology and Diabetology

13 Oberdürrbacher Str. 6

14 97080 Würzburg

15 Telephone: +49-931/201-39740

16 Fax: +49-931/201-6039740

17 Email: Kroiss_M@ukw.de

DE E VERİR. TOTO THE TED MANUS CRÍ DER

18 19 Short title: Hsp90 inhibition in adrenocortical carcinoma

Key words: treatment, in vitro, cell line, heat shock protein

21 Word count (excluding figures and references): 2808

23 Abstract

90kDa heat shock proteins (Hsp90) act as protein chaperones and play a role in modulating endoplasmic reticulum (ER) stress. Hsp90 inhibitors are under clinical investigation as cancer treatment. Mitotane therapy of adrenocortical carcinoma (ACC) has been shown to act through lipid-induced ER-stress. To explore the potential of Hsp90 inhibitors in ACC as a single agent and in combination with mitotane, we analyzed two independent gene expression data sets of adrenal tumors in silico and treated the ACC cell line model NCI-H295 with Hsp90 inhibitors BIIB021 (B) and CCT18159 (C) alone and in combination with mitotane. ER-stress markers were monitored by immunoblotting. Drug synergism was quantified using the median effect model with cell viability as read-out. Cytosolic Hsp90 isoforms AA1 and AB1 were significantly overexpressed in ACC. Viability of H295 cells was impaired by B and C as single agents with an EC50 of 5.7x10-6M and 12.1x10-6M. B but not C dose- dependently increased XBP1 splicing and CHOP expression indicative of ER-stress activation. ER-stress marker expression was enhanced by co-incubation of B with 10uM but not 5uM mitotane. Maximal CHOP expression was induced by 25 uM mitotane alone with no additional effect of B. Combination indices (CI) of B and C with mitotane ranged from 0.64 to 1.38 and 0.68 to 1.30, respectively where CI values <0.5 support clinically-relevant drug synergism. In conclusion, Hsp90 paralogs are differentially expressed in ACC and B but not C activates ER-stress in ACC cells. No meaningful drug synergism of Hsp90 inhibitors with mitotane was observed.

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45 Introduction

46 The treatment of advanced adrenocortical carcinoma (ACC) remains challenging.

47 Mitotane, an orphan drug that counteracts both tumor-related hormone excess and 48 tumor cell growth and proliferation is the only approved drug for ACC treatment.

We have previously demonstrated that mitotane targets sterol-O-acyl transferase 1 (SOAT1) which leads to accumulation of toxic lipids and ultimately activates endoplasmic reticulum stress and apoptosis in the NCI-H295 cell line model of ACC.

It is likely that additional direct (Scheidt, Haralampiev et al. 2016) and indirect (Hescot, Slama et al. 2013) mechanisms contribute to its activity given the high plasma concentrations of mitotane required for clinical activity (Terzolo, Baudin et al. 2013; Kerkhofs, Baudin et al. 2013).

90kDa heat shock proteins (Hsp90) are cellular chaperones which prevent the denaturation and aggregation of proteins in order to maintain protein homeostasis (Nathan, Vos et al. 1997). They participate in multiple functions during cell growth and proliferation. Client proteins include steroid hormone receptors, growth factor receptors, intracellular protein kinases but also oncoproteins like survivin (Butler, Ferraldeschi et al. 2015). Some known client proteins of Hsp90 have been implicated

49 50 51 52 53 54 55 56 57 58 59 60 61 62 in the pathogenesis of ACC (Libe and Bertherat 2005, Almeida, Fragoso et al. 2008, Sbiera, Kroiss et al. 2013).

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Four organelle-specific Hsp90 paralogs exist in humans, of which HSP90AA1 and HSP90AB1 proteins are mainly localized in the cytoplasm whereas HSP90B1 (94- kDa glucose-regulated protein, Grp94) is localized at the endoplasmic reticulum and TNF receptor-associated protein 1 (Trap1) in mitochondria (Seo 2015). Inhibitors of heat shock protein 90 (Hsp90) are a more recent addition to the pharmaceutical tool

69 box of cancer treatment.

