Extra-nuclear and cytoplasmic steroid receptor signalling in hormone dependent cancers

Steroid hormone receptors are key mediators in the execution of hormone action through a combination of genomic and non-genomic action. Since their isolation and characterisation in the early 20th Century much of our understanding of the biological actions of steroid hormones are underpinned by their activated receptor activity. Over the past two decades there has been an acceleration of more omics-based research which has resulted in a major uptick in our comprehension of genomic steroid action. However, it is well understood that steroid hormones can induce very rapid signalling events in tandem with their genomic actions wherein they exert their influence through alterations in gene expression. Thus the totality of genomic and non-genomic steroid action occurs in a simultaneous and reciprocal manner and a greater appreciation of whole cell action is required to fully evaluate steroid hormone activity in vivo. In this mini-review we outline the most recent developments in non-genomic steroid action and cytoplasmic steroid hormone receptor biology in endocrine-related cancers with a focus on the 3-keto steroid receptors, in particular the androgen receptor.


Introduction
Humans and primates are distinguished from other mammals by their unique capacity to produce large amounts of adrenal steroid hormones.These steroid hormones are then further metabolized in peripheral tissues where they bind to specific receptors and mediate diverse physiological responses (reviewed [1]).In total there are 48 mammalian nuclear receptor proteins that can bind to lipophilic molecules, such as steroid hormones, and regulate an array of long-acting, systemic activities throughout the body.One branch of this large family of proteins are the steroid nuclear receptor subfamily (NR3) collectively the estrogen receptors, alpha and beta (NR3A1 and NR3A2), and the 3-keto steroid receptors (3 K-SR) comprising the androgen (AR/ NR3CA4), glucocorticoid receptor (GR/ NR3CA1), mineralocorticoid receptor (MR/ NR3CA2) and the progesterone receptor (PR/ NR3CA3).All of these nuclear steroid hormone receptor (SHR) proteins have a conserved structural architecture that includes an N-terminal regulatory domain, a DNA binding domain (DBD) followed by a flexible hinge region which links the C-terminus ligand binding domain (LBD).Together these proteins play important roles across many processes including development, sexual maturation, reproduction, salt and water balance, metabolism and immune response, hence perturbation of their activities has been associated with a wide range of pathologies including endocrine-related cancers and diseases of sexual development.
Ligand bound SHRs are known to exert their genomic (also referred to as canonical) transcription factor activity by directly binding DNA response elements within enhancer and proximal promoter regions of target genes.In addition, SHRs have also been demonstrated to modulate rapid responses within the cell cytoplasm in a non-canonical manner (reviewed [2]).The precise mechanisms driving non-genomic steroid action have remained somewhat elusive, likely due to the high degree of cross-talk with other signalling pathways.More recently, however, there have been a number of compelling studies that have redirected attention towards the non-genomic steroid receptor action, and also their extra-nuclear function and interactions with other cytosolic organelles [3].For the context of this mini-review, the primary focus will be on SHR activity in endocrine related cancers, which relates to SHR responsive cancers, such as those of the breast, prostate and endometrium, and can oftentimes be extended to cancers of the thyroid and ovary.For clarity, discussion of extra-nuclear steroid hormone action has primarily been grouped into two categories, SHR induced cytoplasmic signalling, and membrane-associate SHR signalling as summarized in the sections below.

