Altered steroid milieu in AI resistant breast cancer ... · 7/9/2019 · 1 1 Altered steroid...

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

response to therapy. 2

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

Laura Creevey1* ([email protected]) 4

Rachel Bleach1* ([email protected]) 5

Stephen F Madden2 ([email protected]) 6

Sinead Toomey3 ([email protected]) 7

Fiona T Bane1 ([email protected]) 8

Damir Varešlija 1 ([email protected]) 9

Arnold D Hill4 ([email protected]) 10

Leonie S Young1 ([email protected]) 11

‡Marie McIlroy1 ([email protected]) 12

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

2. Data Science Centre, RCSI, Dublin 2 14

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

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

* Both authors contributed equally to this manuscript 17

The authors declare there have been no competing interests. 18

‡Corresponding author: M. McIlroy ([email protected]) 19

Endocrine Oncology Research Group. 20

Department of Surgery, 21

Royal College of Surgeons in Ireland, 22

St. Stephens Green, 23

Dublin 2, 24

Ireland. 25

Tel No: 0035314022286 26

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

on October 3, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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

mailto:[email protected]


http://mct.aacrjournals.org/

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Abstract 28

Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor 29

(AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to 30

establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent 31

therapy. 32

This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cell 33

are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were 34

assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated 35

using ChIP and relevance to patient profiles was performed using publicly available datasets. 36

Whilst BEZ235 showed decreased viability across AI sensitive and resistant cell lines, anti-AR treatment elicited a 37

decrease in cell viability only in the AI resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and 38

cAMP-Dependent Protein Kinase Inhibitor β (PKIB), were confirmed to be regulated by 4AD and shown to be 39

mediated by AR; crucially re-exposure to estradiol suppressed expression of these genes. Meta-analysis of 40

transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine 41

therapy (HR=2.551, p=0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not 42

respond to AI therapy. 43

This study highlights the importance of the tumour steroid environment. SGK3 and PKIB are associated with poor 44

response to endocrine therapy and could have utility in tailoring therapeutic approaches. 45

Keywords 46

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

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Introduction 56

ER positive tumours account for approximately 75% of all breast cancer diagnoses [1]. The emergence of selective 57

estrogen receptor modulators (SERM) in the 1970’s proved to be a rubicon in the fight against breast cancer; with 58

all first line therapies thereafter focusing on estrogen driven ER activity [2]. Since the mid -2000s AIs have been 59

recommended as first line therapy for hormone receptor positive, post-menopausal breast cancers [3]. However, 60

the development of resistance to these drugs is a perennial problem with disease recurrence in ~30% of patients 61

[4]. Mechanisms of resistance to anti-estrogen therapy are multifaceted and include alterations in co-activator 62

recruitment [5], dominance of growth factor pathways [6], upregulation of aromatase [7], ER mutations [8] and 63

alterations in steroid handling in breast tumour epithelial cells [9]. 64

The role of AR in breast cancer development and progression is somewhat mired in controversy with evidence 65

suggesting it can either antagonise or promote breast cancer depending on tumour context (reviewed[10]). In ER 66

positive breast cancer the general consensus is that AR protein is a positive prognostic indicator [11]. Conversely, 67

others have shown AR to take on the mantle of a pseudo ER, particularly in the setting of triple negative breast 68

cancer [12]. More recently, a potentiating role of AR in the development of endocrine resistance in ER positive 69

breast cancer has been emerging [13, 14]. Subsequently there is growing interest in targeting the AR with a 70

number of ongoing clinical trials assessing the utility of anti-AR drugs in the treatment of advanced breast cancer. 71

More recently, Aceto et al (2018) identified AR signalling to be activated in circulating tumour cells isolated from a 72

patient with breast to bone metastasis [15]. Nevertheless, when we consider that the majority of breast cancers 73

express AR protein, with some reports of >90% positivity [16], it makes understanding the dichotomous role of AR 74

all the more problematic. It is therefore imperative that we differentiate between AR functions which are 75

protective and those which are tumour promoting; whether this is dependent upon protein interactors, altered 76

steroid levels or mutations remains wholly unknown. 77

PI3K and mTOR signalling has been implicated in mechanisms of resistance to AI therapy in preclinical and clinical 78

trials [17]. In this context, AR in particular, has been established to play a prominent role in mediating PI3K and 79

mTOR signalling in a number of neoplasms [18]. AR mode of action in this setting is known to be quite disparate 80

from transcriptional steroid activity and may contribute to mechanisms of resistance to AI therapy. Indeed, non-81

genomic, sex-nonspecific actions of both estrogen and androgens have been demonstrated to activate intracellular 82

signalling pathways [19]. Here, we show that AR protein levels are elevated in cell line models of letrozole 83

resistance. Significantly, due to the mechanism of action of AI therapy, the intracellular environment becomes 84

saturated with androgens, and will become largely estrogen depleted. The risk that this alteration in the steroid 85

environment may facilitate resistance is supported by clinical evidence which has shown that serum levels of the 86

direct precursor steroid, androstenedione (4AD), are elevated in patients progressing on AI therapy [20, 87

21]. Understanding individual tumour intracrinology will be critical to evaluating this clinically as it is known that 88

elevated levels of androgens (4AD and DHEA) are associated with breast cancer risk >2 years prior to cancer 89




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detection. This suggests that hormone levels effect risk rather than hormone levels being altered by localized 90

steroid production [22]. 4AD is known to bind AR and can induce AR nuclear translocation in vitro, albeit with a 91

lower affinity than 5-Dihydrotestosterone (DHT) [23]. Herein, we report that this chronically altered androgenic 92

steroid environment enhances expression of SGK3 and PKIB transcripts which are both identified as AR/ER 93

regulated genes. SGK3 has previously been identified as a downstream target of both PI3K and AR in prostate 94

cancer [24] , highlighting it as a potential central mediator for both signalling pathways in AI resistant breast cancer 95

[25, 26]. This current study highlights the impact of steroid levels on transcriptional regulation and identified 96

SGK3, in particular as a potential indicator of poor response to AI therapy. As SGK proteins can be targeted 97

pharmacologically this pin-points SGK3 as a potential therapeutic target for ER positive breast cancer that may not 98

respond to conventional endocrine treatment. 99

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Materials and Methods 101

Cell culture. 102

Cell lines were cultured as follows: endocrine-sensitive MCF7 were grown in DMEM (low glucose) with 10% of 103

fetal bovine serum (FBS) and 100 U penicillin/0.1 mg ml−1 streptomycin (Pen-strep) plus 10−8 M 17-β-estradiol 104