HSP90AA1 is strongly upregulated by cellular stress but also fulfills distinct functions

71 in unstressed cells (Zuehlke, Beebe et al. 2015). At variance HSP90AB1 is more

72 constitutively expressed. Few studies have focused on client proteins of the cytosolic 73 74 75 76 isoforms HSP90AA1 and AB1 that are unique to each of them. In cancer cells cytosolic Hsp90 proteins play a central role in maintaining the tyrosine kinase activity of various oncogenic membrane receptors through stabilization of their structure. HSP90AB1 is involved in apoptosis and inflammation and also in the shuttling of 77 steroid hormone receptors into the nucleus. ER-localized HSP90B1 was shown in different cancers to participate in the regulation and maintenance of the folding and secretion of disease-promoting proteins that result from mutations and would lead to ER-stress and cell death in the absence of assisted protein folding (Mclaughlin and 81 Vandenbroeck 2011). Trap1 on the other hand is crucial as a key regulator of mitochondrial bioenergetics in tumor cells with a profound impact on neoplastic growth.

Here we used one of the prototypic Hsp90 inhibitors CCT018159 (Cheung, Matthews et al. 2005, Powers and Workman 2006) and the more recently developed BIIB021 (Zhang, Neely et al. 2010) to explore the therapeutic potential of Hsp90 inhibition in ACC. BIIB021 is not a substrate of the multi-drug resistance P-glycoprotein (P-gp) which is highly expressed in ACC (Flynn, Murren et al. 1992, Haak, van Seters et al. 1993) and hence has been proposed as a treatment of ACC (Zhang, Neely et al. 2010).

Here we aimed to explore the potential of Hsp90 inhibition in adrenocortical carcinoma alone and in combination by combining data mining with in vitro experiments.

Material and methods

In silico analyses

log2 normalized expression data (Human Genome U133 Plus 2.0 Kit, Affymetrix) of Hsp90 paralogs HSP90AA1 (probe 211969_at), HSP90AB1 (probe 200064_at),

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99 HSP90B1 (probe 239451_at) and TRAP1 (probe 201391_at) in 10 normal adrenal glands (nAG), 22 adrenocortical adenomas, and 33 ACC (Giordano, Kuick et al. 2009) were retrieved from the National Center for Biotechnology Information’s Gene Expression Omnibus (GSE10927). In addition, the ratio between the mean expression of cytosolic paralogs HSP90AA1 and HSP90AB1 and the endoplasmic reticulum bound HSP90B1 was calculated. As an independent data set, we used microarray data obtained with the same assay by de Reynies et al (de Reynies, Assie et al. 2009) that were deposited at the European Bioinformatics Institute (EBI) with the accession number E-TABM-311. This data set contains 34 ACC and 58 ACA 108 and was studied with the same gene expression array (Human Genome U133 Plus 2.0 Kit, Affymetrix) and the values from the same oligonucleotide probes were evaluated as described above.

Chemicals

Mitotane was from ISP Chemical Products, CCT018159 from Selleckchem and BIIB021 from Santa-Cruz Biotechnology. Stocks were diluted in ethanol. All buffers and solvents were from Sigma-Aldrich (Steinheim, Germany).

Cell Culture

Adherent variant NCI-H295R cells was obtained from ATCC and cell line identity ascertained by short tandem repeat profiling (Sabine Herterich, Department of Clinical Chemistry and Laboratory Medicine, University Hospital Würzburg). Cells were cultured as described (Kroiss, Sbiera et al. 2016). In short Ham’s F12 medium supplemented with 2.5 % Corning Nu-Serum (Fisher Scientific, Schwerte, Germany), 5.2 ng/ml sodium selenite, 100 µg/ml transferrin and 5 µg/ml insulin was used and the cells were cultured in flasks in a humid atmosphere at 37 ℃ and 5 % CO2.

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123

Cell viability Assay

Viability testing was performed using WST1 reagent according to the manufacturer’s protocol (Roche) by employing a Victor2 multi-plate reader (Perkin-Elmer).

Immunoblot analysis was performed as described (Sbiera, Leich et al. 2015) and the following antibodies used: X-box Binding Protein (XBP1, 1:500, Santa Cruz Biotechnology), Eukaryotic Translation Initiation Factor 2A elF2a (1:1000, D7D3, Cell Signaling), phospho-eIF2a (Ser51) (1:500, Clone D9G8, Cell Signaling), C/EBP- Homologous Protein (CHOP, 1:1000, NBP2-13 172, Novus Biologicals), Glucose- Regulated Protein, 78kDa (GRP78, 1:150, Santa Cruz Biothechnology) and beta actin (ACTB, 1:1000, Clone D6A8, Cell Signaling). HRP-conjugated secondary antibodies was from GE Healthcare (1:5000) and WesternSure Premium chemiluminescence substrate (Li-Cor) used for detection with a C-Digit Instrument (Li-Cor). Immunoblot band intensities were evaluated using ImageJ software 1.48v for Mac (National Institutes of Health, USA).