Cytoplasmic/ extra-nuclear SHR actions
Endocrine therapy is a common treatment for SHR responsive (hormone dependent) cancer, which acts by blocking steroid hormone binding to cognate SHR.Unfortunately, resistance to endocrine therapy can develop, leading to relapse and progression of disease.A number of groups have been very active in the area of targeting non-genomic SHR action in the context of endocrine-resistant cancers, particularly those of breast and prostate [4][5][6].For example, the association of cytoplasmic AR with enhanced migratory capacity was shown to depend on AR/ Filamin interactions that mediate cytoskeletal remodelling events in cancer associated fibroblasts (CAF) within the prostate cancer tumour microenvironment [7].These studies demonstrated that AR /Filamin A, engages with intergrin β1 to regulate the focal adhesion kinase (FAK), paxillin and Rac to facilitate microenvironment remodelling that promotes expansion of prostate cancer organoids.It is well established that androgens can have a dichotomous influence on prostate tumour growth depending on concentration, with supraphysiological levels arresting genomic AR activity and reducing cancer cell growth [8,9].This is relevant as we know AR remains highly expressed in endocrine resistant prostate cancer and influences not only tumour growth but also migration at low levels of androgens (reviewed [10]).The bi-phasic response of cancer cells to high and low androgen levels is also corroborated in work by Nyquist et al., which demonstrated that selectively activating the genomic AR pathway has utility in endocrine resistant prostate cancer [11] and highlights the ligand-dependent duality of SHR function.
Non-genomic steroid hormone actions have been described for all SHR, and these are very often mediated through the activation of second messenger signalling transduction pathways by kinases located within the cytoplasm or in close proximity to the plasma membrane [12].AR, ER and PR have been reported to modulate the Src and PI3K pathways in the cytoplasm and proximal to the plasma membrane [6,13].These signalling transduction pathways are commonly activated in hormone dependant cancers of breast, gynaecological and prostate origin, and are often associated with a more aggressive phenotype and therapy resistance [6,[13][14][15][16][17][18][19][20].Other cytoplasmic signal transduction pathways associated with non-genomic SHR activity include the Ras-Raf, MAP-K/ERK, and JAK-STAT pathways [21][22][23][24][25][26].It is important to note that activation of these pathways can also arise due to SHR-induced transactivation of various membrane receptors including, but not limited to, various receptor tyrosine kinases (RTKs) such as EGFR, IGR1R and PDGFR [27].Of note, cross-talk between genomic and non-genomic SHR have been described [2].Moreover, various pathways, such as the JAK-STAT, MAPK, and FAK, can elicit downstream signalling cascades, resulting in transcriptional modulation (Summarised in Table 1).
Post-translational modifications to SHRs have been highlighted to result in membrane trafficking of SHRs, resulting in cytoplasmic signalling in response to and also independent of steroid hormone stimulus [28,29].Palmitoylation is a common post-translational modification that involves the addition of a long chain fatty acid to proteins, thereby facilitating anchorage to the cell membrane.Recent work by Afrin et al. demonstrated that inhibition of ER palmitoylation reduces cytoplasmic ER and AKT signalling in uterine tissues [30].Coincidentally, Gruslova et al. also demonstrated inhibition of palmitoylation resulted in altered subcellular localisation of ER and reduced proliferation in breast cancer cells resistant to tamoxifen [31].Lipidation of ER has also been described to promote membrane ER localisation and support tumour growth in pituitary cancers [24].It is important to note that whilst membrane anchorage of ER by palmitoylation is associated with aggressive cancer phenotypes, it also plays a role in normal physiological development, as exemplified by Gagnaic et al. in the luminal cells during mammary gland development [32].Palmitoylation has also been described for AR, resulting in increased growth and survival of prostate cancer [28,29].Research by Kim et al. showed that high dietary palmitate is associated with tumour progression in prostate cancer through increased Src activation of MAPK and FAK [33].Whether or not this particular mechanism is driven by the non-genomic action of AR remains elusive, but works by Lin et al and Russo et al. confirmed increased expression of enzymes involved in palmitoylation are associated with an aggressive phenotype in prostate cancer [34,35].
Genetic mutations and variants of SHRs are other well-described mechanisms that have been highlighted to facilitate cytoplasmic signalling, both in response to, and independent of steroid hormone stimulus.In addition to full length ERα and ERβ, other variants such as ERα36 have been described to drive cytoplasmic signalling.ERα36 as a variant of ER that lacks both transactivating domain and possesses a truncated LBD, that has been observed to localise in the cytoplasm, plasma membrane and mitochondria [36][37][38].Non-genomic action of ERα36 has been observed to drive tumour growth, survival, migration, invasion and treatment resistance to tamoxifen [39][40][41][42].Moreover, cytoplasmic localisation of SHRs might act as potential prognostic markers for cancer patients.Ding et al. recently reported that cytoplasmic expression of ERβ is associated with resistance to EGFR-tyrosine kinase inhibitors in metastatic lung cancers [43].Conversely, cytoplasmic localisation of ERα was observed to be positively associated with favourable outcomes in breast cancer patients [44].AR-V7 is a common splice variant of AR that lacks its LBD and has been described to drive ligand-independent genomic and non-genomic AR signalling resulting in increased survival, migration, invasion and treatment resistance in prostate cancer [45][46][47].Of note, recent reports from Konig et al., (2020) observed that cytoplasmic retention of AR-V7 associated with a shorter relapse free survival in prostate cancer patients [48].