(Sigma E8875). MCF7 derived AI-sensitive cells (MCF7-Aro) were developed in-house and cultured in phenol red 105

free MEM (Sigma Aldrich, UK), 10% charcoal dextran stripped FCS, 1% Pen-Strep, 1% L-Glutamine and 200 µg/ml 106

G418 (Geneticin). MCF7-Aro derived letrozole-resistant cells (MCF7-Aro-LetR) were created by long-term 107

treatment of MCF7-Aro with letrozole and 4AD >3 months (Novartis, Basel, Switzerland) in charcoal dextran 108

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 109

morphologic change was noted in the transition of MCF7 cells to AI resistant MCF7aro-LetR with cells displaying a 110

large increase in cell surface area. To further assess the impact of the steroid environment on these cells they were 111

then maintained in medium supplemented with estradiol (10-8 M for 15 weeks) and designated as MCF7aro-LetR-112

Est. After 15 weeks, MCF7aro-LetR-Est cells had reverted to smaller cell sizes with cobblestone morphology, as is 113

observed in MCF7 cells grown in estradiol. 114

ZR75.1 were included as an additional luminal A model that express high AR. They are known to have a PTEN 115

mutation and were cultured in MEM, 10% FCS, 1% Pen-Strep, 1% L-Glutamine [28]. ZR75.1-derived AI-sensitive 116

cells (ZR-Aro) were developed in-house by lentiviral transduction of ZR75.1. ZR-Aro-derived letrozole resistant cells 117

(ZR-Aro-LetR) were then generated by long term treatment of ZR-Aro with 4AD and letrozole >3months using the 118

same culture medium as above and are fully characterized (supplemental figure 2 A, B & C). All cells were 119

maintained in steroid depleted medium for 72 hours before treatment with steroids or drugs. All cells were 120

incubated at 37°C under 5% CO2 in a humidified incubator. In-house cells were authenticated and are routinely 121

verified as endocrine resistant, mycoplasma free and all cells are utilised within 10 passages for triplicate 122




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experiments. The Aro-cell lines from which the AI-resistant cells are derived are generally not used as comparators 123

in these studies as it is well established that CYP19 amplification is a hallmark of endocrine resistance [29] and if 124

cultured in the absence of AI and steroid these cells are even less endocrine sensitive [30-32]. 125

Western blotting 126

Cells were harvested, lysed, electrophoresed, and immunoblotted with specific primary antibodies and 127

corresponding horseradish peroxidase-conjugated secondary antibody (Dako, Den). See supplemental materials 128

and methods for antibody detail. 129

MTS assay 130

For drug treatments, MCF7 and MCF7-Aro-LetR cells were steroid-depleted for 72 hours. MCF7 cells were plated in 131

steroid depleted media supplemented with estradiol 10-8 M to enable cell growth and MCF7-Aro-LetR were seeded 132

in steroid depleted media prior to the addition of BEZ235 (Selleck Chemicals) as per optimal dose concentration, 133

enzalutamide (10µM) (Selleck chemicals) or DMSO vehicle (Sigma Aldrich). Treatments were replenished every 3 134

days. Colorimetric outputs were analyzed by measuring the absorbance at 490nm using a spectrophotometer 135

(Perkin Elmer, USA). Prior to transfection MCF7-Aro-LetR and ZR-Aro-LetR cells were steroid-depleted for 72 136

hours. Cells were transfected with siRNA against SGK3 (L-004162-00, Dharmacon, Colorado, USA) or siRNA 137

negative control (d-001206-13-05) and seeded in a 96-well plate prior to the addition of 4AD (10-7 M). Two-way 138

ANOVA with Bonferroni post-test was used for statistical analysis of results for treatment 1- 3 day. Student’s t-test 139

(two-tailed) was used to compare means for siRNA experiments. 140

Colony forming assay 141

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

RNA extraction, library preparation and RNA-sequencing 143

To assess the transcriptional effects of 4AD in endocrine resistant breast cancer cells, RNA-sequencing was 144

performed using MCF7-Aro-LetR cells which were steroid depleted for 72 hours and treated with either 4AD (10-7 145

M) or vehicle in the presence of letrozole 10-6 M in triplicate. RNA was extracted using an RNeasy Kit (Qiagen, 146

Hilden, Germany). Sequencing was performed on an Illumina HiSeq technology (minimum 10 million clean reads 147

of the RNA-quantification/sample). Three independent biological libraries were prepared for each sample to 148

facilitate the expression detection and variance estimation. 149

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

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




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

For both transcriptomic experiments differential expression was determined using the ebayes function of the R 153

package Limma [33]. In each case a binary comparison was performed between 4AD treated cells and controls. In 154

the case of the microarray experiment the following samples were included, GSM1016474, GSM1016475 and 155

GSM1016476 (controls) and GSM1016486, GSM1016487 and GSM1016488 (treated). A p-value of less than 0.05 156

and a fold change greater than 1.4 fold was considered significant. The package Limma was chosen here for 157

differential expression analysis as it is particularly robust when dealing with small sample sizes in both microarray 158

and RNA-seq experiments [34]. These two lists of differentially regulated genes were overlapped with each other 159

and data from a publically available list of genes associated with acquired endocrine therapy resistance in breast 160

tumours expressing ESR1 but not ERBB2 [35]. The original authors referred to this as their “group 4 set” of genes 161

which exhibited strong hormone responsiveness. A detailed description of how it was generated can be found in 162

the original manuscript. This data was used to generate a candidate genes list for further investigation. All 163

calculations were carried out in the R statistical environment (https://cran.r-project.org/). 164

165

RNA-seq validation 166

A panel of breast cancer cell lines (MCF7, MCF7-Aro-LetR, MCF7-Aro-LetR-Est, ZR75.1 and ZRaro-LetR) were steroid 167

depleted for 72 hrs followed by treatment with 4AD (10-7 M) and ethanol vehicle (0.0001% (v/v)) for 24hr. 168

Letrozole 10-6 M was also added to AI resistant cells lines. RNA was extracted using an RNeasy Kit (Qiagen, Hilden, 169