124 125 126 Immunoblotting 127 128 129 130 131 132 133 134 135 136 137 138 Statistical Analyses 139 140 141 142 143 144 145 were determined by applying curve fit nonlinear regression analyses.

Prism 6.0 software (GraphPad) for Mac was used for all statistical analyses. For group comparison of gene expression data, Kruskal Wallis test with Dunn’s post hoc correction for multiple comparisons was used. For cell viability comparisons, repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey’s multiple comparisons test was applied. p<0.05 was considered statistically significant. Single drug concentrations inducing 50 % (EC50) of the observed effect

Assessment of pharmacodynamics drug interaction

146 147 For assessment of drug interaction parameters, the median effect model proposed 148 by Chou et al. (Chou 2006) was applied using Compusyn Software (Version 1.0,

downloaded from www.combosyn.com) as described previously (Kroiss, Sbiera et al.

2016). Cell viability measured by WST-1 testing was used as a read-out and 25 uM

mitotane was considered as the maximum effect. Normalization was carried out using Prism Version 6.0 (GraphPad). To determine cooperativity indices, drug concentrations of 5-25 uM mitotane and 1-25 uM CCT018159 and BIIB021, respectively were used.

Results

In silico analysis of HSP90 mRNA expression in adrenocortical tissues.

mRNA expression of cytosolic HSP90AA1 and HSP90AB1 was significantly higher in ACC and adrenocortical adenomas compared to normal adrenal glands (HSP90AA1: 4.37±0.08, ACC; 4.36±0.04, ACA vs. 4.30±0.04, nAG; ACA vs. NAG p<0.05, ACC vs. nAG p<0.01; HSP90AB1: 4.35±0.09, ACC; 4.37±0.03, ACA vs. 4.26±0.06, nAG; ACA vs. NAG p<0.05, ACC vs. nAG p<0.01) (Fig.1A and B). At variance, the mRNA expression of HSP90B1 (Fig.1C), the protein product of which has a predominant ER localization, was significantly less expressed in the ACC versus the normal and benign adrenocortical tissues (2.63+0.23, ACC; 2.94±0.26, ACA vs. 2.91±0.21, nAG; ACC vs. ACA or nAG p<0.01). Considering the ratio between the mRNA expression of the two cytoplasmic Hsp90 isoforms and the ER isoform (Fig. 1D), there was clear increase of the ratio in favour of the cytosolic isoforms in ACCs compared to the two comparators (1.66±0.13, ACC; 1.49±0.14, ACA vs. 1.48±0.11, p<0.05). Furthermore, TRAP1 had a trend towards higher expression in ACA than in nAG but was expressed at significantly higher levels in ACC compared to nAG (2.54+0.14, ACC; 2.48±0.12, ACA vs. 2.33±0.13, nAG; ACC vs. nAG p<0.001).

To confirm these data, we analysed an independent data set (de Reynies, Assie et al. 2009), which however does not report normal adrenal gland expression. We found expression differences between adrenocortical carcinomas and adenomas of HSP90AA1 (11.07±0.46 vs. 10.85±0.32, p<0.05), HSP90AB1 (10.93±0.45 vs.

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10.49±0.52, p<0.001). Although the difference in HSP90B1 mRNA expression missed statistical significance alone (9.76±0.47 vs. 9.82+0.30, p=n.s.), the ratio of cytosolic vs. ER-bound Hsp90 differed in a similar manner between ACC and ACA (1.20±0.08 vs. 1.16±0.03, p<0.05) as observed in the first data set. TRAP1 mRNA showed a similar trend towards higher expression in ACC than in the ACA (4.64±0.31 vs. 4.53+0.23, p=0.08).