Membrane-associated steroid receptors
In addition to cytoplasmic signalling induced by SHRs, steroid hormones have also been reported to induce membrane-initiated steroid signalling (MISS) through membrane-localised receptors, often referred to as membrane-associated steroid receptors (mSR).This class of receptor deviates from their SHR counterparts as they are localised to the plasma membrane and possess different molecular/structural architectures; lacking both DBD, LBD, and the ability to translocate into the nucleus.Similar to SHRs, mSRs are modulated by steroid ligands (i.e.androgens, estrogens, progestins, and corticosteroids) and have been implicated in the regulation of cytoplasmic signalling cascades that are involved in cell growth and proliferation, migration, metabolism, and treatment resistance.For example, progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein that has been shown to bind progesterone, resulting in the induction of spectral and conformational changes [59].However, despite the description of MISS for all mSR and their emerging importance in various cancer types, the current research on these mSRs has been comparatively limited when compared to SHRs (See Table 2 for an up to date summary of recent mSR associated functions).Moreover, the precise mechanism by which steroid ligands induce MISS has not been fully elucidated for most mSRs, and recent efforts to investigate ligand-binding affinity have been limited.

Membrane-associated progesterone receptors (mPR)
The discovery of mPR over two decades ago has expanded our understanding of how progesterone exerts its non-genomic steroid effects beyond its well-established genomic action [60,61].These receptors play a role in mediating various rapid cellular responses, and their function is an active area of research in female health and reproduction.mPRs are generally classified into two main categories: mPR and progesterone receptor membrane component 1/2 (PGRMC1/2) [62].Additionally, mPRs can be further segregated into five subtypes such as mPRα, mPRβ, and mPRγ, mPRδ, mPRε.While the current research on these mPRs is still somewhat limited, various studies have reported mPRs to be associated with G-coupled proteins, increased cAMP and calcium signalling and have been demonstrated to modulate intracellular signalling kinases such as MAPK, AKT and STAT3 signalling pathways [63][64][65][66][67][68][69].mPRs are also associated with migration, proliferation, differentiation and treatment resistance to chemotherapy [69][70][71].
PGRMC1 is a membrane progesterone receptor that has been observed to play a role in mediating various biological processes including cell growth and survival [72,73].Although PGRMC1 is known to localise to the plasma membrane, it has been demonstrated to reside in various cellular compartments including the endoplasmic reticulum, mitochondria, and the cytoplasm [74].Recent studies by Ponikwicka-Tyszko et al. demonstrated that non-canonical action of progesterone is associated with tumour growth, proliferation, and migration in ovarian cancer, facilitated through PGRMC1 [75].Coincidentally, research by other groups has also shown similar findings in breast cancer models [76][77][78].Moreover, PGRMC1 has been reported to modulate PI3K/AKT and EGFR signalling in triple negative breast cancer cells [79], and is associated with poor prognosis in breast and gynaecological cancers [73,80].Although the current research surrounding PGRMC1 is primarily focused in female cancers (breast and gynaecological), PGRMC1 has also been demonstrated to promote tumour progression in colorectal cancer [81], glioblastomas [82,83], and has also been reported to modulate metabolic perturbation in endocrine disorders such as polycystic ovary syndrome (PCOS) and metabolic syndrome [84,85].

