Altered Steroid Milieu in AI Resistant Breast Cancer Facilitates AR Mediated Gene Expression Associated with Poor

Author Manuscript Published OnlineFirst on July 9, 2019; DOI: 10.1158/1535-7163.MCT-18-0791 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Altered steroid milieu in AI resistant breast cancer facilitates AR mediated gene expression associated with poor

2 response to therapy.

3 Short title: Androstenedione drives AR mediated gene expression in AI resistance.

4 Laura Creevey1* ([email protected])

5 Rachel Bleach1* ([email protected])

6 Stephen F Madden2 ([email protected])

7 Sinead Toomey3 ([email protected])

8 Fiona T Bane1 ([email protected])

9 Damir Varešlija 1 ([email protected])

10 Arnold D Hill4 ([email protected])

11 Leonie S Young1 ([email protected])

12 ‡Marie McIlroy1 ([email protected])

13 1. Endocrine Oncology Research Group, Department of Surgery, RCSI, Dublin 2

14 2. Data Science Centre, RCSI, Dublin 2

15 3. Department of Oncology, RCSI, Beaumont Hospital, Dublin 9

16 4. Department of Surgery, RCSI, Beaumont Hospital, Dublin 9

17 * Both authors contributed equally to this manuscript

18 The authors declare there have been no competing interests. 19 ‡Corresponding author: M. McIlroy ([email protected])

20 Endocrine Oncology Research Group.

21 Department of Surgery,

22 Royal College of Surgeons in Ireland,

23 St. Stephens Green,

24 Dublin 2,

25 Ireland.

26 Tel No: 0035314022286

27 Funding: Health Research Board (HRA-POR-2013-276) (MMcI) and BHCRDT (MMcI).

Downloaded from mct.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 9, 2019; DOI: 10.1158/1535-7163.MCT-18-0791 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

28 Abstract

29 Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor 30 (AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to 31 establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent 32 therapy.

33 This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cell 34 are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were 35 assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated 36 using ChIP and relevance to patient profiles was performed using publicly available datasets.

37 Whilst BEZ235 showed decreased viability across AI sensitive and resistant cell lines, anti-AR treatment elicited a 38 decrease in cell viability only in the AI resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and 39 cAMP-Dependent Protein Kinase Inhibitor β (PKIB), were confirmed to be regulated by 4AD and shown to be 40 mediated by AR; crucially re-exposure to estradiol suppressed expression of these genes. Meta-analysis of 41 transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine 42 therapy (HR=2.551, p=0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not 43 respond to AI therapy.

44 This study highlights the importance of the tumour steroid environment. SGK3 and PKIB are associated with poor 45 response to endocrine therapy and could have utility in tailoring therapeutic approaches.

46 Keywords

47 Androgen receptor, aromatase inhibitor, breast cancer, enzalutamide, SGK3.

56 Introduction

57 ER positive tumours account for approximately 75% of all breast cancer diagnoses [1]. The emergence of selective 58 estrogen receptor modulators (SERM) in the 1970’s proved to be a rubicon in the fight against breast cancer; with 59 all first line therapies thereafter focusing on estrogen driven ER activity [2]. Since the mid -2000s AIs have been 60 recommended as first line therapy for hormone receptor positive, post-menopausal breast cancers [3]. However, 61 the development of resistance to these drugs is a perennial problem with disease recurrence in ~30% of patients 62 [4]. Mechanisms of resistance to anti-estrogen therapy are multifaceted and include alterations in co-activator 63 recruitment [5], dominance of growth factor pathways [6], upregulation of aromatase [7], ER mutations [8] and 64 alterations in steroid handling in breast tumour epithelial cells [9].

65 The role of AR in breast cancer development and progression is somewhat mired in controversy with evidence 66 suggesting it can either antagonise or promote breast cancer depending on tumour context (reviewed[10]). In ER 67 positive breast cancer the general consensus is that AR protein is a positive prognostic indicator [11]. Conversely, 68 others have shown AR to take on the mantle of a pseudo ER, particularly in the setting of triple negative breast 69 cancer [12]. More recently, a potentiating role of AR in the development of endocrine resistance in ER positive 70 breast cancer has been emerging [13, 14]. Subsequently there is growing interest in targeting the AR with a 71 number of ongoing clinical trials assessing the utility of anti-AR drugs in the treatment of advanced breast cancer. 72 More recently, Aceto et al (2018) identified AR signalling to be activated in circulating tumour cells isolated from a 73 patient with breast to bone metastasis [15]. Nevertheless, when we consider that the majority of breast cancers 74 express AR protein, with some reports of >90% positivity [16], it makes understanding the dichotomous role of AR 75 all the more problematic. It is therefore imperative that we differentiate between AR functions which are 76 protective and those which are tumour promoting; whether this is dependent upon protein interactors, altered 77 steroid levels or mutations remains wholly unknown.

78 PI3K and mTOR signalling has been implicated in mechanisms of resistance to AI therapy in preclinical and clinical 79 trials [17]. In this context, AR in particular, has been established to play a prominent role in mediating PI3K and 80 mTOR signalling in a number of neoplasms [18]. AR mode of action in this setting is known to be quite disparate 81 from transcriptional steroid activity and may contribute to mechanisms of resistance to AI therapy. Indeed, non- 82 genomic, sex-nonspecific actions of both estrogen and androgens have been demonstrated to activate intracellular 83 signalling pathways [19]. Here, we show that AR protein levels are elevated in cell line models of letrozole 84 resistance. Significantly, due to the mechanism of action of AI therapy, the intracellular environment becomes 85 saturated with androgens, and will become largely estrogen depleted. The risk that this alteration in the steroid 86 environment may facilitate resistance is supported by clinical evidence which has shown that serum levels of the 87 direct precursor steroid, androstenedione (4AD), are elevated in patients progressing on AI therapy [20, 88 21]. Understanding individual tumour intracrinology will be critical to evaluating this clinically as it is known that 89 elevated levels of androgens (4AD and DHEA) are associated with breast cancer risk >2 years prior to cancer

