Investigating progestin-mediated crosstalk between the androgen receptor and estrogen receptor subtypes in breast cancer cell lines

Brink, Danielle (2019-02)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Estrogen and the estrogen receptor alpha (ERα) are traditionally considered as the main etiological factors in breast cancer. However, other members of the steroid receptor family, including ERβ and the androgen receptor (AR) have also been shown to play a role. It is known that ERα drives breast cancer cell proliferation, while ERβ antagonizes ERα-mediated effects. Moreover, the AR, which is expressed in the majority of ER-positive breast cancer tumours, has been shown to inhibit the transcriptional activity of ERα and increase ERβ expression when activated by the potent natural androgen, 5α-dihydrotestosterone (DHT). Together, this suggests that the AR is associated with a good prognosis in ER-positive breast cancer. The question that arises is whether all agonists binding to the AR would elicit similar effects. This is particularly relevant to progestins used by millions of women in contraception and menopausal hormone therapy (HT), as a number of progestins are known to bind to the AR, with some displaying androgenic properties similar to DHT and others displaying anti-androgenic properties. For example, progestins like medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), and levonorgestrel (LNG) have been shown to be as potent and efficacious as the natural androgen DHT, while others like nestorone (NES) and nomegesterol acetate (NOMAC) display anti-androgenic properties similar to the natural progestogen, progesterone (P4). It is noteworthy that MPA, NET-A and LNG have all been associated with an increased risk of breast cancer. However, the underlying mechanisms whereby these progestins contribute to increased breast cancer risk has not been established. In this study, our main aim was to investigate whether androgenic progestins, unlike anti-androgenic progestins, would elicit similar effects as DHT and the synthetic androgen, mibolerone (Mib), on ERβ and ERα expression in human breast cancer cell lines. First however, we used mammalian two-hybrid assays to investigate the ability of the progestins to induce the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C interaction) of the AR, and showed that progestins elicit different conformations in the receptor. Western blot analysis showed that unlike the androgens that increased AR protein levels, the progestins did not influence AR protein levels in the MCF-7 BUS or MDA-MB-453 breast cancer cells. Quantitative real-time PCR (qPCR) showed that like the androgens, MPA, NET-A and LNG all increased ERβ mRNA expression in the MDA-MB-453 cell line, while P4, NES and NOMAC did not. Moreover, by using the AR antagonist, hydroxyflutamide, we showed that these effects were mediated by the AR. Although these results suggest another mechanism by which the AR may inhibit breast cancer cell growth, the results should be interpreted with caution as we show that the ARmediated effects of the androgens and androgenic progestins, in fact, increased proliferation of the MCF-7 BUS and T47D breast cancer cell lines. Unlike, Mib and DHT, which decreased ERα mRNA expression via the AR, MPA, NET-A and LNG had no effect on ERα expression. Although the precise physiological implications of these preliminary results remain to be determined, our findings highlight the fact that the role of progestins in breast cancer is not straightforward. Moreover, these findings contribute to our understanding of crosstalk between the AR and ER subtypes in breast cancer.

AFRIKAANSE OPSOMMING: Estrogeen en die estrogeenreseptor alfa (ERα) word tradisioneel beskou as die hoof etiologiese faktore in borskanker. Ander lede van die steroïedreseptor familie, insluitende ERβ en die androgen reseptor (AR), speel egter ook 'n rol in borskanker. Dit is bekend dat ERα borskankerproliferasie dryf, terwyl ERβ hierdie ERα-gemedieërde effekte teenwerk. Verder is dit bewys dat die AR, wat in die meerderheid van ER-positiewe borskankergewasse uitgedruk word, die transkripsionele aktiwiteit van ERα inhibeer en die uitdrukking van ERβ verhoog wanneer dit geaktiveer word deur die natuurlike androgeen, 5α-dihidrotestosteroon (DHT). Saam dui dit daarop dat die AR geassosieer word met 'n goeie prognose in ER-positiewe borskanker. Die vraag wat egter ontstaan is of alle agoniste wat aan die AR bind soortgelyke effekte sal ontlok. Hierdie vraag is veral van toepassing op progestiene wat deur miljoene vroue gebruik word in voorbehoedmiddels en menopausale hormoonterapie (HT), aangesien baie van hierdie progestiene aan die AR kan bind, en óf androgen eienskappe soortgelyk aan DHT vertoon, óf anti-androgeniese eienskappe. Byvoorbeeld, progestiene soos medroksieprogesteroon asetaat (MPA), noretisteroon asetaat (NET-A) en levonorgestrel (LNG) is bewys om ewe sterk en doeltreffend as die natuurlike androgeen DHT te wees, terwyl ander soos nesteroon (NES) en nomegesterolasetaat (NOMAC) anti-androgeniese eienskappe soortgelyk aan die natuurlike progestogeen progesteroon (P4) vertoon. Dit is opmerklik dat MPA, NET-A en LNG almal verband hou met 'n verhoogde risiko van borskanker. Die onderliggende meganismes waardeur hierdie progestiene bydra tot 'n verhoogde borskanker risiko is egter nog nie vasgestel nie. In hierdie studie was ons hoofdoel om vas te stel of androgeniese progestiene, in teenstelling met antiandrogeniese progestiene, soortgelyke effekte as DHT en die sintetiese androgeen, miboleroon (Mib), op ERβ- en ERα-uitdrukking in menslike borskanker sellyne sal hê. Eerstens het ons die vermoë van die progestiene ondersoek om die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale domeine (N/C interaksie) van die AR te induseer. Ons het gewys dat progestiene verskillende konformasies in die reseptor ontlok. Deur gebruik te maak van westernklad-analise het ons gewys dat progestiene, in teenstelling met die androgene wat AR-proteïenvlakke verhoog het, geen invloed op AR-proteïenvlakke in die MCF-7 BUS of MDA-MB-453 borskankerselle gehad het nie. Kwantitatiewe intydse PKR (qPKR) het getoon dat MPA, NET-A en LNG, nes die androgene, ERβ mRNA-uitdrukking in die MDA-MB-453-sellyn verhoog, terwyl P4, NES en NOMAC geen effek getoon het nie. Verder, deur die gebruik van die AR antagonis, hidroksieflutamied, het ons getoon dat hierdie effekte deur die AR bemiddel is. Alhoewel hierdie resultate 'n ander meganisme voorstel waarmee die AR borskankergroei kan inhibeer, moet dié resultate met omsigtigheid geïnterpreteer word, aangesien ons toon dat die AR-gemedieërde effekte van die androgene en androgenise progestiene inderdaad die proliferasie van die MCF-7 en T47D-sellyne verhoog. In teenstelling met Mib en DHT, wat via die AR die ERα mRNA uitdrukking verlaag, het MPA, NET-A en LNG geen effek op ERα-uitdrukking gehad nie. Alhoewel die presiese fisiologiese implikasies van hierdie voorlopige resultate nog bepaal moet word, beklemtoon ons bevindings die feit dat die rol van progestiene in borskanker nie eenvoudig is nie. Daarbenewens dra hierdie bevindinge by tot ons begrip van wisselwerking tussen die AR en ER subtipes in borskanker.

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