Membrane-associated estrogen receptors (mER)
The discovery of mER has facilitated research to further our understanding of non-genomic estrogenic action.G protein-coupled receptor (GPCR) estrogen receptors (GPER) have been studied as rapid responders to estrogens [86].GPER, also known as GPR30 is a transmembrane receptor that plays a role in mediating myriad biological processes, including cell proliferation, calcium signalling and migration.GPER is ubiquitously expressed across multiple tissue types, and it mediates diverse physiological processes depending on cell type.Additionally, the role of GPER action in cancers has been debated, with various reports suggesting conflicting pro-and anti-tumour activity.Recent clinical data from various research groups have highlighted a correlation between GPER expression and poor prognosis in breast, gynaecological, gastric, and lung cancers [87][88][89][90][91][92].Conversely, other studies have suggested GPER expression to be potentially associated with good clinical outcome in breast and cervical cancer [93,94].The precise mechanism of estrogen-induced GPER intracellular signalling is still under investigation, however, there is evidence that GPER regulates invasion, migration and adhesion through FAK/PI3K signalling [95].

Membrane associated mineralocorticoid (mMR) & glucocorticoid receptor (mGR)
Research surrounding mMRs and mGRs to date has been somewhat limited.Earlier research has demonstrated corticosteroids to induce rapid biological responses, and recent efforts by Weiss et al. and Karst et al. have shown MR localisation to the plasma membrane in response to corticosteroids [96,97].However, novel mMR have not been described, and this can be attributed to crosstalk between mineralocorticoids, corticosteroids and other mSR.Research by various groups have demonstrated aldosterone initiating MISS through GPER, which has previously been described as a mER [98][99][100].Moreover, recent work by Ping et al. demonstrated structural based evidence of interactions between corticosteroids and GPR97, which is a GCPR that plays a role in adhesion and migration [101].