90 detection. This suggests that hormone levels effect risk rather than hormone levels being altered by localized 91 steroid production [22]. 4AD is known to bind AR and can induce AR nuclear translocation in vitro, albeit with a 92 lower affinity than 5-Dihydrotestosterone (DHT) [23]. Herein, we report that this chronically altered androgenic 93 steroid environment enhances expression of SGK3 and PKIB transcripts which are both identified as AR/ER 94 regulated genes. SGK3 has previously been identified as a downstream target of both PI3K and AR in prostate 95 cancer [24] , highlighting it as a potential central mediator for both signalling pathways in AI resistant breast cancer 96 [25, 26]. This current study highlights the impact of steroid levels on transcriptional regulation and identified 97 SGK3, in particular as a potential indicator of poor response to AI therapy. As SGK proteins can be targeted 98 pharmacologically this pin-points SGK3 as a potential therapeutic target for ER positive breast cancer that may not 99 respond to conventional endocrine treatment. 100

101 Materials and Methods

102 Cell culture.

103 Cell lines were cultured as follows: endocrine-sensitive MCF7 were grown in DMEM (low glucose) with 10% of 104 fetal bovine serum (FBS) and 100 U penicillin/0.1 mg ml−1 streptomycin (Pen-strep) plus 10−8 M 17-β-estradiol 105 (Sigma E8875). MCF7 derived AI-sensitive cells (MCF7-Aro) were developed in-house and cultured in phenol red 106 free MEM (Sigma Aldrich, UK), 10% charcoal dextran stripped FCS, 1% Pen-Strep, 1% L-Glutamine and 200 µg/ml 107 G418 (Geneticin). MCF7-Aro derived letrozole-resistant cells (MCF7-Aro-LetR) were created by long-term 108 treatment of MCF7-Aro with letrozole and 4AD >3 months (Novartis, Basel, Switzerland) in charcoal dextran 109 stripped FCS, 1% Pen-Strep, 1% L-Glutamine, 200 µg/ml G418, 2.5-8 M 4AD and 10-6 M letrozole [27]). An obvious 110 morphologic change was noted in the transition of MCF7 cells to AI resistant MCF7aro-LetR with cells displaying a 111 large increase in cell surface area. To further assess the impact of the steroid environment on these cells they were 112 then maintained in medium supplemented with estradiol (10-8 M for 15 weeks) and designated as MCF7aro-LetR- 113 Est. After 15 weeks, MCF7aro-LetR-Est cells had reverted to smaller cell sizes with cobblestone morphology, as is 114 observed in MCF7 cells grown in estradiol.

115 ZR75.1 were included as an additional luminal A model that express high AR. They are known to have a PTEN 116 mutation and were cultured in MEM, 10% FCS, 1% Pen-Strep, 1% L-Glutamine [28]. ZR75.1-derived AI-sensitive 117 cells (ZR-Aro) were developed in-house by lentiviral transduction of ZR75.1. ZR-Aro-derived letrozole resistant cells 118 (ZR-Aro-LetR) were then generated by long term treatment of ZR-Aro with 4AD and letrozole >3months using the 119 same culture medium as above and are fully characterized (supplemental figure 2 A, B & C). All cells were 120 maintained in steroid depleted medium for 72 hours before treatment with steroids or drugs. All cells were

121 incubated at 37°C under 5% CO2 in a humidified incubator. In-house cells were authenticated and are routinely 122 verified as endocrine resistant, mycoplasma free and all cells are utilised within 10 passages for triplicate

123 experiments. The Aro-cell lines from which the AI-resistant cells are derived are generally not used as comparators 124 in these studies as it is well established that CYP19 amplification is a hallmark of endocrine resistance [29] and if 125 cultured in the absence of AI and steroid these cells are even less endocrine sensitive [30-32].

126 Western blotting

127 Cells were harvested, lysed, electrophoresed, and immunoblotted with specific primary antibodies and 128 corresponding horseradish peroxidase-conjugated secondary antibody (Dako, Den). See supplemental materials 129 and methods for antibody detail.

130 MTS assay

131 For drug treatments, MCF7 and MCF7-Aro-LetR cells were steroid-depleted for 72 hours. MCF7 cells were plated in 132 steroid depleted media supplemented with estradiol 10-8 M to enable cell growth and MCF7-Aro-LetR were seeded 133 in steroid depleted media prior to the addition of BEZ235 (Selleck Chemicals) as per optimal dose concentration, 134 enzalutamide (10µM) (Selleck chemicals) or DMSO vehicle (Sigma Aldrich). Treatments were replenished every 3 135 days. Colorimetric outputs were analyzed by measuring the absorbance at 490nm using a spectrophotometer 136 (Perkin Elmer, USA). Prior to transfection MCF7-Aro-LetR and ZR-Aro-LetR cells were steroid-depleted for 72 137 hours. Cells were transfected with siRNA against SGK3 (L-004162-00, Dharmacon, Colorado, USA) or siRNA 138 negative control (d-001206-13-05) and seeded in a 96-well plate prior to the addition of 4AD (10-7 M). Two-way 139 ANOVA with Bonferroni post-test was used for statistical analysis of results for treatment 1- 3 day. Student’s t-test 140 (two-tailed) was used to compare means for siRNA experiments.

141 Colony forming assay

142 Assays were performed as per previously described [14].

143 RNA extraction, library preparation and RNA-sequencing

144 To assess the transcriptional effects of 4AD in endocrine resistant breast cancer cells, RNA-sequencing was 145 performed using MCF7-Aro-LetR cells which were steroid depleted for 72 hours and treated with either 4AD (10-7 146 M) or vehicle in the presence of letrozole 10-6 M in triplicate. RNA was extracted using an RNeasy Kit (Qiagen, 147 Hilden, Germany). Sequencing was performed on an Illumina HiSeq technology (minimum 10 million clean reads 148 of the RNA-quantification/sample). Three independent biological libraries were prepared for each sample to 149 facilitate the expression detection and variance estimation.

150 Transcriptomic analysis (RNA-seq analysis and microarray analysis)

151 RNA seq pre-processing: see supplemental material and methods.

152 Microarray pre-processing: see supplemental material and methods.

153 For both transcriptomic experiments differential expression was determined using the ebayes function of the R 154 package Limma [33]. In each case a binary comparison was performed between 4AD treated cells and controls. In 155 the case of the microarray experiment the following samples were included, GSM1016474, GSM1016475 and 156 GSM1016476 (controls) and GSM1016486, GSM1016487 and GSM1016488 (treated). A p-value of less than 0.05 157 and a fold change greater than 1.4 fold was considered significant. The package Limma was chosen here for 158 differential expression analysis as it is particularly robust when dealing with small sample sizes in both microarray 159 and RNA-seq experiments [34]. These two lists of differentially regulated genes were overlapped with each other 160 and data from a publically available list of genes associated with acquired endocrine therapy resistance in breast 161 tumours expressing ESR1 but not ERBB2 [35]. The original authors referred to this as their “group 4 set” of genes 162 which exhibited strong hormone responsiveness. A detailed description of how it was generated can be found in 163 the original manuscript. This data was used to generate a candidate genes list for further investigation. All 164 calculations were carried out in the R statistical environment (https://cran.r-project.org/).