In vitro activity of single agent and combined treatment with CCT018159 and mitotane

We next analysed the impact of Hsp90 inhibition on ER-stress markers alone and in combination with mitotane. By using the classical Hsp90 inhibitor CCT018159 we found a dose-dependent increase in expression of the protein product of spliced XBP1 mRNA (XBPs) at the expense of the protein product of unspliced XBP1 mRNA (4.04+0.28-fold change for 10uM, p<0.01; Fig. 2 A, Fig. S2 A). Phosphorylation of eIF2a protein was evident only at the highest concentration tested (8.0±5.28-fold for 50 µM, p<0.05) as a single agent (Fig. 2 A, Fig. S2 B) with total elF2x (Fig. 2 A, Fig. S2 C), and GRP78 (Fig. 2 A, Fig. S2 D) protein remaining unchanged. Of note, we only found only moderate increase of ER-stress effector CHOP protein expression with CCT018159 alone (2.39±0.30-fold change for 10uM CCT018159 compared to 3.23±0.45-fold change for 10uM mitotane; Fig. 2 A, Fig. S2 E). Despite marginal induction of ER-stress initiation through XBP1 splicing and elF2a phorphorylation alone, combination with 5 uM and 10 uM mitotane led to additive CHOP expression (e.g. 7.96+0.88-fold change with 10uM CCT018159 and 10uM mitotane, p<0.01; Fig. 2 A, Fig. S2 E) whereas 25 uM mitotane alone led to both highly activated elF2x phosphorylation and CHOP expression.

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In vitro activity of single agent and combined treatment with BIIB021 and mitotane

We next assessed the fully synthetic Hsp90 inhibitor BIIB021. We found this compound to increase XBP1s/ XBP1_ ratio 2.66±0.60-fold for 10uM and 5.07+0.84- fold at 50 uM (Fig. 2 B, Fig. S3 A). Phosphorylation of eIF2x protein was increased 1.47±0.24 -fold at 10uM and 4.51+0.01-fold at 50uM (Fig. 2 B, Fig. S3 B) with total eIF2a protein remaining unchanged (Fig. 2 B, Fig. S3 C). We saw a dose dependent increase of ER-stress effector CHOP protein expression by BIIB021 that paralleled XBP1 splicing. The effect on CHOP protein expression was stronger for BIIB021 (6.89±1.79 -fold increase, p<0.01) compared to mitotane (1.51+0.38 -fold increase, n.s.) at the same concentration (10uM). ER-resident heat shock protein GRP78 (Fig. 2 B, Fig. S3 D) expression was not affected. Mitotane alone activated XBP1 splicing already at 5uM to an extent that was similar to that seen with maximal BIIB021 dose (2.73+0.17 -fold for 5uM mitotane and 1.50±0.57 -fold for 50uM BIIB021) and dose- dependently increased elF2a phosphorylation (Fig. 2B, Fig. S3 B). Interestingly, combination treatment of BIIB021 with mitotane demonstrated additive CHOP protein expression at 5 uM and 10 uM mitotane whereas 25 uM mitotane alone fully activated CHOP expression and also upstream elF2x phosphorylation (Fig. 2A, Fig. S3 B and E). Co-treatment had a synergistic effect on XBP-1 splicing in the lower range of mitotane concentrations (e.g. 6.44+1.33 -fold change with 10UM of both substances). Of note, XBP1 splicing was strongly activated by 25 UM mitotane alone but showed a trend towards lesser activation when combined with BIIB021 (160.4 fold change for 25M mitotane alone vs 97.2- fold change when 10UM BIIB021 was also added).

Limited drug synergism of Hsp90 inhibitors with mitotane

We next investigated the impact of ER-stress modulation by Hsp90 inhibitors and mitotane on viability of NCI-H295 cells and found a dose-dependent impairment after treatment with single agent CCT018159 (EC50: 11.5 x10-6 M; Fig. 3A) and BIIB021

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(5.7 x10-6 M, Fig. 4A). EC50 of mitotane was 12.1 x10-6 M consistent with previous results (Sbiera, Fassnacht et al. 2015).

To determine the value of combining Hsp90 inhibitors with mitotane in vitro, we used

cell viability measured by WST-1 assay as a read out and applied individual compounds at concentrations in the range of their respective EC50 for NCI-H295R cells. Drug synergism was determined using algorithms developed by Chou et al (Chou 2006) with cooperativity indices (CI) below 1 indicating additivity, and values between 0.3 and 0.7 being considered synergistic.