Membrane associated androgen receptor (mAR)
In addition to their genomic SHR, androgens have been described to mediate rapid MISS through mAR, namely via Oxoeicosanoid Receptor 1 (OXER1), Zinc Transporter Member 9 (ZIP9), the G protein-coupled receptor family C group 6 member A (GPRC6A), and Ca 2+ channel Transient Receptor Potential Cation Channel Subfamily M (Melastatin) Member 8 (TRPM8) [86,102].OXER1 is a GCPR that plays a role in regulating various cellular and physiological processes including immune responses and inflammation, migration, cell growth and survival [103][104][105].Although OXER1 is a 5-oxoeicosatretraenoic acid-inducible membrane receptor, recent reports have highlighted androgens as a potential novel antagonist of OXER1 [106,107].Androgen-induced OXER1 signalling results in inhibition of cAMP production, resulting in cell migration and actin cytoplasmic reorganisation through PI3K and FAK [105][106][107].Furthermore, increased expression of OXER1 is associated with poor survival in breast cancer [108].The current research surrounding OXER1 is ongoing, and will help elucidate the exact mechanism by which androgens modulate OXER1 intracellular signalling.Similarly to OXER1, ZIP9 is a GCPR that plays a role in maintaining cellular homeostasis [109].Although ZIP9 has been highlighted as an androgen-inducible mAR, investigations regarding the mechanism of ligand binding and intracellular signalling has been limited.In addition, early research on androgen-induced ZIP9 has been observed to modulate MAPK and Erk1/2 signalling [109,110], and has been associated with migration in prostate and breast cancer cells [111,112].GPRC6A is another GCPR that has been demonstrated to exert non-genomic androgenic steroid action, and is involved in regulating various cellular processes including cell proliferation and metabolism [113].Investigation into the mechanism of androgen-induced signalling has been somewhat controversial, as GPRC6A has been demonstrated to be activated by various other ligands, however, recent evidence by Ye et al. demonstrated a dose response of GPRC6A-induced MAPK, ERK and mTORc signalling in response to testosterone [114].
TRPM8 is one of several mammalian homologues of the transient proliferation and migration [117][118][119][120][121][122][123] receptor potential (TRP) family that plays a role in ion channel regulation and is involved in regulation sematosensation [115].TRPM8 is localised on the plasma membrane, but has also been observed to localise in the endoplasmic reticulum [102].The majority of recent investigations into non-genomic androgen action in cancers has been centred on TRPM8, most notably in prostate cancer where TRPM8 has been considered as a potential novel biomarker.Recent studies by Genovesi et al. have illustrated differential expression profiles of TRPM8 in prostate cancer progression [116].Efforts by various groups including Di Donato et al. and Di Sarno et al. have also explored the utility of TMPM8 as novel target in AR-dependent prostate cancer cells [117,118].Similar to other mARs, the exact mechanism by which androgens induce intracellular signalling through TMPM8 has not been fully elucidated.Earlier evidence has shown TRPM8 response to androgens such as testosterone in a dose dependent manner, and recent work by Grolez et al. demonstrated interactions between AR and TRPM8 on the plasma membrane as another mechanism of AR-mediated cytoplasmic signalling through this mAR [119].TRPM8 has also been reported to be associated with migration and proliferation in prostate cancer, potentially through FAK/PI3K modulation, however, this exact mechanism remains unclear and further studies are needed to confirm this mechanism [119,120].More recent evidence has implicated TRPM8 in regulating proliferation and migration in bladder cancer cells [121], and breast cancer cells through the AMPK pathway [122], and is associated with poor prognosis in colorectal cancer [123].

Classical SHR have structural features that dictate their propensity to exist in a variety of mono and oligomeric states
The ER and 3 K-SR (AR, PR, GR and MR) family of receptors evolved from a clade of Ancestral Steroid Receptors (AncSR) that have been conserved over 450 million years primarily driven by their unique suitability to exert physiological control [124].And whilst 3 K-SR are derived from the same phylogenetic branch as the ER proteins they did evolve at a later stage and have some distinctive features that set them apart [125].Whereas ER nuclear receptors are hardwired to exist as homodimers, the 3 K-SR have a unique C-terminal extension (CTE) that permits interaction within the same ligand binding domain (LBD) enabling stabilisation of the protein in a monomeric state [126].Furthermore, not only can 3 K-SR protein allostery be mediated by the degree of ligand affinity but also by the degree of DNA affinity; with contributions from both elements ultimately determining the activity of receptor [127].How important the affinity of the ligand binding is to this process is still unclear, however, compelling data from the ARV7 variant lacking a LBD provides some insight.ARV7 is constitutively active, driving a similar cistrome to that of the full length AR receptor in the absence of ligand, and is therefore a major driver of androgen deprivation therapy resistancein low androgen environments [128,129].Unlike full length AR, ARV7 is dependent upon the dimerization box (D-Box) domain for dimer formation and subsequent nuclear translocation.It has been shown to adopt a 'hit and run' mode of DNA binding and of transcription initiation with cursory chromatin residency [130].Intriguingly this particular mode of DNA hypermobility is more characteristic of repressive mechanisms.Similar to AR, GR has also been reported to exist in a range of mono, oligo and tetrameric states and can yield non-canonical dimer structures dependent on the presence of various ligands [126,[131][132][133].Most recently GR has been reported to engage with RAS in a ligand-free cytoplasmic manner, thus in the absence of glucocorticoids it may manifest as an inhibitor of solid tumour growth (lung, breast, colorectal) which has wide-ranging implications for the management of chemotherapy patients [23,134].
Similarly, AR is present as a homodimer within the cytoplasm and must disassociate into a monomeric form upon ligand binding so that nuclear translocation can occur [135].However, the post-translational modification, Ser815 AR, stabilises the homomeric form of the receptor, perturbing genomic activation even in the presence of a potent ligand and resulting in cytoplasmic retention of the SHR [136].Further studies have shown Ser815 AR localises to the endoplasmic reticulum and potentially plays an important role on the mediation of stress response via suppression of the AKT pathway again highlighting the relevance of non-canonical SHR action in prostate cancer [136,137].Further studies will be required to fully elucidate, not only the implications of these various mono and multimeric states on steroid hormone action, but also to determine what ligands are responsible for their formation and whether they regulate differential transcriptional programmes.