165

166 RNA-seq validation

167 A panel of breast cancer cell lines (MCF7, MCF7-Aro-LetR, MCF7-Aro-LetR-Est, ZR75.1 and ZRaro-LetR) were steroid 168 depleted for 72 hrs followed by treatment with 4AD (10-7 M) and ethanol vehicle (0.0001% (v/v)) for 24hr. 169 Letrozole 10-6 M was also added to AI resistant cells lines. RNA was extracted using an RNeasy Kit (Qiagen, Hilden, 170 Germany). cDNA synthesis was performed using Superscript III First Strand Synthesis System (Life Sciences). See 171 supplemental material and methods for primer details.

172 MassArray analysis

173 See supplemental material and methods.

174 siRNA transfection

175 AR, ESR1 and SGK3 were silenced by transient transfection using experimentally verified pools of siRNA. AR was 176 silenced using siRNA pools SMARTpool (catalog number L-003400-00) (30 nM), SGK3 SMARTpool (catalog number 177 L-004162-00) (30 nM) and ESR1 SMARTpool (catalog number L-003401-00-0005) (30 nM), all purchased from 178 Dharmacon. All transfections were carried out using Lipofectamine 2000 transfection reagent according to 179 manufacturer’s instructions (Invitrogen, UK) and a non-targeting siRNA pool negative control (Dharmacon) was 180 used as a control for all siRNA experiments. Confirmation of mRNA knockdown was performed using primers for 181 AR (F: 5’-AGCGACTTCACCGCACCTCA-3’, R: 5’-CAGTCTCCAAACGCATGTCCCCG-3’), ESR1 (F: 5’

182 TGTACCTGGACAGCAGCAAG-3’, R: 5’-TCTCCAGGTAGTAGGGCACC-3’) 24 hours post transfection. siRNA efficacy 183 was also confirmed at protein level (Supplemental Figure 2D&E).

184 Chromatin immunoprecipitation.

185 MCF7-Aro-LetR cells were treated with 4AD 10-7 M (30 mins), estradiol 10-8 M (45 mins) or ethanol vehicle. ChIP 186 was performed as previously described [27]. Rabbit anti-AR (3 ug, sc-816, SCBT), anti-ESR1 (6 ug, SC-543, SCBT) or 187 mouse IgG (6 ug)/rabbit (3 ug) (Dako) was added to the supernatant fraction and incubated overnight at 4°C with 188 rotation. Proteins were un-crosslinked, DNA extracted and specific primers used to amplify the DNA of the SGK3 189 proximal promoter. SGK3 proximal promoter primers: Forward 5’-GACTTGTGTAACATGGTCTCTTTCA-3’ and reverse 190 5’-CAAGTTCAATCTGACCCTCATATCT-3’ [24].

191 Meta-analysis of SGK3 mRNA expression and breast cancer patient survival.

192 BreastMark [36] is an algorithm that enables the identification of subsets of gene transcript/miRNAs that are 193 associated with disease progression and survival in breast cancer and its subtypes. Using this resource the 194 association of expression levels of SGK3 mRNA and survival were evaluated in endocrine-treated/untreated 195 datasets.

196 Evaluation of SGK3 in clinical cohorts and responsiveness to endocrine therapy.

197 Series GSE59515 (Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer)[37] was 198 used to assess mRNA expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post- 199 menopausal breast cancer patients (n=25). USCS Xena browser (https://xenabrowser.net/heatmap/) was used to 200 interrogate TCGA Breast cancer datasets filtered to focus analysis on pre and post-menopausal, endocrine therapy 201 treated patients (n=415). Copy number variation for SGK3 was evaluated and cohorts were stratified by masked 202 copy number repeat to eliminate sex chromosome and germ-line artifacts. Kaplan Meier graphs (quartiles) were 203 generated from these data to ascertain association of copy number amplification and overall survival.

204 RNA extraction from FFPE tissues

205 Breast cancer patients (n=6) who were responsive (n=3) or non-responsive (n=3) to AI therapy were selected. 206 Informed written consent was received from all patients, and the study was approved by institutional review board 207 Royal College of Surgeons in Ireland (CTI09/07). Hematoxylin and eosin stained sections of formalin fixed, paraffin 208 embedded (FFPE) tumor tissues were analyzed by a pathologist for histological and tumor cellularity classifications. 209 Tumor content was annotated on sections and RNA was extracted from these areas using Qiagen AllPrep DNA/RNA 210 FFPE kit according to manufacturer instructions.

211 Statistical analysis

212 A two-tailed Student’s t test was used to compare means. Two-way ANOVA with Bonferroni post-test was used to 213 compare replicate means. Dose response curves were normalized to 100% and 0% viability based on the lowest 214 and highest drug concentrations (GraphPad Prism v8).

215 Data accession code

216 RNA-seq data are available at the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/), provisional 217 accession code: SRP148035.

218

219 Results

220 AI-sensitive MCF7 and AI-resistant MCF7Aro-LetR cells exhibit decreased cell viability when treated with BEZ235, 221 however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR therapy. Estradiol 222 dependent parental MCF7 cells were found to harbor a PIK3CA E545K missense mutation (Figure 1A and B (i)) 223 which is a known hotspot mutation in breast cancer [38]. Analysis of the letrozole resistant MCF7 derivative cell- 224 line (MCF7-Aro-LetR) confirmed they retained the same mutation (Figure 1A and B (ii)). The cell lines exhibited 225 varied levels of AR protein expression with low amounts in the MCF7 and higher levels in the MCF7-Aro-LetR 226 (Figure 1C and supplemental Figure 1A (i)). This was mirrored in the ZR75.1 letrozole resistant derivative cell line 227 (ZR75aro-LetR) when compared to their parental cell line; ER protein levels did not vary across the panel of cell 228 lines (Figure 1C and supplemental Figure 1A (ii)). Dose response analysis was used to establish sensitivity to 229 PI3K/mTOR inhibition by the pan class inhibitor BEZ235 (Sellekchem) and to a αδ PI3K inhibitor, Pictilisib 230 (Sellekchem) (Supplemental Figure 1B (i-iv) & C (i-ii)).