By using this model, we found CI between 0.59 and 1.30 for CCT018159 (Fig. 3 B, C) with resulting dose reduction indices (DRI) only at low fractional activity (Fa) pointing to negligible drug synergism between mitotane and CCT018159.

238 Similarly, cooperativity between BIIB021 and mitotane was limited with cooperativity indices insignificantly different from 1 and resulting DRI with maximal values of 9.54 and 3.52 for mitotane and BIIB, respectively at low Fa of both drugs (Fig. 4 B, C).

Discussion

Heat shock proteins (Hsp) are required to enable proper folding of numerous proteins involved in a broad array of functional processes. The Hsp90 family comprises the cytosolic HSP90AA1 (HSP90alpha, HSP86) and HSP90AB1 (HSP90beta, Hsp84), the ER-localized HSP90B1 (Grp94/96) and mitochondrial Trap1 (TNF receptor- associated protein) proteins.

Hsp90 inhibition has been explored pre-clinically in vitro and in vivo for treatment of various types of cancer (Pandey, Prasad et al. 2016) and several compounds have been studied in clinical trials (Cheung, Matthews et al. 2005, Butler, Ferraldeschi et al. 2015).

Our in silico analyses of two large mRNA expression datasets in adrenocortical tissues revealed significant alteration of Hsp90 mRNA expression especially in ACC. Of note, the overexpression of the cytosolic forms occurs at the expense of the ER-

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bound Hsp90 proteins suggesting that probably in the ACC the cytosolic activity plays a preponderant role in maintaining proper folding of proteins. Given that the majority of adrenocortical carcinoma cases actively secretes steroid hormones such as cortisol and androgens, it is conceivable that Hsp90 proteins are relevant to maintain the synthetic capacity of the cell, especially by assisting in the proper folding of steroidogenic enzymes. Whereas it has been demonstrated that different types of Hsp90 family members are necessary for glucocorticoid receptor signaling (Morishima, Mehta et al. 2018), this has not been investigated for steroidogenic enzymes.

Furthermore, gradual increase in TRAP1 in benign and - more pronouncedly - in the malignant adrenocortical tumors indicates a requirement of maintaining mitochondrial energetic homeostasis and resistance to reactive oxygen species (ROS) in the tumors.

We have previously demonstrated that mitotane, the only drug approved specifically for the treatment of ACC, leads to apoptosis in the NCI-H295 cell line model of ACC through induction of ER-stress (Sbiera, Fassnacht et al. 2015). This finding led us to the idea that further increasing the ER-stress through additional pharmacological input may increase the therapeutic potential of mitotane which could than be used at much lower and less side effects inducing concentrations. We have already shown that bortezomib and carfilzomib, two proteasome inhibitors, could also activate ER- stress and displayed synergism with mitotane to induce ACC cell death in vitro (Kroiss, Sbiera et al. 2016).

Along the same lines we here have tested two further drugs, BIIB021 and CCT018159, both specific inhibitors of Hsp90 ATPase activity which thus inhibit both the cytosolic and ER forms of Hsp90 proteins (Frey, Leskovar et al. 2007, Richter, Reinstein et al. 2007).

We observed that BIIB021 as a monotherapy induced ER-stress in H295 cells similar to mitotane as indicated by similar level increase in typical ER-stress markers like

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283 284 285 286 spliced XBP1, eIF2a phosphorylation and CHOP expression while CCT018159 alone induced ER-stress only marginally. Despite this difference, both substances were able to enhance the effect on ER-stress of mitotane in the lower concentration rage, which shows that small disturbances might be sufficient to tip the balance towards 287 ER-stress. Surprisingly, at higher concentrations, mitotane alone strongly activated 288 ER-stress such that any possible additive effect by the two Hsp90 inhibitors would be blunted. We therefore conclude that mitotane treatment alone at clinically relevant concentrations leads to complete activation of ER-stress and mitotane and Hsp90 inhibitors likely induce ER-stress through the same pathway.

