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). 1 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. 48 49 50 51 52 53 54 55 2 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. 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 3 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. 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.