231 Endocrine sensitive (MCF7) and AI resistant cells (MCF7-Aro-LetR) were treated with BEZ235 or enzalutamide (anti- 232 AR) as single or combination treatment followed by an evaluation of cell viability. BEZ235 treatment significantly 233 decreased cell viability in all cell lines tested compared to vehicle (Figure 1D and 1E). Of note, the AI-resistant 234 MCF7-Aro-LetR cells showed a significant decrease in levels of cell viability following enzalutamide treatment 235 (Figure 1E), this was in contrast to the parental MCF7 cells, in which viability was sustained when AR was targeted 236 compared to vehicle (Figure 1D). Combined BEZ235 and enzalutamide treatment resulted in a significant decrease 237 in viability across all cell lines, however, this effect was most likely carried by BEZ235 (Figure 1D and 1E). This 238 result was mirrored by colony forming assays suggesting that it is cell proliferation that is being affected (Figure 1F 239 (i-ii)).

240 4AD upregulates genes associated with steroid and PI3K signalling.

241 Transcriptomic analysis enabled the identification of genes differentially regulated when AI resistant cells are 242 exposed to 4AD in the presence of letrozole to recapitulate the steroid environment under AI therapy

243 (Supplemental table 1, RNAseq Data). This in-house generated gene list was further supported by comparison with 244 genes identified in a publically available dataset of MCF7-Aro cell treated with 4AD without AI. This helped define 245 gene expression changes attributed solely to the effects of 4AD and provided evidence of putative 4AD specific 246 regulation in AI-resistance. Subsequent comparison with the ‘Group 4’ genes associated with ER+, HER2- endocrine 247 resistance (GSE8140, [35]) yielded a subset of 8 genes (Figure 2A). As using data from independently published 248 datasets introduces confounding factors the genes identified were validated in-house in two genetically disparate 249 AI resistant models.

250 Chronic adaptation to the steroid environment induces a unique transcriptome that can be reverted by re- 251 introduction of estradiol.

252 Validation of the transcriptomic analysis was performed by treating endocrine sensitive MCF7 and ZR75.1 cells and 253 their endocrine resistant derivatives (MCF7-Aro-LetR and ZR-Aro-LetR cells) with 4AD and investigating the impact 254 on the transcript levels of the top 4 genes in our list: growth regulation by estrogen in breast cancer 1 (GREB1), 255 serum /glucocorticoid regulated kinase family member 3 (SGK3), cAMP-dependant kinase inhibitor β (PKIB) and 256 MYB proto-oncogene like 1 (MYBL1). Each of these genes validated in both AI-resistant cell lines, MCF7-Aro-LetR 257 and ZR-Aro-LetR, with significant increases in GREB1, SGK3, PKIB and MYBL1 in response to 4AD treatment (Figure 258 2B (i-viii)). GREB1, SGK3, PKIB and MYBL1 did not increase in response to 4AD treatment in endocrine sensitive 259 MCF7 cells and no change was observed for 3 out of 4 of these genes in endocrine sensitive ZR75.1 cells with the 260 exception of PKIB which demonstrated a very modest increase (Figure 2C (i-viii)). Overexpression of the aromatase 261 gene was used to generate both models of AI resistance and has also been a feature observed in the development 262 of AI resistance in the clinic. In order to address whether CYP19A overexpression was a driving factor in the 263 increased expression of SGK3, PKIB, MYBL1 and GREB1 a series of experiments using MCF7 cells overexpressing 264 aromatase and cultured in the presence of 4AD were performed. Data from these experiments confirmed that 265 aromatase overexpression alone did not induce increased levels of these genes (Supplemental figure 3A (i-iv)). 266 Further evidence that aromatase overexpression is inconsequential for these genes is provided via analysis of GSE 267 10911 [39] and from TCGA cohorts (Supplemental figure 3B and supplemental figure 6). The former study focused 268 on MCF7-Aro cells cultured in the absence of steroid, with testosterone (T) or with T plus AI. SGK3 levels were 269 increased in the MCF7aro plus T compared to steroid unstimulated cells, most interestingly, there was a further 270 significant increase in SGK3 mRNA in cells cultured with T and AI. Reversion of the MCF7-Aro-LetR transcriptional 271 response to 4AD could be achieved by culturing MCF7-Aro-LetR in estradiol for 15 weeks. Cell growth initially 272 decreased with notable alteration in cell morphology to the cobblestone appearance of estradiol dependent MCF7 273 and smaller cell size (see Figure 6A for illustrative images). Treatment of these cells with 4AD (24 hours) produced 274 very minimal effects on gene expression in comparison to those observed in the MCF7-Aro-LetR from which they 275 were derived (Figure 3A (i-iv), 3B & 6A).

276 Androstenedione stimulated induction of target gene mRNA is reduced post siRNA knockdown of either AR or 277 ER; both AR and ER are recruited to a common region on each gene promoter.

278 As it has been established that many transcripts are under cooperative AR:ER regulation [13] we then wished to 279 establish whether the genes identified in this current study were regulated by ER and/or AR. To determine if AR 280 and/or ER are integral to the 4AD mediated upregulation of GREB1, SGK3, PKIB and MYBL1, siRNA inhibition of AR 281 and ER was performed, and siRNA efficacy was confirmed (supplemental Figure 2D (i-ii) & 2E (i-ii)). The impact of 282 knockdown of nuclear receptors (NRs) on target gene expression in MCF7-Aro-LetR cells was evaluated. siRNA 283 knockdown of AR combined with 4AD treatment resulted in a significant decrease in SGK3 and PKIB transcript 284 levels but had no significant impact on GREB1 or MYBL1 (Figure 4A (i-iv)). siRNA knockdown of ER combined with 285 4AD treatment resulted in significantly reduced levels of all four targets (Figure 4B (i-iv)). Regulation of SGK3 286 protein by AR and ER was then confirmed at the level of protein expression (supplemental figure 3C (i-ii)). AR and 287 ER recruitment to these target genes was then evaluated in a publically available dataset (GSE104399) [40]. 288 Although primarily a male breast cancer dataset it contains ChIP-seq data for both AR and ER recruitment in a 289 female breast cancer patient (patient 8: ER+, PR+, HER2-, post-menopausal) (Figure 4C, upper panels). Using the 290 ChIP-seq peak information published by the original authors we were able to confirm the binding of ER to the 291 proximal promoter of SGK3, GREB1, MYBL and PKIB and AR to GREB1 and PKIB in this patient sample (see 292 supplemental table 2 and supplemental figure 4 for additional detail for binding locations). Enhanced AR 293 recruitment and ER occupancy of the SGK3 promoter was confirmed using ChIP in 4AD stimulated MCF7-Aro-LetR 294 (Figure 4C and 4D). Whilst this analysis was limited to one patient, it does suggest that AR and ER bind at regions 295 enriched with ESR1 motifs. ER ChIP-seq data for the MCF7-Aro-LetR confirmed ER recruitment to the proximal 296 promoters of all 4 genes in the presence of 4AD plus letrozole (Figure 4C, lower panel; supplemental table 2 and 297 supplemental figure 4).