More importantly, all three substances affected the ACC cell viability in a similar concentration range. Hence, at least CCT018159 is likely to counteract cell proliferation independently of the ER-stress. Maybe this is also the reason why we failed to see therapeutically meaningful synergism between mitotane and both Hsp90 inhibitors despite the fact that the ER-stress induction synergism between mitotane and BIIB021 did not reflect also on cell viability. This may be partly because the ER- bound form of Hsp90 (HSP90B1) is significantly down-regulated in ACC compared to the normal and benign adrenal tissues as shown by in silico analyses. This is likely to reduce the impact of Hsp90 inhibitors on assisted protein folding at the ER. Despite the fact that these inhibitors to a certain extent have comparable effects on ER-stress markers as mitotane monotherapy in vitro, the relatively high concentrations required do not suggest to clinically investigate these drugs with priority in the treatment of ACC.

Note added in proof

During the review process of this manuscript the group of Dr. Lalli showed (Ruggiero, Doghman-Bouguerran et al. 2018) that the novel inhibitor of glucose regulated protein/Binding immunoglobulin protein (GRP78/BiP) HA15 exhibits pronounced synergism with mitotane in NCI-H295 cells. From our point of view this demonstrates

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that therapeutic interference with endoplasmic reticulum stress pathway may be of use in adrenocortical carcinoma even if - as demonstrated in this manuscript - inhibition of Hsp90 appears to be a less suitable way to do so.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft within the CRC/Transregio 205/1 „The Adrenal: Central Relay in Health and Disease“, Project B16 (M.F. and M.K.) and grants KR-4371/1-2 to M.K. and FA 466/4-2 to M.F. The Else Kroner-Fresenius-Stiftung supported this work with grant 2016_A96 to S.S. and M.K. We thank Sabine Herterich from the Department of Clinical Chemistry and Laboratory Medicine, University Hospital Würzburg for performing the STR identification of the cell lines used for the in vitro experiments.

Figure Legends.

Figure 1. In silico analysis of mRNA expression of the different Hsp90 paralogs in adrenocortical tissues. mRNA expression of the cytosolic HSP90 AA1 (A) and AB1 (B) and the predominantly endoplasmic reticulum localised HSP90 B1 (C) in normal adrenal glands (nAG), adrenocortical adenomas (ACA) and adrenocortical carcinomas (ACC) was specifically analysed in previously published data (Giordano et al. 2009). The ratio between the two cytoplasmic Hsp90 and the ER paralog is shown in (D). Expression of mitochondrial Hsp90 paralog TRAP1 (E). Horizontal bars represent the mean and standard error of the mean (SEM). Kruskal Wallis test with Dunn’s post hoc correction for multiple comparisons was used to assess the statistical significance of differential expression. * p<0.05; ** p<0.01; *** p<0.001.

Figure 2. Influence of co-treatment with mitotane and Hsp90 inhibitors on protein expression of ER-stress markers. Immunoblotting of indicated marker proteins in NCI-H295R cells treated with different concentrations of mitotane and

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CCT018159 (A) or BIIB021 (B). XBP1-u=unspliced form of XBP1, XBP1-s=spliced form of XBP1, P-eIF2a=phosphorylated form of elF2a.

Figure 3. Influence of co-treatment with mitotane and Hsp90 inhibitor CCT018159 on NCI-H295R adrenocortical cell-line viability. WST-1 viability measurements of co-treatment (A). Normalised isobologram (B) and dose-effect table (C) of the combinatorial treatment effect of mitotane and CCT018159. CI=combination index, CI<1 (green fields and upward pointing arrows) indicates additivity and synergism (Cl<0.5). Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey’s multiple comparisons test were used to assess the statistical significance of differential expression. * p<0.05; ** p<0.01; *** p<0.001.

Figure 4. Influence of co-treatment with mitotane and Hsp90 inhibitor BIIB021 on NCI-H295R adrenocortical cell-line viability. WST-1 viability measurements of co-treatment (A). Normalised isobologram (B) and dose-effect table (C) of the combinatorial treatment effect of mitotane and BIIB021. CI=combination index, CI<1 (green fields and upward pointing arrows) indicates additivity and synergism (Cl<0.5). Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey’s multiple comparisons test were used to assess the statistical significance of differential expression. * p<0.05, ** p<0.01, *** p<0.001.