The Androgen Receptor is structurally adapted to exist in a more flexible state; implications for monomeric stability and degeneracy of DNA binding
More recently there have been a number of studies focused on the action of AR within the cytoplasm specifically, highlighting the importance of localisation within the cell (plasma membrane, mitochondria, endosome) [138,139].Much of this data is from studies in the field of endocrinology and type 2 diabetes militus, nevertheless, the information generated from these studies has shown the relevance of exploring steroid action in a wider context, incorporating metabolism, exocytosis, organelle biogenesis alongside their impact on global gene expression [3].Hormone-related cancers, in particular, are very much associated with the development of metabolic syndrome emphasizing the relevance of these non-genomic actions on cell metabolism [140].In the study investigating the insulinotropic action of the extra-nuclear AR in pancreatic beta-cells, the authors clearly show the wide-ranging actions and influence on cell metabolism with consequences for the body as a whole [3].
Interesting data from the Claessens and Estébanez-Perpiñá labs have highlighted the influence of perturbation of AR dimerization on the functionality of the receptor.AR homodimers can be mediated via DBD, N-C termini interaction and also by LBD interactions, the latter of which shed light on some of the more elusive functions of the receptor.Structural biology data show that the large LBD interaction protein surface potentially harbours up to 40 commonly mutated sites with implication in diseases such as Androgen Insensitivity Syndrome (AIS) through to endocrine resistant prostate cancer [141].The development of a mouse model harbouring a W731R point mutation within the AR LBD (AR Lmon/Y ) has also provided unprecedented insight with regards the perturbation of ligand binding dimerization events in particular [142].This in vivo model closely phenocopies the signs and symptoms of Partial Androgen Insensitivity Syndrome (PAIS) and demonstrates that an androgen excess phenotype accompanies impaired AR dimer status, increased levels of cytoplasmic AR and altered co-regulator binding.This is in direct contrast to in vitro evidence which showed that impaired AR LBD dimerization could still activate reporter assays, translocate to the nucleus and bind DNA; suggesting that engagement with co-regulatory complexes is impaired in vivo resulting in compromised binding to androgen response elements within chromatin structures.Indeed, AR LBD mutations outside the ligand binding pocket also impact the activity of the receptor by altering the equilibrium of monomeric and oligomeric forms, post translational modifications and subsequent variation in co-regulator recruitment [133] (See Fig. 1 for summary -AR activity is mediated by the degree of ligand affinity but also by the degree of DNA affinity; with contributions from both elements ultimately determining the activity of receptor).

