Androgenic steroid action in breast cancer

Androgenic steroid action in breast cancer

By Rachel Bleach, PhD Student, RCSI

By the age of 79 years, 1 in 14 women will develop breast cancer (Global Burden of Disease Cancer 2017). Beatson reported on the utility of removing the ovaries in the treatment of advanced breast cancer in 1896, leading to the observation that estrogen is one of the driving factors in breast cancer. This discovery revolutionised the field for breast cancer treatments and since then, there has been a firm focus on estrogen ablation as a therapy (Love and Philips 2002). Initially therapies such as selective estrogen receptor modulators/degraders were developed to target the estrogen receptor (ER) protein. More recently, targeting of the enzyme CYP19 (aromatase), responsible for the synthesis of estogens from androgens, led to the development of aromatase inhibitor (AI) therapies which have become a standard treatment for ER+ve breast cancer. Both of these therapeutic approaches have been very successful in the clinic, and consequently the mortality rate of breast cancer has been greatly reduced. However, unfortunately approximately one third of women will suffer recurrence of the disease.

Aromatase inhibitor therapies

AI therapy is the first line therapy for postmenopausal women with breast cancer. Their mechanism of action inhibits the conversion of androgens, and as a consequence they create an unopposed highly androgenic steroid tumour environment. The aim of my PhD is to gain an understanding of how the androgen receptor (AR), by way of signalling mechanisms and novel protein interactions, plays a role in the adaptability of a tumour cell to the altered steroid environment resulting from AI treatment.

The AR, like ER, is a member of the steroid nuclear receptor superfamily which are ligand activated transcription factors. The majority of nuclear receptors contain a DNA binding domain (DBD), a ligand binding domain (LBD) and control the expression of a multitude of genes. The AR is the most commonly expressed sex steroid receptor in all stages of breast cancer; its expression is detected in up to 90% of primary tumours and 75% of metastatic tumours (Birrell, Bentel et al. 1995, Moinfar, Okcu et al. 2003).

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Figure 1: In postmenopausal women circulating androgens, androstenedione (4AD) and testosterone, are produced in the ovaries and adrenal glands (left). They are converted to estrogens in peripheral and breast tissue by the aromatase enzyme (centre). Aromatase inhibitor drugs (represented by red x) work by blocking the action of the aromatase enzyme thus inhibiting the conversion of androgens to estrogen. In premenopausal women the ovaries are the main source of estrogen biosynthesis and this is regulated by the hypothalamus and pituitary gland (right). Adapted from: Mechanisms of aromatase inhibitor resistance Ma C.X., Reinert T., Chmielewska I., Ellis M.J. 2015, Nature reviews cancer.

Dr. Jekyll and Mr.Hyde aka the androgen receptor

There is a lot of controversy surrounding the role of AR in breast cancer. Some studies report its expression is associated with a good prognosis (Kim, Jae et al. 2015), while others report its association with a decrease in disease free survival (Choi, Kang et al. 2015). Much like the split personality of Jekyll and Hyde, the androgen receptor can have dual functions depending on the cellular environment and therefore, its association with prognosis is very much context dependent. Studies have shown that alterations in the antagonist relationship between ER and AR may play a role in the development of breast cancer, and in particular, AR:ER expression ratio may be a common feature that influences the development of resistance to ER-directed therapy (Liao and Dickson 2002, Labrie 2006, Cochrane, Bernales et al. 2014). One hypothesis is that in the absence, or low abundance of estrogen, such as in the case of long term estrogen deprivation seen with AI therapy, AR levels increase and it becomes a substitute for ER to drive tumour growth. This mechanism is recapitulated in the cell line model developed in our lab, where ER+ve MCF7 cells were cultured with an AI for >3 months to make them AI resistant. This resulted in an increase in AR expression. Furthermore, proliferation assays demonstrate their growth shifts to become AR dependent as the anti-AR therapy enzalutamide inhibits their growth. These AI resistant cells also develop more stem cell – like characteristics as confirmed via mammosphere formation and self-renewal capabilities which we have demonstrated require AR receptor expression.