298

299 Knockdown of SGK3 inhibits AI resistant cell viability in the presence of androstenedione or estradiol.

300 SGK3 has previously been documented as a downstream target of AR and ER (α and β isoforms) in prostate and 301 ERα in breast cancer [24, 25] and is also associated with stabilization of endoplasmic reticulum stress. Here, we 302 have shown that SGK3 mRNA and protein levels are significantly increased in MCF7-Aro-LetR cultured in the 303 presence of 4AD (Figure 5A (i-ii)). Treatment with BEZ235 or a combination of the anti-AR therapy, bicalutamide, 304 with BEZ235 decreased SGK3 mRNA expression (Figure 5B). We found that when SGK3 levels are abrogated by 305 siRNA targeting SGK3 (Figure 5C (i-iii)) in the absence of steroid there was no change in cell viability (Figure 5D (i)), 306 in contrast, when SGK3 was targeted for degradation in the presence of 4AD there was a significant decrease in cell 307 viability (Figure 5D (ii)). This result was also reflected in cell counts of MCF7-Aro-LetR cells exposed to siRNA 308 targeting SGK3 (Figure 5D (iii)). Further validation was carried out in ZR-Aro-LetR cells (Supplemental figure 5A (i-

309 iii)) Follow-up siSGK3 experiments using a variety of steroids demonstrated that decreased viability is only evident 310 when cells are exposed to 4AD or estradiol but there is no alteration observed when cells are cultured in the 311 potent androgen, R1881, or the control, cholesterol (Supplemental figure 5B).

312 SGK3 and PKIB are associated with poor recurrence free survival (RFS) in the post-menopausal, endocrine 313 treated breast cancer patient population (ER+ PR+). 314 In vitro data presented has shown the impact of the steroid microenvironment upon target gene expression 315 identified by RNA-seq (schematic overview: Figure 6A). In post-menopausal women 100% of sex hormones are 316 synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 317 levels of 4AD to be elevated in patients whose disease progresses on AI therapy [19, 20] and more recent data has 318 shown 4AD dominates breast tumour intracrinology [41]. In contrast serum and tissue levels of estradiol are 319 markedly reduced in patients treated with an aromatase inhibitor [3]. Our study identified SGK3 and PKIB as 4AD 320 regulated transcripts mediated in part by AR in collaboration with ER. This supports the hypothesis that 321 transcriptional reprogramming as a result of the steroid environment can facilitate resistance to AI therapy 322 (summarized: Figure 6B). The BreastMark meta-analysis resource was used to evaluate the impact of SGK3 and 323 PKIB transcript levels on endocrine treated breast cancer patient outcome [36]. The high expression group in each 324 plot (blue) accounts for the upper quartile of expression levels for a particular transcript and the low expression 325 group (red) the remaining 75%. This was applied to each of the individual datasets and the information was then 326 combined to perform a global pooled survival analysis. Meta-analysis of the AR mediated genes, SGK3 and PKIB, 327 showed that these genes have no impact on RFS in treatment naïve, postmenopausal ER positive breast cancer, 328 however, when we evaluate their impact in an endocrine treated population it is clear that patients with high 329 levels of these transcripts do not benefit from endocrine therapy (Figure 6C (i-ii)).

330 SGK3 levels are sustained in breast cancer patients who do not respond to AI therapy.

331 To evaluate these observations further, SGK3 levels were assessed in a clinical cohort of breast cancer patients 332 defined as responsive or non-responsive to AI therapy. Data Series GSE59515 [37] was used to assess mRNA 333 expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post-menopausal breast 334 cancer patients (n=25). SGK3 levels are lower in responsive patients post therapy (drop in mean expression: 16.75 335  0.563, one-tailed t-test: p=0.0015 ) (Figure 7A(i)). Conversely, SGK3 mRNA in non-responders is somewhat 336 sustained (drop in mean expression 16.64  6.44, one-tailed t-test: not significant) (Figure 7A(ii)). This was 337 mirrored by levels of AR mRNA with a significant drop in responders (one-tailed t-test: p=0.0196) compared with 338 sustained levels in non-responders to AI therapy (one-tailed t-test: not significant) (Figure 7B (i-ii)). These data 339 were validated in a second cohort of AI responders and non-responders (n=6). SGK3 mRNA was only detectable in 340 post-menopausal non-responders (Figure 7A(iii), a result that is mirrored by the levels of AR mRNA (Figure 7B(iii). 341 Of note, the only patient non-responsive to AI with no detectable SGK3 or AR transcript was pre-menopausal; 342 highlighting the post-menopausal hormone state as being crucial to the expression of these target genes. Whilst

343 exploring the expression of SGK3 transcript in clinical cohorts, it was noted that there is a large percentage (16%) 344 of breast cancer patients with an alteration in the SGK3 gene. The impact of SGK3 copy number amplification was 345 then evaluated in post-menopausal, breast cancer patients treated with endocrine therapy (n=132). Kaplan Meier 346 survival curves showed that copy number amplification of SGK3 significantly associates with poor survival in the 347 post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort yielded no association, 348 however, it should be noted that numbers in this cohort were <50 (Figure 7C).

349

350 Discussion

351 PI3K signalling as a driver of endocrine resistance in ER positive breast cancer has been validated in many previous 352 studies and clinical trials [17, 42]. Given that the combination of the αδ-PI3Kinase inhibitor pictilisib plus 353 fulvestrant does not improve survival in AI resistant breast cancer, suggests that the PI3K pathway alone or in 354 combination with ER is not the sole driver of AI resistance [43]. Our cell line models of AI resistant breast cancer 355 are cultured in letrozole and 4AD which results in elevation of AR protein levels in contrast to parental cell lines. In 356 light of the abundance of data from the field of prostate cancer which has elucidated mechanisms by which AR can 357 act as a mediator of second messenger signalling, a potentiating role for AR in our model of AI resistance was 358 evaluated [44]. In this study we have utilised transcriptomic analysis to elucidate gene expression changes 359 associated with resistance to AI therapy.