New insightstechnological advances
Intracrinology is a term first coined by Labrie in 1988 which describes the mechanism that permits specific and local production of steroids within cells that do not significantly impact circulating levels [143].A major deficit in our understanding of steroid hormone action within tumours is their subsequent metabolism within target cells yielding an unknown tumour steroid milieu.The experimental application of steroid conjugates over the years has informed much of our knowledge of rapid response to steroid hormones, that are independent of intracellular SHR ligand binding [144][145][146].In recent years there have been rapid advances in our ability to explore the tumour intracrine environment via various chromatography tandem mass-spectrometry approaches [147,148] and mass-spectrometry imaging [149,150].There are also live cell imaging technologies such as massively parallel fluorescence correlation spectroscopy (mpFCS) that can be utilised to track and map the directionality of nuclear-cytoplasmic SHR translocation, providing detailed information on the trafficking of the ligand bound receptor [151].These technologies permit unprecedented insight into the localisation of the ligand bound SHR within tissue and cells, that can then be mapped to dynamic receptor localisation and directionality.Data of this nature will facilitate a much deeper appreciation of SHR action in the context of the ligand driver, something which thus far has been pre-determined based on accepted enzyme kinetics.How far these kinetics deviate from the norm in different pathologies is somewhat poorly understood.Significantly, recent data has reported alterations in intracellular acidification rates on exposure to DHT; resultant shifts in intracellular pH may subsequently cause alterations in the mean lifetimes of metabolic co-factors such as flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] which may have implications for reaction kinetics [3,152].

Relevance to endocrine-related cancers
Incidence rates of endocrine-related cancers such as those of breast and prostate cancer are predicted to continue to rise over coming decades, and whilst many therapies have been developed to target steroid hormone signalling, resistance remains a clinical concern impacting ~30 % of patients.Selective SHR modulators/ degraders have been the therapeutic mainstay for breast cancers (selective ER modulators/ degraders (SERM/D)) for many decades, and more recently in the treatment of prostate cancer (selective androgen receptor modulators/ degraders (SARM/D)).However, the inability to identify patients who will benefit from these drugs at point of diagnosis has remained a clinical conundrum.Clinical determinants of therapeutic selection for SERM are often solely based on presence (>10 %) of SHR protein expression within the primary tumour.In the context of breast cancer, molecular profiling approaches such as Mammaprint and Oncotype DX that are indicative of receptor activity have entered the clinical space, however, the efficacy of these platforms have been shown to be vulnerable to agerelated endocrine factors highlighting the need to explore steroid receptor activity across the entire spectrum of their influence both genomic and non-genomic [153,154].
The interplay between the estrogen and 3 K-SR receptors has drawn much interest over the past few years.Notably, there has been a wealth of studies in breast cancer in particular that have demonstrated quenching of ER genomic action by the 3 K-SR receptor family members, specifically AR [155] and GR ( [156,157].However, what is perhaps not as well explored is the potential for non-genomic 3 K-SR activity to act as drivers of tumourigenesis especially in the context of anti-estrogen/ ER targeting therapy resistance [158].There is strong epidemiological and clinical evidence that estrogens alone may not be responsible for breast cancer growth and development, particularly in the setting of post-menopausal breast cancer [105,[116][117][118][119]. Furthermore, with improvements in steroid hormone detection it is now possible to evaluate intracrine levels of steroid hormones within cancer tissue in a highly specific and sensitive manner using gas chromatography tandem mass spectrometry.Of note, it has been reported that adrenal steroid prohormones are the most abundant overall in post-menopausal breast cancer tissue and their uptake is also reported to be associated with poor outcome in prostate cancer [147,159].More in-depth exploration of the precursor to potent steroid ratio within post-menopausal breast cancer tissue reveals a huge dichotomy, with ~50 % of the specimens exhibiting abundant adrenal androgens with no detection of estrogen; whilst the other half exhibit androgen prohormone levels below the level of quantification with corresponding elevated levels of estrogens [147].In contrast to the predominately nuclear residency of the ER, the 3 K-SR receptor members are much more commonly found to be in a dynamic state of translocation between the cytosolic and nuclear compartments.This is borne out by the variable response rates to antagonists and agonists of AR [155,[160][161][162], GR [163,164] and PR in breast cancer studies.These newer steroid quantification studies highlight the potential utility of patient specific profiles in determining response to therapy and may have implications for the activity and localisation of SHR.
It is likely that the steroid hormone milieu and tumour intracrinology will be the major determinants of associated kinetic trafficking of SHR.Our appreciation for the complexity of the steroid hormone landscape had been hampered by inferior detection methods until relatively recently.The utility of more sensitive gas/ liquid tandem mass spectrometry approaches have allowed us to more completely understand the nature of systemic steroid levels as well as tumour intracrinology; often with somewhat surprising results such as those reported by Moon et al., wherein many breast tumours show a preponderance of androgens with low conversion to estrogens [147].More recently, inspiring research by a number of groups have explored the existence of an 11-oxygenated adrenal androgen subclass of steroids with unknown biological action [165][166][167][168]. Evidence to date shows that these 11-oxygenated androgens do not diminish with age as observed with classical androgens that are significantly depleted by the 7th decade of life [166].Excess androgens and glucocorticoids in particular are associated with poor outcome in the vast majority of cancers.Whether these are due to perturbation of metabolism or result from alterations in body composition is uncertain, however, both are likely to contribute to some degree.Considering the unique hormone profile of humans, characterised by extended periods of sexual maturation, cyclical fertility and a protracted stage of low reproductive activity/ capacity in old-age, it is imperative that we take a more holistic approach to understanding steroid hormone action by incorporating the ligand profile in analysis.