Germany). cDNA synthesis was performed using Superscript III First Strand Synthesis System (Life Sciences). See 170

supplemental material and methods for primer details. 171

MassArray analysis 172

See supplemental material and methods. 173

siRNA transfection 174

AR, ESR1 and SGK3 were silenced by transient transfection using experimentally verified pools of siRNA. AR was 175

silenced using siRNA pools SMARTpool (catalog number L-003400-00) (30 nM), SGK3 SMARTpool (catalog number 176

L-004162-00) (30 nM) and ESR1 SMARTpool (catalog number L-003401-00-0005) (30 nM), all purchased from 177

Dharmacon. All transfections were carried out using Lipofectamine 2000 transfection reagent according to 178

manufacturer’s instructions (Invitrogen, UK) and a non-targeting siRNA pool negative control (Dharmacon) was 179

used as a control for all siRNA experiments. Confirmation of mRNA knockdown was performed using primers for 180

AR (F: 5’-AGCGACTTCACCGCACCTCA-3’, R: 5’-CAGTCTCCAAACGCATGTCCCCG-3’), ESR1 (F: 5’ 181



https://cran.r-project.org/


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TGTACCTGGACAGCAGCAAG-3’, R: 5’-TCTCCAGGTAGTAGGGCACC-3’) 24 hours post transfection. siRNA efficacy 182

was also confirmed at protein level (Supplemental Figure 2D&E). 183

Chromatin immunoprecipitation. 184

MCF7-Aro-LetR cells were treated with 4AD 10-7 M (30 mins), estradiol 10-8 M (45 mins) or ethanol vehicle. ChIP 185

was performed as previously described [27]. Rabbit anti-AR (3 ug, sc-816, SCBT), anti-ESR1 (6 ug, SC-543, SCBT) or 186

mouse IgG (6 ug)/rabbit (3 ug) (Dako) was added to the supernatant fraction and incubated overnight at 4°C with 187

rotation. Proteins were un-crosslinked, DNA extracted and specific primers used to amplify the DNA of the SGK3 188

proximal promoter. SGK3 proximal promoter primers: Forward 5’-GACTTGTGTAACATGGTCTCTTTCA-3’ and reverse 189

5’-CAAGTTCAATCTGACCCTCATATCT-3’ [24]. 190

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

BreastMark [36] is an algorithm that enables the identification of subsets of gene transcript/miRNAs that are 192

associated with disease progression and survival in breast cancer and its subtypes. Using this resource the 193

association of expression levels of SGK3 mRNA and survival were evaluated in endocrine-treated/untreated 194

datasets. 195

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

Series GSE59515 (Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer)[37] was 197

used to assess mRNA expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post-198

menopausal breast cancer patients (n=25). USCS Xena browser (https://xenabrowser.net/heatmap/) was used to 199

interrogate TCGA Breast cancer datasets filtered to focus analysis on pre and post-menopausal, endocrine therapy 200

treated patients (n=415). Copy number variation for SGK3 was evaluated and cohorts were stratified by masked 201

copy number repeat to eliminate sex chromosome and germ-line artifacts. Kaplan Meier graphs (quartiles) were 202

generated from these data to ascertain association of copy number amplification and overall survival. 203

RNA extraction from FFPE tissues 204

Breast cancer patients (n=6) who were responsive (n=3) or non-responsive (n=3) to AI therapy were selected. 205

Informed written consent was received from all patients, and the study was approved by institutional review board 206

Royal College of Surgeons in Ireland (CTI09/07). Hematoxylin and eosin stained sections of formalin fixed, paraffin 207

embedded (FFPE) tumor tissues were analyzed by a pathologist for histological and tumor cellularity classifications. 208

Tumor content was annotated on sections and RNA was extracted from these areas using Qiagen AllPrep DNA/RNA 209

FFPE kit according to manufacturer instructions. 210

Statistical analysis 211



https://xenabrowser.net/heatmap/


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A two-tailed Student’s t test was used to compare means. Two-way ANOVA with Bonferroni post-test was used to 212

compare replicate means. Dose response curves were normalized to 100% and 0% viability based on the lowest 213

and highest drug concentrations (GraphPad Prism v8). 214

Data accession code 215

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

accession code: SRP148035. 217

218

Results 219

AI-sensitive MCF7 and AI-resistant MCF7Aro-LetR cells exhibit decreased cell viability when treated with BEZ235, 220

however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR therapy. Estradiol 221

dependent parental MCF7 cells were found to harbor a PIK3CA E545K missense mutation (Figure 1A and B (i)) 222

which is a known hotspot mutation in breast cancer [38]. Analysis of the letrozole resistant MCF7 derivative cell-223

line (MCF7-Aro-LetR) confirmed they retained the same mutation (Figure 1A and B (ii)). The cell lines exhibited 224

varied levels of AR protein expression with low amounts in the MCF7 and higher levels in the MCF7-Aro-LetR 225

(Figure 1C and supplemental Figure 1A (i)). This was mirrored in the ZR75.1 letrozole resistant derivative cell line 226

(ZR75aro-LetR) when compared to their parental cell line; ER protein levels did not vary across the panel of cell 227

lines (Figure 1C and supplemental Figure 1A (ii)). Dose response analysis was used to establish sensitivity to 228

PI3K/mTOR inhibition by the pan class inhibitor BEZ235 (Sellekchem) and to a αδ PI3K inhibitor, Pictilisib 229

(Sellekchem) (Supplemental Figure 1B (i-iv) & C (i-ii)). 230

Endocrine sensitive (MCF7) and AI resistant cells (MCF7-Aro-LetR) were treated with BEZ235 or enzalutamide (anti-231

AR) as single or combination treatment followed by an evaluation of cell viability. BEZ235 treatment significantly 232

decreased cell viability in all cell lines tested compared to vehicle (Figure 1D and 1E). Of note, the AI-resistant 233

MCF7-Aro-LetR cells showed a significant decrease in levels of cell viability following enzalutamide treatment 234

(Figure 1E), this was in contrast to the parental MCF7 cells, in which viability was sustained when AR was targeted 235

compared to vehicle (Figure 1D). Combined BEZ235 and enzalutamide treatment resulted in a significant decrease 236

in viability across all cell lines, however, this effect was most likely carried by BEZ235 (Figure 1D and 1E). This 237

result was mirrored by colony forming assays suggesting that it is cell proliferation that is being affected (Figure 1F 238