360 AI therapy creates a unique androgenic environment that permits exploration of steroid drivers of resistance that 361 may not be wholly dependent upon a functional ER; this is exemplified clinically by the similar overall survival 362 achieved when AI or selective estrogen receptor degrader (SERD) are used as second line therapy after recurrence 363 on AI [45]. AR is normally associated with good patient outcome [11, 16] and the pro-survival response observed 364 in the enzalutamide treated MCF7 cells in this study would fully support this role. However, the strongly opposing 365 action of the same drug in the isogenic MCF7-Aro-LetR cells highlights the importance of the steroid 366 microenvironment in directing the action of AR. As we have observed contrasting impact on cell growth with anti- 367 AR therapy we may presume the response is heavily influenced by the steroid driver (estradiol in MCF7 and 368 androstenedione in MCF7-Aro-LetR). Our study is not without limitations as the cell line models used are 369 engineered to overexpress CYP19 (amplification of which is known to be associated with AI resistance [29]); 370 however, control experiments clearly show that aromatase overexpression in this system does not increase 371 expression of the 4AD target genes. Furthermore, analyses of clinical TCGA datasets show no association of CYP19 372 amplification and SGK3 expression, suggesting that this is a disparate mechanism of resistance. It is therefore 373 plausible that estrogen depletion as a consequence of AI therapy may be permissive of AR mediated 374 transcriptomic alterations.

375 Elevated levels of sex steroids and their prohormones have long been associated with increased breast cancer risk 376 primarily via epidemiological and clinical observation [20, 46]. In this study we have focused on the effects of 4AD 377 arising from the adrenal gland and ovarial interstitial cells which acts as the direct sex-steroid precursor for both 378 androgens and estrogens [47, 48]. Disappointingly , clinical trials of the CYP17 inhibitor, AA (abiraterone), alone or 379 in combination with AI have shown no difference in progression free survival between modalities; results that are 380 perhaps confounded by an associated rise in progesterone and the potential of AA to act as an ER agonist in breast 381 cancer [49, 50]. It is also plausible that 4AD will be metabolized to other steroids such as 5α-androstanedione or 382 3β-androstanediol, themselves known to act as drivers of breast cancer proliferation [51]. Interestingly, a 383 proliferative role of androstene-3b,17b-diol has recently been suggested for ER positive breast cancer with low 384 intratumoural estradiol levels [52]. Whilst weaker sex steroids are incapable of inducing the conformational 385 change required for classical NR genomic action, they and/or their metabolites may play a role in driving non- 386 genomic activity. Indeed, the sheer overabundance of weaker steroids and the formation of transient nuclear 387 receptor (NR) associations may well be able to initiate second messenger signalling which is not dependent upon 388 low disassociation constants [19]. It has often been reported that male and female sex steroids are associated 389 with both ER positive and ER negative breast cancers, highlighting the importance of the steroid drivers which may 390 be acting independently of the classical ER action in some tumours [46].

391 As the majority of breast cancers express AR it is of interest to understand how the estradiol depleted steroid 392 environment, which accompanies AI therapy, modifies AR action. Many studies have focused on the 393 transcriptomic role of AR in this setting with much of the data indicating a co-operative AR:ER dynamic [13, 40]. 394 Indeed from the RNA sequencing data in our current study the main network of genes altered in response to 4AD 395 treatment were also found to be transcriptionally regulated by ER alpha. Of note, SGK3 and PKIB were also 396 confirmed to be regulated, in part, by AR. Recruitment of both NRs to regions harboring classical EREs in the 397 proximal promoter region was further confirmed in ChIP sequencing data from a female breast cancer patient in 398 the Severson study [40]. Importantly we have established that SGK3 is indeed a steroid regulated, AR target gene 399 and that its expression is enhanced when estrogen synthesis is inhibited in models of AI resistance.

400 The most interesting observation made in this study was the shift in gene expression induced by 4AD which was 401 shown, not only in AI resistant cells derived from MCF7, but also those generated from ZR75.1. This was in stark 402 contrast to the unresponsive parental cell lines, suggesting that chronic alterations in steroid bioavailability can 403 induce transcriptomic reprogramming. This was further supported by data from the long-term estradiol treated 404 MCF7-Aro-LetR, whose gene expression response to 4AD was completely reverted to reflect that of estradiol 405 dependent MCF7. SGK3, in particular, has recently been associated with poor response to AI therapy in breast 406 cancer and has been demonstrated to play a role in stabilization of the endoplasmic reticulum during cell stress 407 [25]. SGK3 has a high degree of similarity to AKT with both kinases targeting many of the same substrates. The SGK 408 family has been implicated in breast cancer resistance to PI3K inhibition in numerous in vitro studies and in a

409 clinical trial [26]; of note SGK3 can be activated independently of Class I PI3K [53]. In our study, reduction in SGK3 410 transcript resulted in a loss of cell viability but only in the presence of 4AD or estradiol. This mirrors the finding 411 reported by Wang et al (2017), wherein they observed complete loss in viability when SGK3 is depleted; the more 412 pronounced effect reported was likely due to the increased efficacy of knockdown achieved and also because of 413 the different steroid environments (testosterone versus 4AD) [25].