Supplementary Figure 1. In silico analysis of mRNA expression of the different Hsp90 paralogs in adrenocortical tissues. mRNA expression of the cytosolic HSP90AA1 (A) and AB1 (B) and the predominantly endoplasmic reticulum localised HSP90B1 (C) in normal adrenal glands, adrenocortical adenomas and ACCs was specifically analysed in published data sets (de Reynies, Assie et al. 2009). The ratio between the two cytoplasmic Hsp90 and the ER paralog is displayed (D). Expression

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of mitochondrial Hsp90 paralog TRAP1 (E). Horizontal bars represent the mean and standard error of the mean (SEM). Kruskal Wallis test with Dunn’s post hoc correction for multiple comparisons was used to assess the statistical significance of differential expression. * p<0.05; ** p<0.01; *** p<0.001; n.s., not statistically significant.

Supplementary Figure 2. Evaluation of protein expression of ER-stress markers after co-treatment with mitotane and Hsp90 inhibitor CCT018159. Quantitative evaluation of immunoblotting results for the spliced/unspliced XBP-1 ratio (XBP1-s/u; A), phosphorylated (B) and total elF2a (C), GRP-78 (C) and CHOP (E) protein in NCI-H295R cells treated with different concentrations of mitotane and CCT018159.

Supplementary Figure 3. Evaluation of protein expression of ER-stress markers after co-treatment with mitotane and Hsp90 inhibitor BIIB021. Quantitative evaluation of immunoblotting results for the spliced/unspliced XBP-1 ratio (XBP1-s/u; A), phosphorylated (B) and total elF2a (C), GRP-78 (C) and CHOP (E) protein in NCI-H295R cells treated with different concentrations of mitotane and BIIB021.

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472 473 474 475 476 477 478 479 480 481 482

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Figure 1

€8

HSP90 AB1 (cytosole)

ACA

ACC

nAG

ACA

ACC

BAG

ACA

ACC

me HSPS0 AA1; AB1 (cytosol)/ O

1.5

TRAP1 (mae sendnie)

nAG

ACA

ACC

1.5

ACA

AGG

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XBP1-u

XBP1-S

elF2a

P-eIF2a

CHOP

GRP-78

ß-actin

CCT018159

0.1

μΜ

Mitotane

μΜ

Figure 2

XBP1-u

XBP1-S

elF2a

P-eIF2a

CHOP

GRP-78

ß-actin

BIIB021

0.1

μΜ

Mitotane

μΜ

ACCEPTE

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viability (A.U.)

CCT018159

0.1

μΜ

Mitotane

μΜ

CCT018159

0.5

Mitotane

	Mitotane [μM]	CCT018159 [μM]	Effect	CI
0	5	1	0.50	1 0.59
H	5	5	0.48	>> 0.94
A	5	10	0.34	1.07
A	5	25	0.05	1 0.68
0	10	1	0.52	L 1.13
X	10	5	0.43	I 1.30
++	10	10	0.26	I 1.20
0	10	25	0.04	₼ 0.73
.	25	1	0.07	៛ 0.89
A	25	5	0.04	î 0.81
V	25	10	0.03	î 0.77

Figure 3

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viability (A.U.)

BIIB021 Mitotane

0.1

5 10 25 50 0 1

μΜ

25 UM

BIIB021

1.5

0.5

Mitotane

Dose Mito	Dose B2B	Effect
O 5	1	0.33 1 0.64
0 5	5	0.11 0.90
A 5	10	0.05 , 1.03
Y 5	25	0.02 I 1.38
10	1	0.32 0.98
10	5	0.12 1.15 ៛
+ 10	10	0.05 1.17 1
6 10	25	0.02 I 1.36
25	1	0.05 Î 0.84
1 25	5	0.03 0.91
P 25	10	0.03 I 1.24
0 25	25	0.01 L 1.31

Figure 4

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Highlights:

· endoplasmic reticulum (ER) stress is known to be induced by mitotane in ACC

. inhibition of Hsp90 proteins may activate ER-stress in tumor cells

· up-regulation of mRNAs encoding cytoplasmic Hsp90 in published ACC data sets

· Hsp90 inhibitors CCT018159 and BIIB021 activate markers of ER-stress

. no meaningful drug synergism was observed when cell viability was used as a read-out

. combination of mitotane and current Hsp90 inhibitors is likely not useful for ACC treatment

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Quartz 4

Explorer

30315857

Accepted Manuscript

Material and methods

Chemicals

Cell Culture

Results

Limited drug synergism of Hsp90 inhibitors with mitotane

Discussion

Note added in proof

Acknowledgments

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Highlights:

Graph View

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