(i-ii)). 239

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

Transcriptomic analysis enabled the identification of genes differentially regulated when AI resistant cells are 241

exposed to 4AD in the presence of letrozole to recapitulate the steroid environment under AI therapy 242



https://www.ncbi.nlm.nih.gov/sra/


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(Supplemental table 1, RNAseq Data). This in-house generated gene list was further supported by comparison with 243

genes identified in a publically available dataset of MCF7-Aro cell treated with 4AD without AI. This helped define 244

gene expression changes attributed solely to the effects of 4AD and provided evidence of putative 4AD specific 245

regulation in AI-resistance. Subsequent comparison with the ‘Group 4’ genes associated with ER+, HER2- endocrine 246

resistance (GSE8140, [35]) yielded a subset of 8 genes (Figure 2A). As using data from independently published 247

datasets introduces confounding factors the genes identified were validated in-house in two genetically disparate 248

AI resistant models. 249

Chronic adaptation to the steroid environment induces a unique transcriptome that can be reverted by re-250

introduction of estradiol. 251

Validation of the transcriptomic analysis was performed by treating endocrine sensitive MCF7 and ZR75.1 cells and 252

their endocrine resistant derivatives (MCF7-Aro-LetR and ZR-Aro-LetR cells) with 4AD and investigating the impact 253

on the transcript levels of the top 4 genes in our list: growth regulation by estrogen in breast cancer 1 (GREB1), 254

serum /glucocorticoid regulated kinase family member 3 (SGK3), cAMP-dependant kinase inhibitor β (PKIB) and 255

MYB proto-oncogene like 1 (MYBL1). Each of these genes validated in both AI-resistant cell lines, MCF7-Aro-LetR 256

and ZR-Aro-LetR, with significant increases in GREB1, SGK3, PKIB and MYBL1 in response to 4AD treatment (Figure 257

2B (i-viii)). GREB1, SGK3, PKIB and MYBL1 did not increase in response to 4AD treatment in endocrine sensitive 258

MCF7 cells and no change was observed for 3 out of 4 of these genes in endocrine sensitive ZR75.1 cells with the 259

exception of PKIB which demonstrated a very modest increase (Figure 2C (i-viii)). Overexpression of the aromatase 260

gene was used to generate both models of AI resistance and has also been a feature observed in the development 261

of AI resistance in the clinic. In order to address whether CYP19A overexpression was a driving factor in the 262

increased expression of SGK3, PKIB, MYBL1 and GREB1 a series of experiments using MCF7 cells overexpressing 263

aromatase and cultured in the presence of 4AD were performed. Data from these experiments confirmed that 264

aromatase overexpression alone did not induce increased levels of these genes (Supplemental figure 3A (i-iv)). 265

Further evidence that aromatase overexpression is inconsequential for these genes is provided via analysis of GSE 266

10911 [39] and from TCGA cohorts (Supplemental figure 3B and supplemental figure 6). The former study focused 267

on MCF7-Aro cells cultured in the absence of steroid, with testosterone (T) or with T plus AI. SGK3 levels were 268

increased in the MCF7aro plus T compared to steroid unstimulated cells, most interestingly, there was a further 269

significant increase in SGK3 mRNA in cells cultured with T and AI. Reversion of the MCF7-Aro-LetR transcriptional 270

response to 4AD could be achieved by culturing MCF7-Aro-LetR in estradiol for 15 weeks. Cell growth initially 271

decreased with notable alteration in cell morphology to the cobblestone appearance of estradiol dependent MCF7 272

and smaller cell size (see Figure 6A for illustrative images). Treatment of these cells with 4AD (24 hours) produced 273

very minimal effects on gene expression in comparison to those observed in the MCF7-Aro-LetR from which they 274

were derived (Figure 3A (i-iv), 3B & 6A). 275




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Androstenedione stimulated induction of target gene mRNA is reduced post siRNA knockdown of either AR or 276

ER; both AR and ER are recruited to a common region on each gene promoter. 277

As it has been established that many transcripts are under cooperative AR:ER regulation [13] we then wished to 278

establish whether the genes identified in this current study were regulated by ER and/or AR. To determine if AR 279

and/or ER are integral to the 4AD mediated upregulation of GREB1, SGK3, PKIB and MYBL1, siRNA inhibition of AR 280

and ER was performed, and siRNA efficacy was confirmed (supplemental Figure 2D (i-ii) & 2E (i-ii)). The impact of 281

knockdown of nuclear receptors (NRs) on target gene expression in MCF7-Aro-LetR cells was evaluated. siRNA 282

knockdown of AR combined with 4AD treatment resulted in a significant decrease in SGK3 and PKIB transcript 283

levels but had no significant impact on GREB1 or MYBL1 (Figure 4A (i-iv)). siRNA knockdown of ER combined with 284

4AD treatment resulted in significantly reduced levels of all four targets (Figure 4B (i-iv)). Regulation of SGK3 285

protein by AR and ER was then confirmed at the level of protein expression (supplemental figure 3C (i-ii)). AR and 286

ER recruitment to these target genes was then evaluated in a publically available dataset (GSE104399) [40]. 287

Although primarily a male breast cancer dataset it contains ChIP-seq data for both AR and ER recruitment in a 288

female breast cancer patient (patient 8: ER+, PR+, HER2-, post-menopausal) (Figure 4C, upper panels). Using the 289

ChIP-seq peak information published by the original authors we were able to confirm the binding of ER to the 290

proximal promoter of SGK3, GREB1, MYBL and PKIB and AR to GREB1 and PKIB in this patient sample (see 291

supplemental table 2 and supplemental figure 4 for additional detail for binding locations). Enhanced AR 292

recruitment and ER occupancy of the SGK3 promoter was confirmed using ChIP in 4AD stimulated MCF7-Aro-LetR 293

(Figure 4C and 4D). Whilst this analysis was limited to one patient, it does suggest that AR and ER bind at regions 294

enriched with ESR1 motifs. ER ChIP-seq data for the MCF7-Aro-LetR confirmed ER recruitment to the proximal 295

promoters of all 4 genes in the presence of 4AD plus letrozole (Figure 4C, lower panel; supplemental table 2 and 296

supplemental figure 4). 297

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

SGK3 has previously been documented as a downstream target of AR and ER (α and β isoforms) in prostate and 300