Considerations for human and health and disease
There is evolutionary evidence that the ancestral origin of modern day SHRs were in fact environmental sensors, this coupled with the concept of the 'soft-key and lock' in the presence of super-abundant ligand highlights how important it is to consider the activity of these receptors in the context of their steroid milieu [169].The relevance of pathologic cytoplasmic SHR action has been known for many decades, however when considering the emerging risk of potent endocrine disrupting chemicals (some endogenous and other xenobiotic) it is an area of SHR biology that has not be fully explored.This is particularly important when we consider that investigation of the endocrine disrupting potential of steroid ligands and their metabolites is primarily based on genomic reporter assays.In light of recent advances in our understanding of steroid hormone action with regards human metabolism means that it is very probable that we do not have a comprehensive platform to determine whether something is endocrine disrupting from a non-genomic, extra-nuclear SHR perspective.
The activation of SHRs is complex and context dependent.Over the past 5 years we have observed an expansion in our understanding around the mono and oligomeric states of these receptors, the various dimerization modes and how flexibility in dimer formation can have an impact on the degeneracy of the DNA motifs to which they are bound.In vivo exploration of the SHR LBD mutations highlight a crucial aspect of SHR biology and its interconnectedness with the hypothalamicpituitaryadrenal (HPA) axis which oftentimes is not apparent via in vitro/ cell free systems.It is very often these aspects of SHR biology that are most challenging to recapitulate due to the unique nature of human endocrinology.Steroid hormones act in concert throughout development and aging and exert systemic, long-lasting effects on reproduction, metabolism, immune response and chronicity, all of which are known to play a role in the development of endocrine-related cancers.Twenty years ago Stephen Hammes authored a paper that stated "the nature of a steroid-induced signal (genomic vs. nongenomic) may depend on the type of target cell, the receptor location within cells, as well as the ligand itself"; this is more relevant today than ever and expanding our appreciation of total steroid action in endocrine-related cancer is still very much a work in progress [170].

Fig. 1 .
Fig. 1.AR activity is mediated by ligand and DNA binding affinity.Androgen receptor protein allostery is mediated by the degree of ligand affinity/ abundance but also by the degree of DNA affinity constrained by the stereochemical properties of the dimer.ERG -ETS Transcription Factor ERG.SWI/SNF -SWItch/Sucrose Non-Fermentable chromatin remeodelling factors, C/PAIS -Complete/ Partial Androgen Insensitivity Syndrome, ARE -Androgen Response Element, DHTdihydroxytestosterone, ARV7 -AR variant 7, LBDligand binding domain, DBD -DNA Binding Domain, ARE* -Androgen Response Element (palindromic, inverted, direct repeats), half-ARE $ -half Androgen Response Element [126,130,136,142].

Table 1
Biological consequence of non-genomic SHR crosstalk activity in cancer.
S. Agbana and M. McIlroy

Table 2
Summary of mSRs.