414 To understand the clinical relevance of these findings, a meta-analysis of the AR mediated genes, SGK3 and PKIB, 415 was performed. This analysis showed that these genes have no impact on RFS in treatment naïve, 416 postmenopausal, ER positive breast cancer, however, when we evaluate their impact in an endocrine treated 417 population it is clear that patients with high levels of these transcripts do not benefit from endocrine therapy. 418 When this was evaluated in AI responsive versus non-responsive patients it was clear that levels of SGK3 and AR 419 mRNA are sustained in patients failing on therapy. It was also noted that many breast cancer patients have 420 elevation of SGK3 copy number; whether this is as a consequence of an altered steroid microenvironment is an 421 intriguing possibility that is yet to be determined. We would purport, from these observations, that AI treated 422 breast cancers adapt to utilize bioavailable steroids such as those of adrenal origin. As highlighted in this study, 423 evaluating the implications of steroid alterations in the clinical management of disease is warranted. 424 425 Acknowledgements: Author contributions: LC carried out RNA-seq sample preparation, EC50 assays, MTS viability 426 assays, ChIP, qRT-PCR, participated in study design, analysed results and assisted in manuscript preparation. RB 427 assisted in validation of RNA-seq, MTS assays, western blotting, analysed results and generated graphics and 428 assisted in manuscript preparation. SM carried out bioinformatic analysis, assisted in the study design and 429 manuscript preparation. ST carried out MassArray analysis of cell line PI3K mutational status, assisted with study 430 design and preparation of manuscript. DV generated the ZR-Aro-LetR cell line, conducted the related motility and 431 PS2 expression analysis and assisted with preparation of manuscript. ADKH identified appropriate clinical samples, 432 prepared samples for analysis and assisted with study design. LY contributed to data analysis and manuscript 433 preparation. MMcI conceived study design, performed siRNA experiments, data analysis, generated graphics and 434 wrote the manuscript. All authors read and approved the final version of the manuscript. Many thanks to Dr 435 Katherine Sheehan, Tony O’Grady and Joanna Fay (Department of Pathology), Lance Hudson and Aisling Hegarty 436 for their guidance and help with sample preparation. Funding: Health Research Board (HRA-POR-2013-276) 437 (MMcI) and BHCRDT (MMcI).

438 Availability of data: The datasets generated and/or analyzed during the current study are available in the Gene 439 Expression Omnibus repository, [Accession code: SRP148035].

440 Ethics approval and informed written consent to participate/ consent for publication – CTI 09/07

441

442

443

444 Abbreviations

445 androgen receptor (AR)

446 aromatase inhibitor (AI)

447 estrogen receptor (ER)

448 androstenedione (4AD)

449 dehydroepiandrosterone (DHEA)

450 5-Dihydrotestosterone (DHT)

451 tissue microarray (TMA)

452 serum and glucocorticoid-regulated kinase 3 (SGK3)

453 cAMP-Dependent Protein Kinase Inhibitor β (PKIB)

454 selective estrogen receptor modulators (SERM)

455 growth regulation by estrogen in breast cancer 1 (GREB1)

456 MYB proto-oncogene like 1 (MYBL1)

457 nuclear receptors (NRs)

458 chromatin immunoprecipitation (ChIP)

459 formalin fixed paraffin embedded (FFPE)

460 recurrence free survival (RFS)

461 progression free survival (PFS)

462 abiraterone (AA)

463 testosterone (T)

464

465

466

467 References

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646 Figure legends.

647 Figure 1: AI-sensitive MCF7 and AI-resistant MCF7-Aro-LetR cells exhibit decreased cell viability when treated 648 with BEZ235, however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR 649 therapy. A. Tabulated details on two sets of paired isogenic breast cancer cells lines (endocrine sensitive MCF7, 650 ZR75.1 and their AI-resistant derivatives MCF7-Aro-LetR and ZR-Aro-LetR) including luminal type, AR protein 651 expression and mutations impacting PI3K signalling. B. (i) Traces from the Sequenom MassArray show that 652 endocrine sensitive MCF7 cells have a heterozygous PIK3CA E545K mutation and (ii) Endocrine resistant MCF7-Aro- 653 LetR cells have a heterozygous PIK3CA E545K mutation. C. Western blot analysis was used to evaluate AR and ER 654 expression across five breast cancer cell lines (MCF7, MCF7-Aro-LetR, ZR75.1 and ZR-Aro-LetR) D & E. The effect of 655 pan class PI3K/mTOR inhibitor (BEZ235) and anti-AR (enzalutamide) on cell viability was assessed using MTS and 656 colony formation assay in endocrine sensitive and endocrine resistant cells. D MTS of MCF7 response to BEZ235 or 657 to a combination of BEZ235 plus enzalutamide compared to vehicle control. F (i) Colony formation assays reflect 658 MTS results in MCF7 cells. E MTS of MCF7-Aro-LetR cells in response to single agents BEZ235 and enzalutamide and 659 also to a combination treatment. F (ii) Colony formation assays reflect MTS results in MCF7-Aro-LetR cells. Graphs 660 representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). Two-way 661 ANOVA with Bonferroni post-test to compare replicate means was used in MTS day 1 to day 9 readings to 662 determine significance. Student’s paired, 2-tailed t-test established significance to compare means * p<0.05, ** 663 p<0.01, *** p<0.001.

664 Figure 2. 4AD upregulates genes associated with steroid and PI3K signalling in two models of AI resistance but 665 not in the parental cell lines. A. RNA sequencing was performed following androstenedione (4AD 10-7M) 666 treatment plus letrozole for 24 hrs in AI resistant MCF7-Aro-LetR cells versus vehicle. This data was then 667 compared with array data for MCF7aro treated with 4AD versus vehicle to identify 4AD specific transcripts. These 668 genes were then integrated with the ‘Group 4 ‘ gene set of endocrine resistant genes associated with ER+, HER2 – 669 ve disease yielding a list of 8 genes. B. (i-viii) qRT-PCR validated differentially expressed genes (GREB1, SGK3, PKIB 670 and MYBL1) in AI resistant cell models MCF7-Aro-LetR and ZR-Aro-LetR cells. C. (i-viii) qRT-PCR showed expression 671 of these genes in parental MCF7 and ZR75.1 cells on exposure to 4AD . Graphs representative of n=3. Error bars 672 are representative of mean ± standard error of the mean (SEM). Student’s paired, 2-tailed t-test established 673 significance * p<0.05, ** p<0.01, *** p<0.001. ¶ [32], $ [36].

674 Figure 3. Culturing AI resistant cells (MCF7-Aro-LetR) in estradiol for 15 weeks reverts 4AD driven gene 675 expression. A. (i) & (ii) qRT-PCR of SGK3 mRNA displays a very modest increase, whereas GREB1 is significantly 676 decreased compared to vehicle control. A (iii) & (iv) PKIB and MYBL1 expression is unchanged in the presence of 677 4AD. Graphs representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). B. 678 Gene expression (SGK3, GREB1, PKIB & MYBL1) after 24 hrs 10-7M 4AD treatment in MCF7-Aro-LetR cells long 679 term cultured in 4AD compared to MCF7-Aro-LetR cells long term cultured in 10-8M estradiol (MCF7aroLetR-Est). 680 Student’s paired, 2-tailed t-test was carried out to compared individual gene expression across both cell lines.