ERα in breast cancer [24, 25] and is also associated with stabilization of endoplasmic reticulum stress. Here, we 301

have shown that SGK3 mRNA and protein levels are significantly increased in MCF7-Aro-LetR cultured in the 302

presence of 4AD (Figure 5A (i-ii)). Treatment with BEZ235 or a combination of the anti-AR therapy, bicalutamide, 303

with BEZ235 decreased SGK3 mRNA expression (Figure 5B). We found that when SGK3 levels are abrogated by 304

siRNA targeting SGK3 (Figure 5C (i-iii)) in the absence of steroid there was no change in cell viability (Figure 5D (i)), 305

in contrast, when SGK3 was targeted for degradation in the presence of 4AD there was a significant decrease in cell 306

viability (Figure 5D (ii)). This result was also reflected in cell counts of MCF7-Aro-LetR cells exposed to siRNA 307

targeting SGK3 (Figure 5D (iii)). Further validation was carried out in ZR-Aro-LetR cells (Supplemental figure 5A (i-308




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iii)) Follow-up siSGK3 experiments using a variety of steroids demonstrated that decreased viability is only evident 309

when cells are exposed to 4AD or estradiol but there is no alteration observed when cells are cultured in the 310

potent androgen, R1881, or the control, cholesterol (Supplemental figure 5B). 311

SGK3 and PKIB are associated with poor recurrence free survival (RFS) in the post-menopausal, endocrine 312

treated breast cancer patient population (ER+ PR+). 313

In vitro data presented has shown the impact of the steroid microenvironment upon target gene expression 314

identified by RNA-seq (schematic overview: Figure 6A). In post-menopausal women 100% of sex hormones are 315

synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 316

levels of 4AD to be elevated in patients whose disease progresses on AI therapy [19, 20] and more recent data has 317

shown 4AD dominates breast tumour intracrinology [41]. In contrast serum and tissue levels of estradiol are 318

markedly reduced in patients treated with an aromatase inhibitor [3]. Our study identified SGK3 and PKIB as 4AD 319

regulated transcripts mediated in part by AR in collaboration with ER. This supports the hypothesis that 320

transcriptional reprogramming as a result of the steroid environment can facilitate resistance to AI therapy 321

(summarized: Figure 6B). The BreastMark meta-analysis resource was used to evaluate the impact of SGK3 and 322

PKIB transcript levels on endocrine treated breast cancer patient outcome [36]. The high expression group in each 323

plot (blue) accounts for the upper quartile of expression levels for a particular transcript and the low expression 324

group (red) the remaining 75%. This was applied to each of the individual datasets and the information was then 325

combined to perform a global pooled survival analysis. Meta-analysis of the AR mediated genes, SGK3 and PKIB, 326

showed that these genes have no impact on RFS in treatment naïve, postmenopausal ER positive breast cancer, 327

however, when we evaluate their impact in an endocrine treated population it is clear that patients with high 328

levels of these transcripts do not benefit from endocrine therapy (Figure 6C (i-ii)). 329

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

To evaluate these observations further, SGK3 levels were assessed in a clinical cohort of breast cancer patients 331

defined as responsive or non-responsive to AI therapy. Data Series GSE59515 [37] was used to assess mRNA 332

expression levels of SGK3 and AR pre and 3 months post AI treatment in a cohort of post-menopausal breast 333

cancer patients (n=25). SGK3 levels are lower in responsive patients post therapy (drop in mean expression: 16.75 334

0.563, one-tailed t-test: p=0.0015 ) (Figure 7A(i)). Conversely, SGK3 mRNA in non-responders is somewhat 335

sustained (drop in mean expression 16.64 6.44, one-tailed t-test: not significant) (Figure 7A(ii)). This was 336

mirrored by levels of AR mRNA with a significant drop in responders (one-tailed t-test: p=0.0196) compared with 337

sustained levels in non-responders to AI therapy (one-tailed t-test: not significant) (Figure 7B (i-ii)). These data 338

were validated in a second cohort of AI responders and non-responders (n=6). SGK3 mRNA was only detectable in 339

post-menopausal non-responders (Figure 7A(iii), a result that is mirrored by the levels of AR mRNA (Figure 7B(iii). 340

Of note, the only patient non-responsive to AI with no detectable SGK3 or AR transcript was pre-menopausal; 341

highlighting the post-menopausal hormone state as being crucial to the expression of these target genes. Whilst 342




12

exploring the expression of SGK3 transcript in clinical cohorts, it was noted that there is a large percentage (16%) 343

of breast cancer patients with an alteration in the SGK3 gene. The impact of SGK3 copy number amplification was 344

then evaluated in post-menopausal, breast cancer patients treated with endocrine therapy (n=132). Kaplan Meier 345

survival curves showed that copy number amplification of SGK3 significantly associates with poor survival in the 346

post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort yielded no association, 347

however, it should be noted that numbers in this cohort were <50 (Figure 7C). 348

349

Discussion 350

PI3K signalling as a driver of endocrine resistance in ER positive breast cancer has been validated in many previous 351

studies and clinical trials [17, 42]. Given that the combination of the αδ-PI3Kinase inhibitor pictilisib plus 352

fulvestrant does not improve survival in AI resistant breast cancer, suggests that the PI3K pathway alone or in 353

combination with ER is not the sole driver of AI resistance [43]. Our cell line models of AI resistant breast cancer 354

are cultured in letrozole and 4AD which results in elevation of AR protein levels in contrast to parental cell lines. In 355

light of the abundance of data from the field of prostate cancer which has elucidated mechanisms by which AR can 356

act as a mediator of second messenger signalling, a potentiating role for AR in our model of AI resistance was 357

evaluated [44]. In this study we have utilised transcriptomic analysis to elucidate gene expression changes 358

associated with resistance to AI therapy. 359

AI therapy creates a unique androgenic environment that permits exploration of steroid drivers of resistance that 360

may not be wholly dependent upon a functional ER; this is exemplified clinically by the similar overall survival 361

achieved when AI or selective estrogen receptor degrader (SERD) are used as second line therapy after recurrence 362

on AI [45]. AR is normally associated with good patient outcome [11, 16] and the pro-survival response observed 363

in the enzalutamide treated MCF7 cells in this study would fully support this role. However, the strongly opposing 364

action of the same drug in the isogenic MCF7-Aro-LetR cells highlights the importance of the steroid 365

microenvironment in directing the action of AR. As we have observed contrasting impact on cell growth with anti-366