Sex(y) Steroids

Understanding the role of steroids is of fundamental importance to the advancement of breast cancer treatments. They are the endogenous ligands to nuclear receptors and therefore their availability and binding is the vital stimulus that drives receptor activation and cellular responses. Studying steroid biochemistry is probably not the sexiest science subject and this is reflected in the void of knowledge on tumour steroid intracrinology. However, to quote from an interesting review last year “It is important to obtain a better understanding of steroid hormone producing enzymes in breast carcinoma tissues, as the ratio of steroids and their metabolites promoting cancer versus those inhibiting cancer will likely determine the status and/or progression of breast cancer tumours” (Africander and Storbeck 2017). Endogenous ligands for AR include dehydroepiandrosterone sulphate (DHEAS), DHEA, androstenedione (4AD), testosterone and 5α-dihydrotestosterone (DHT). The majority of DHEA, 4AD, testosterone and DHEAS is secreted by the adrenal glands, in females testosterone, DHEA and 4AD are also produced in the ovaries (Gao, Bohl et al. 2005). At menopause the ovaries cease estrogen production. However, along with the adrenal gland they continue to produce androgens (Fogle, Stanczyk et al. 2007). During the postmenopausal period the predominant metabolic pathway of estrogen synthesis is from 4AD, however AI therapies prevent this conversion from taking place (Chetrite, Cortes-Prieto et al. 2000). Therefore, all our in vitro work is carried out using the estrogen precursor 4AD as its physiology is the most relevant to the postmenopausal AI resistant setting, and as little is known of tumour intracrinology we cannot assume its conversion to other metabolites.

AR actions – genomic, non-genomic or both?

Androgens bind to the ligand binding domain of the AR and induce a conformational change to an active state heralded by disassociation of the chaperone heat-shock proteins (HSPs). When AR is in this active state in the cytoplasm it can homodimerize (with AR), heterodimerize (for example with ER) or interact with other proteins to initiate rapid second messenger signalling cascades. These actions results in either the canonical – genomic signalling, or non-genomic signalling mechanisms. Genomic signalling involves AR binding to androgen response elements on the DNA, with recruitment of coactivators and corepressors resulting in transcriptional regulation of AR target genes such as prostate specific antigen (PSA). Studies in prostate cancer have shown rapid non genomic second messenger signalling following exposure to androgens (Peterziel H. 1999, Heinlein and Chang 2002). This second messenger signalling cascade occurs within minutes and does not require AR binding to the DNA to induce its effects (Foradori, Weiser et al. 2008). Our research suggests this is also a possible mechanism in AI resistant breast cancer. Initiating these signal transduction pathways can also influence cellular processes such as proliferation, invasion and migration. It is exciting to postulate that this is the mechanism behind the aggressive phenotype we observe in our AI resistant compared to sensitive cell line. So genomic, non-genomic or both? The answer is certainly both actions are occurring, but it is possibly initiated by different ligands and may induce different cellular effects.

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Figure 2: Diagram showing mechanism of genomic and non-genomic signalling of AR in prostate cancer. (A) Genomic signalling requires AR translocation to the nucleus and binding to the DNA (B) Non-genomic signalling which involves AR interacting with signalling molecules in the cytoplasm. Androgen receptor mediated non-genomic regulation of prostate cancer cell proliferation, Liao R.S., Ma S., Miao L., Li R., Yin Y., Raj G.V. 2013, Translational andrology and urology

Since it is known that ligand-activated protein-protein interactions can affect the intracellular mobility of nuclear receptors, it is critical to evaluate both nuclear and cytoplasmic complexes formed by AR in breast cancer. The innovative focus of my project will be to identify novel AR interacting proteins unique to our resistant cell models using state-of –the art mass spectrometry. Characterisation of AR interactors will help elucidate mechanisms of resistance to AI therapy, and in turn these novel AR protein partners will aid the identification of patients who would benefit from anti-AR therapy.


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9th Mar 2021 Rachel Bleach, PhD Student, RCSI

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