681 Figure 4: Androstenedione stimulated transcript levels of SGK3 and PKIB are reduced in response to siRNA 682 knockdown of either AR or ESR1; in addition, AR and/or ER are recruited to these target genes in vivo and in 683 vitro. A. qRT-PCR of MCF7-Aro-LetR transfected with siRNA-AR treated with 4AD shows a significant decrease in 684 SGK3 and PKIB transcript levels (i-ii) but does not impact transcript levels of GREB1 or MYBL1 (iii-iv). B. qRT-PCR of 685 MCF7-Aro-LetR transfected with siRNA–ESR1 in MCF7-Aro-LetR cells treated with 4AD results in a significant 686 decrease in transcript levels of the SGK3, GREB1, PKIB and MYBL1 (i-iv). C. Recruitment of AR and ER to target 687 gene promoters was evaluated using publically available data from a breast cancer patient ChIP-sequencing study, 688 and ER recruitment in the MCF7-Aro-LetR cell line via ChIP-sequencing. ARϕ recruitment to the SGK3 and MYBL1 689 promoter was validated via ChIP in MCF7-Aro-LetR D. (i) Validation of the putative AR target gene SGK3 was 690 confirmed by performing ChIP to determine recruitment of AR to the promoter of SGK3 in MCF7-Aro-LetR cells in 691 the presence of 4AD. (ii) ChIP experiments were performed to determine recruitment of ER to the promoter of 692 SGK3 in MCF7-Aro-LetR cells treated with 4AD. All graphs representative of three experimental replicates. Error 693 bars are representative of mean ± standard error of the mean (SEM) of n=3. Student’s paired, 2-tailed t-test 694 established significance * p<0.05, ** p<0.01, *** p<0.001.

695 Figure 5: BEZ235 or combination of BEZ235 with the anti-AR Bicalutamide decreases SGK3 mRNA expression. 696 Knockdown of SGK3 inhibits AI resistant cell proliferation only in the presence of androstenedione. A (i) SGK3 697 protein expression is increased in the presence of 10-7M 4AD in MCF7-Aro-LetR cells. (ii) Densitometry analysis of 3 698 independent western blots confirmed significant increase in SGK3 protein expression with 4AD treatment. B. qRT- 699 PCR evaluation of SGK3 mRNA expression with BEZ235 +/- anti-AR therapy bicalutamide in MCF7-Aro-LetR cells. C 700 (i) Western blot of SGK3 protein levels following siSGK3in MCF7-Aro-LetR. (iI) Densitometry analysis shows SGK3 701 protein levels following siSGK3. (iii) qRT-PCR of SGK3 mRNA following siSGK3 in MCF7aro-LetR D (i) MTS of 702 MCF7Aro-LetR cell viability following knockdown of SGK3 in the absence of steroid. (ii) MTS assay of MCF7aro-LetR 703 cell viability following siSGK3 combined with 4AD treatment . (iii) Cell counts confirmed decreased cell viability in 704 MCF7Aro-LetR cells following siSGK3. Graphs representative of n=3. Error bars are representative of mean ± 705 standard error of the mean (SEM). Student’s 2-tailed t-test established significance for A, B and D.

706 Figure 6: SGK3, PKIB and GREB1 are 4AD regulated transcripts mediated in part by AR in collaboration with ER. 707 Associated outcome in clinical cohorts highlights a significant impact on therapeutic response to endocrine

708 therapy. A Schematic overview of key study findings highlight the morphologic and transcriptional changes driven 709 by chronic exposure of MCF7 to 4AD in the presence of letrozole when stably overexpressing CYP19. These same 710 cells, when maintained in estradiol, revert to their original morphology and no longer exhibit alterations in 4AD 711 mediated gene expression. B. Study summary: 1. In post-menopausal women 100% of sex hormones are 712 synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 713 levels of 4AD to be elevated in patients whose disease progresses on AI therapy and more recent data has shown 714 4AD dominates breast tumour intracrinology. 2. In contrast serum and tissue levels of estradiol are markedly 715 reduced in patients treated with an aromatase inhibitor. 3. Our study identified SGK3 and PKIB as 4AD regulated 716 transcripts mediated in part by AR in collaboration with ER. C (i) Meta-analysis of 4AD regulated genes SGK3 and 717 PKIB showed that there is no impact on recurrence-free survival in the endocrine untreated population (ER+ PR+, 718 n=379, HR= 1.23 (0.76 - 2.08), logrank p=0.37). (ii) Meta-analysis of 4AD regulated genes SGK3 and PKIB showed 719 that SGK3 and PKIB is associated with poor recurrence-free survival in the endocrine treated population (ER+ PR+, 720 n=231, HR=2.55 (1.34-4.85), logrank p=0.003).

721

722 Figure 7: Validation of SGK3 expression in clinical cohorts as an indicator of poor response to AI therapy.

723 A (i) Evaluation of GSE59515 shows SGK3 mRNA decreases significantly in patients who are responsive to AI 724 therapy (mean expression: 16.75  0.563, one-tailed t-test: p=0.0015). A (ii) Conversely, SGK3 mRNA in non- 725 responders is somewhat sustained (mean expression 16.64  6.44, one-tailed t-test:not significant). A (iii) 726 Validation was performed in a second cohort of patients who were either responsive or non-responsive to AI 727 therapy. SGK3 mRNA was only detectable in non-responders. B (i) This was mirrored by levels of AR mRNA with a 728 significant drop in responders (mean 43.7822.07 one-tailed t-test: p=0.0196) compared with sustained levels in 729 B (ii) non-responders to AI therapy (mean 22.8729.98, one-tailed t-test: not significant). B (iii) Validation was 730 performed in a second cohort of patients who were either responsive or non-responsive to AI therapy. AR mRNA 731 was only detectable in non-responders. C. UCSC Xena browser was used to interrogate TCGA breast cancer data. 732 Kaplan Meier survival curves showed that copy number amplification of SGK3 significantly associates with poor 733 survival in endocrine treated, post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort 734 yielded no association, however, it should be noted that numbers in this cohort were <50.

735

736

Altered steroid milieu in AI resistant breast cancer facilitates AR mediated gene expression associated with poor response to therapy.

Laura Creevey, Rachel Bleach, Stephen F Madden, et al.

Mol Cancer Ther Published OnlineFirst July 9, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-0791

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