AR therapy we may presume the response is heavily influenced by the steroid driver (estradiol in MCF7 and 367

androstenedione in MCF7-Aro-LetR). Our study is not without limitations as the cell line models used are 368

engineered to overexpress CYP19 (amplification of which is known to be associated with AI resistance [29]); 369

however, control experiments clearly show that aromatase overexpression in this system does not increase 370

expression of the 4AD target genes. Furthermore, analyses of clinical TCGA datasets show no association of CYP19 371

amplification and SGK3 expression, suggesting that this is a disparate mechanism of resistance. It is therefore 372

plausible that estrogen depletion as a consequence of AI therapy may be permissive of AR mediated 373

transcriptomic alterations. 374




13

Elevated levels of sex steroids and their prohormones have long been associated with increased breast cancer risk 375

primarily via epidemiological and clinical observation [20, 46]. In this study we have focused on the effects of 4AD 376

arising from the adrenal gland and ovarial interstitial cells which acts as the direct sex-steroid precursor for both 377

androgens and estrogens [47, 48]. Disappointingly , clinical trials of the CYP17 inhibitor, AA (abiraterone), alone or 378

in combination with AI have shown no difference in progression free survival between modalities; results that are 379

perhaps confounded by an associated rise in progesterone and the potential of AA to act as an ER agonist in breast 380

cancer [49, 50]. It is also plausible that 4AD will be metabolized to other steroids such as 5α-androstanedione or 381

3β-androstanediol, themselves known to act as drivers of breast cancer proliferation [51]. Interestingly, a 382

proliferative role of androstene-3b,17b-diol has recently been suggested for ER positive breast cancer with low 383

intratumoural estradiol levels [52]. Whilst weaker sex steroids are incapable of inducing the conformational 384

change required for classical NR genomic action, they and/or their metabolites may play a role in driving non-385

genomic activity. Indeed, the sheer overabundance of weaker steroids and the formation of transient nuclear 386

receptor (NR) associations may well be able to initiate second messenger signalling which is not dependent upon 387

low disassociation constants [19]. It has often been reported that male and female sex steroids are associated 388

with both ER positive and ER negative breast cancers, highlighting the importance of the steroid drivers which may 389

be acting independently of the classical ER action in some tumours [46]. 390

As the majority of breast cancers express AR it is of interest to understand how the estradiol depleted steroid 391

environment, which accompanies AI therapy, modifies AR action. Many studies have focused on the 392

transcriptomic role of AR in this setting with much of the data indicating a co-operative AR:ER dynamic [13, 40]. 393

Indeed from the RNA sequencing data in our current study the main network of genes altered in response to 4AD 394

treatment were also found to be transcriptionally regulated by ER alpha. Of note, SGK3 and PKIB were also 395

confirmed to be regulated, in part, by AR. Recruitment of both NRs to regions harboring classical EREs in the 396

proximal promoter region was further confirmed in ChIP sequencing data from a female breast cancer patient in 397

the Severson study [40]. Importantly we have established that SGK3 is indeed a steroid regulated, AR target gene 398

and that its expression is enhanced when estrogen synthesis is inhibited in models of AI resistance. 399

The most interesting observation made in this study was the shift in gene expression induced by 4AD which was 400

shown, not only in AI resistant cells derived from MCF7, but also those generated from ZR75.1. This was in stark 401

contrast to the unresponsive parental cell lines, suggesting that chronic alterations in steroid bioavailability can 402

induce transcriptomic reprogramming. This was further supported by data from the long-term estradiol treated 403

MCF7-Aro-LetR, whose gene expression response to 4AD was completely reverted to reflect that of estradiol 404

dependent MCF7. SGK3, in particular, has recently been associated with poor response to AI therapy in breast 405

cancer and has been demonstrated to play a role in stabilization of the endoplasmic reticulum during cell stress 406

[25]. SGK3 has a high degree of similarity to AKT with both kinases targeting many of the same substrates. The SGK 407

family has been implicated in breast cancer resistance to PI3K inhibition in numerous in vitro studies and in a 408




14

clinical trial [26]; of note SGK3 can be activated independently of Class I PI3K [53]. In our study, reduction in SGK3 409

transcript resulted in a loss of cell viability but only in the presence of 4AD or estradiol. This mirrors the finding 410

reported by Wang et al (2017), wherein they observed complete loss in viability when SGK3 is depleted; the more 411

pronounced effect reported was likely due to the increased efficacy of knockdown achieved and also because of 412

the different steroid environments (testosterone versus 4AD) [25]. 413

To understand the clinical relevance of these findings, a meta-analysis of the AR mediated genes, SGK3 and PKIB, 414

was performed. This analysis showed that these genes have no impact on RFS in treatment naïve, 415

postmenopausal, ER positive breast cancer, however, when we evaluate their impact in an endocrine treated 416

population it is clear that patients with high levels of these transcripts do not benefit from endocrine therapy. 417

When this was evaluated in AI responsive versus non-responsive patients it was clear that levels of SGK3 and AR 418

mRNA are sustained in patients failing on therapy. It was also noted that many breast cancer patients have 419

elevation of SGK3 copy number; whether this is as a consequence of an altered steroid microenvironment is an 420

intriguing possibility that is yet to be determined. We would purport, from these observations, that AI treated 421

breast cancers adapt to utilize bioavailable steroids such as those of adrenal origin. As highlighted in this study, 422

evaluating the implications of steroid alterations in the clinical management of disease is warranted. 423

424

Acknowledgements: Author contributions: LC carried out RNA-seq sample preparation, EC50 assays, MTS viability 425

assays, ChIP, qRT-PCR, participated in study design, analysed results and assisted in manuscript preparation. RB 426

assisted in validation of RNA-seq, MTS assays, western blotting, analysed results and generated graphics and 427

assisted in manuscript preparation. SM carried out bioinformatic analysis, assisted in the study design and 428

manuscript preparation. ST carried out MassArray analysis of cell line PI3K mutational status, assisted with study 429

design and preparation of manuscript. DV generated the ZR-Aro-LetR cell line, conducted the related motility and 430

PS2 expression analysis and assisted with preparation of manuscript. ADKH identified appropriate clinical samples, 431

prepared samples for analysis and assisted with study design. LY contributed to data analysis and manuscript 432

preparation. MMcI conceived study design, performed siRNA experiments, data analysis, generated graphics and 433

wrote the manuscript. All authors read and approved the final version of the manuscript. Many thanks to Dr 434

Katherine Sheehan, Tony O’Grady and Joanna Fay (Department of Pathology), Lance Hudson and Aisling Hegarty 435

for their guidance and help with sample preparation. Funding: Health Research Board (HRA-POR-2013-276) 436

(MMcI) and BHCRDT (MMcI). 437

Availability of data: The datasets generated and/or analyzed during the current study are available in the Gene 438

Expression Omnibus repository, [Accession code: SRP148035]. 439

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

441




15

442

443

Abbreviations 444

androgen receptor (AR) 445

aromatase inhibitor (AI) 446

estrogen receptor (ER) 447

androstenedione (4AD) 448

dehydroepiandrosterone (DHEA) 449

5-Dihydrotestosterone (DHT) 450

tissue microarray (TMA) 451

serum and glucocorticoid-regulated kinase 3 (SGK3) 452

cAMP-Dependent Protein Kinase Inhibitor β (PKIB) 453

selective estrogen receptor modulators (SERM) 454

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

MYB proto-oncogene like 1 (MYBL1) 456

nuclear receptors (NRs) 457

chromatin immunoprecipitation (ChIP) 458

formalin fixed paraffin embedded (FFPE) 459

recurrence free survival (RFS) 460

progression free survival (PFS) 461

abiraterone (AA) 462

testosterone (T) 463

464




16

465

466

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53. Bago R, Malik N, Munson MJ, Prescott AR, Davies P, Sommer E, Shpiro N, Ward R, Cross D, 633 Ganley IG et al: Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the 634 phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class 635 III phosphoinositide 3-kinase. Biochem J 2014, 463(3):413-427. 636

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645

Figure legends. 646

Figure 1: AI-sensitive MCF7 and AI-resistant MCF7-Aro-LetR cells exhibit decreased cell viability when treated 647

with BEZ235, however only MCF7-Aro-LetR cells display decreased cell viability when treated with anti-AR 648

therapy. A. Tabulated details on two sets of paired isogenic breast cancer cells lines (endocrine sensitive MCF7, 649

ZR75.1 and their AI-resistant derivatives MCF7-Aro-LetR and ZR-Aro-LetR) including luminal type, AR protein 650

expression and mutations impacting PI3K signalling. B. (i) Traces from the Sequenom MassArray show that 651

endocrine sensitive MCF7 cells have a heterozygous PIK3CA E545K mutation and (ii) Endocrine resistant MCF7-Aro-652

LetR cells have a heterozygous PIK3CA E545K mutation. C. Western blot analysis was used to evaluate AR and ER 653

expression across five breast cancer cell lines (MCF7, MCF7-Aro-LetR, ZR75.1 and ZR-Aro-LetR) D & E. The effect of 654

pan class PI3K/mTOR inhibitor (BEZ235) and anti-AR (enzalutamide) on cell viability was assessed using MTS and 655

colony formation assay in endocrine sensitive and endocrine resistant cells. D MTS of MCF7 response to BEZ235 or 656

to a combination of BEZ235 plus enzalutamide compared to vehicle control. F (i) Colony formation assays reflect 657

MTS results in MCF7 cells. E MTS of MCF7-Aro-LetR cells in response to single agents BEZ235 and enzalutamide and 658

also to a combination treatment. F (ii) Colony formation assays reflect MTS results in MCF7-Aro-LetR cells. Graphs 659

representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). Two-way 660

ANOVA with Bonferroni post-test to compare replicate means was used in MTS day 1 to day 9 readings to 661

determine significance. Student’s paired, 2-tailed t-test established significance to compare means * p<0.05, ** 662

p<0.01, *** p<0.001. 663

Figure 2. 4AD upregulates genes associated with steroid and PI3K signalling in two models of AI resistance but 664

not in the parental cell lines. A. RNA sequencing was performed following androstenedione (4AD 10-7M) 665

treatment plus letrozole for 24 hrs in AI resistant MCF7-Aro-LetR cells versus vehicle. This data was then 666

compared with array data for MCF7aro treated with 4AD versus vehicle to identify 4AD specific transcripts. These 667

genes were then integrated with the ‘Group 4 ‘ gene set of endocrine resistant genes associated with ER+, HER2 –668

ve disease yielding a list of 8 genes. B. (i-viii) qRT-PCR validated differentially expressed genes (GREB1, SGK3, PKIB 669

and MYBL1) in AI resistant cell models MCF7-Aro-LetR and ZR-Aro-LetR cells. C. (i-viii) qRT-PCR showed expression 670

of these genes in parental MCF7 and ZR75.1 cells on exposure to 4AD . Graphs representative of n=3. Error bars 671

are representative of mean ± standard error of the mean (SEM). Student’s paired, 2-tailed t-test established 672

significance * p<0.05, ** p<0.01, *** p<0.001. ¶ [32], $ [36]. 673




21

Figure 3. Culturing AI resistant cells (MCF7-Aro-LetR) in estradiol for 15 weeks reverts 4AD driven gene 674

expression. A. (i) & (ii) qRT-PCR of SGK3 mRNA displays a very modest increase, whereas GREB1 is significantly 675

decreased compared to vehicle control. A (iii) & (iv) PKIB and MYBL1 expression is unchanged in the presence of 676

4AD. Graphs representative of n=3. Error bars are representative of mean ± standard error of the mean (SEM). B. 677

Gene expression (SGK3, GREB1, PKIB & MYBL1) after 24 hrs 10-7M 4AD treatment in MCF7-Aro-LetR cells long 678

term cultured in 4AD compared to MCF7-Aro-LetR cells long term cultured in 10-8M estradiol (MCF7aroLetR-Est). 679

Student’s paired, 2-tailed t-test was carried out to compared individual gene expression across both cell lines. 680

Figure 4: Androstenedione stimulated transcript levels of SGK3 and PKIB are reduced in response to siRNA 681

knockdown of either AR or ESR1; in addition, AR and/or ER are recruited to these target genes in vivo and in 682

vitro. A. qRT-PCR of MCF7-Aro-LetR transfected with siRNA-AR treated with 4AD shows a significant decrease in 683

SGK3 and PKIB transcript levels (i-ii) but does not impact transcript levels of GREB1 or MYBL1 (iii-iv). B. qRT-PCR of 684

MCF7-Aro-LetR transfected with siRNA–ESR1 in MCF7-Aro-LetR cells treated with 4AD results in a significant 685

decrease in transcript levels of the SGK3, GREB1, PKIB and MYBL1 (i-iv). C. Recruitment of AR and ER to target 686

gene promoters was evaluated using publically available data from a breast cancer patient ChIP-sequencing study, 687

and ER recruitment in the MCF7-Aro-LetR cell line via ChIP-sequencing. ARϕ recruitment to the SGK3 and MYBL1 688

promoter was validated via ChIP in MCF7-Aro-LetR D. (i) Validation of the putative AR target gene SGK3 was 689

confirmed by performing ChIP to determine recruitment of AR to the promoter of SGK3 in MCF7-Aro-LetR cells in 690

the presence of 4AD. (ii) ChIP experiments were performed to determine recruitment of ER to the promoter of 691

SGK3 in MCF7-Aro-LetR cells treated with 4AD. All graphs representative of three experimental replicates. Error 692

bars are representative of mean ± standard error of the mean (SEM) of n=3. Student’s paired, 2-tailed t-test 693

established significance * p<0.05, ** p<0.01, *** p<0.001. 694

Figure 5: BEZ235 or combination of BEZ235 with the anti-AR Bicalutamide decreases SGK3 mRNA expression. 695

Knockdown of SGK3 inhibits AI resistant cell proliferation only in the presence of androstenedione. A (i) SGK3 696

protein expression is increased in the presence of 10-7M 4AD in MCF7-Aro-LetR cells. (ii) Densitometry analysis of 3 697

independent western blots confirmed significant increase in SGK3 protein expression with 4AD treatment. B. qRT-698

PCR evaluation of SGK3 mRNA expression with BEZ235 +/- anti-AR therapy bicalutamide in MCF7-Aro-LetR cells. C 699

(i) Western blot of SGK3 protein levels following siSGK3in MCF7-Aro-LetR. (iI) Densitometry analysis shows SGK3 700

protein levels following siSGK3. (iii) qRT-PCR of SGK3 mRNA following siSGK3 in MCF7aro-LetR D (i) MTS of 701

MCF7Aro-LetR cell viability following knockdown of SGK3 in the absence of steroid. (ii) MTS assay of MCF7aro-LetR 702

cell viability following siSGK3 combined with 4AD treatment . (iii) Cell counts confirmed decreased cell viability in 703

MCF7Aro-LetR cells following siSGK3. Graphs representative of n=3. Error bars are representative of mean ± 704

standard error of the mean (SEM). Student’s 2-tailed t-test established significance for A, B and D. 705

Figure 6: SGK3, PKIB and GREB1 are 4AD regulated transcripts mediated in part by AR in collaboration with ER. 706

Associated outcome in clinical cohorts highlights a significant impact on therapeutic response to endocrine 707




22

therapy. A Schematic overview of key study findings highlight the morphologic and transcriptional changes driven 708

by chronic exposure of MCF7 to 4AD in the presence of letrozole when stably overexpressing CYP19. These same 709

cells, when maintained in estradiol, revert to their original morphology and no longer exhibit alterations in 4AD 710

mediated gene expression. B. Study summary: 1. In post-menopausal women 100% of sex hormones are 711

synthesized in peripheral tissues from circulating adrenal and ovarian precursors steroids. Clinical data has shown 712

levels of 4AD to be elevated in patients whose disease progresses on AI therapy and more recent data has shown 713

4AD dominates breast tumour intracrinology. 2. In contrast serum and tissue levels of estradiol are markedly 714

reduced in patients treated with an aromatase inhibitor. 3. Our study identified SGK3 and PKIB as 4AD regulated 715

transcripts mediated in part by AR in collaboration with ER. C (i) Meta-analysis of 4AD regulated genes SGK3 and 716

PKIB showed that there is no impact on recurrence-free survival in the endocrine untreated population (ER+ PR+, 717

n=379, HR= 1.23 (0.76 - 2.08), logrank p=0.37). (ii) Meta-analysis of 4AD regulated genes SGK3 and PKIB showed 718

that SGK3 and PKIB is associated with poor recurrence-free survival in the endocrine treated population (ER+ PR+, 719

n=231, HR=2.55 (1.34-4.85), logrank p=0.003). 720

721

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

A (i) Evaluation of GSE59515 shows SGK3 mRNA decreases significantly in patients who are responsive to AI 723

therapy (mean expression: 16.75 0.563, one-tailed t-test: p=0.0015). A (ii) Conversely, SGK3 mRNA in non-724

responders is somewhat sustained (mean expression 16.64 6.44, one-tailed t-test:not significant). A (iii) 725

Validation was performed in a second cohort of patients who were either responsive or non-responsive to AI 726

therapy. SGK3 mRNA was only detectable in non-responders. B (i) This was mirrored by levels of AR mRNA with a 727

significant drop in responders (mean 43.7822.07 one-tailed t-test: p=0.0196) compared with sustained levels in 728

B (ii) non-responders to AI therapy (mean 22.8729.98, one-tailed t-test: not significant). B (iii) Validation was 729

performed in a second cohort of patients who were either responsive or non-responsive to AI therapy. AR mRNA 730

was only detectable in non-responders. C. UCSC Xena browser was used to interrogate TCGA breast cancer data. 731

Kaplan Meier survival curves showed that copy number amplification of SGK3 significantly associates with poor 732

survival in endocrine treated, post-menopausal patients (p=0.016). Analysis of the pre-menopausal patient cohort 733

yielded no association, however, it should be noted that numbers in this cohort were <50. 734

735

736




Published OnlineFirst July 9, 2019.Mol Cancer Ther Laura Creevey, Rachel Bleach, Stephen F Madden, et al. therapy.mediated gene expression associated with poor response to Altered steroid milieu in AI resistant breast cancer facilitates AR

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