Investigating the influence of the progesterone receptor isoforms on androgen receptor activity in breast and prostate cancer cell lines

Rademeyer, Anina Zenda (2021-04)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Breast cancer growth and survival are primarily dependent on signalling of estrogens via the estrogen receptor (ER), while for prostate cancer it is dependent on androgens acting via the androgen receptor (AR). However, other steroid receptors such as the progesterone receptor (PR) have also been found in both breast and prostate cancer tumours. Results from a previous study in our laboratory, have suggested crosstalk between the AR and both the PR isoforms, PRA and PRB, in both breast and prostate cancer cell lines. This study also showed that, in addition to transactivation on an androgen response element (ARE), the AR could also transactivate an estrogen response element (ERE), which is the binding site for the ER. As the aforementioned study used synthetic ARE- and ERE-containing promoters in reporter assays, the aim of this study was to assess whether similar responses would also be observed on endogenous AR- and ER-regulated genes in the MDA-MB-231 breast cancer cell line. As a proof of concept, a preliminary experiment was also performed in the PC3 prostate cancer cell line. Whether the AR and PRA or PRB occur in a molecular complex was also investigated using co-immunoprecipitation (Co-IP) assays. Realtime quantitative PCR (qPCR) results showed that both PRA and PRB, modulate the activity of the AR on the expression of the endogenous AR-regulated gene, prostate specific antigen (PSA) and on the ER-regulated genes, cathepsin-D (CTSD) and PR. More specifically, both the unliganded and progesterone (P4)-liganded PR, further increased the dihydrotestosterone (DHT)-induced expression of the AR-regulated PSA gene in the MDA-MB-231 breast cancer cell line. While neither the unliganded PR isoforms, nor liganded PRB, modulated the AR-mediated downregulation of the ER-regulated CTSD gene by DHT, the P4-activated PRA, when expressed in excess, reversed this downregulation. Ligand-activated AR also decreased PR mRNA expression, while both the unliganded and liganded PRA and PRB reversed this decrease. Preliminary results in the PC3 prostate cancer cell line show that ligand activated AR downregulated the expression of the AR-regulated TMPRSS2 gene. Unliganded PRA, expressed at equivalent levels to the AR, lifted this decrease, while when either unliganded or liganded PRA and PRB was present in excess relative to AR, TMRPSS2 mRNA expression was increased. CTSD mRNA expression was increased in MDA-MB-231 breast cancer cells transfected with PRA, suggesting that PRA can activate transcription of an ER-regulated gene. In contrast, PRB decreased expression of an ER-regulated gene, as indicated by the decreased CTSD mRNA expression in MDA-MB-231 cells transfected with PRB. Under the experimental conditions used in this study, we could not show a molecular interaction between the AR and PRA or PRB in COS-1 or MDA-MB-231 cells. However, collectively, the realtime qPCR results support previous findings with promoter-reporters showing crosstalk between the AR and PR in breast cancer, and contribute to our knowledge of steroid receptor crosstalk in breast cancer.

AFRIKAANSE OPSOMMING: Die groei en oorlewing van borskanker is hoofsaaklik afhanklik van estrogeenseine via die estrogeenreseptor (ER), terwyl prostaatkanker afhanklik is van androgene wat via die androgeenreseptor (AR) funksioneer. Ander steroïedreseptore soos die progesteroonreseptor (PR) word egter ook in bors- en prostaatkankergewasse gevind. Resultate van 'n vorige studie in ons laboratorium, het voorgestel dat wisselwerkingsmeganismes tussen die AR en beide die PR isoforme, PRA en PRB, in beide bors- en prostaatkankersellyne voorkom. Hierdie studie het ook getoon dat die AR, benewens transaktivering op 'n androgeenresponselement (ARE), ook 'n estrogeenresponselement (ERE) kan transaktiveer, wat die bindingsplek vir die ER is. Aangesien die voorafgenoemde studie sintetiese ARE- en ERE-bevattende promotors in promotor-rapporteerder toetse gebruik het, was die doel van hierdie studie om te bepaal of soortgelyke response ook op endogene AR- en ER-gereguleerde gene in die MDA-MB-231 borskankersellyn waargeneem sou word. As bewys van die konsep is 'n voorlopige eksperiment ook in die PC3 prostaatkankersellyn uitgevoer. Of AR en PRA of PRB in 'n molekulêre kompleks voorkom, was ook geondersoek met behulp van ko-immunopresipitasie (Co-IP) toetse. Kwantitatiewe intydse PKR (qPKR) resultate het getoon dat beide PRA en PRB die aktiwiteit van die AR op die uitdrukking van die endogene AR-gereguleerde geen, prostaat- spesifieke antigeen (PSA) en op die ER-gereguleerde gene, katepsien-D (CTSD) en PR, moduleer. Meer spesifiek, beide die ligandlose en progesteroon (P4) ligand-gebonde PR, het die 5α-dihidrotestosteroon (DHT) geïnduseerde uitdrukking van die AR-gereguleerde PSA geen in die MDA-MB-231 borskankersellyn verder verhoog. Alhoewel die ligandlose PR isoforme, en ligand-gebonde PRB, nie die AR-gemedieerde afregulering van die ER- gereguleerde CTSD-geen deur DHT gemoduleer het nie, het die P4-geaktiveerde PRA hierdie afregulering omgekeer, wanneer dit in oormaat uitgedruk is. Ligand-geaktiveerde AR het ook die uitdrukking van PR mRNA-uitdrukking verminder, terwyl beide die ligandlose en ligand- gebonde PRA en PRB hierdie afname omgekeer het. Voorlopige resultate in die PC3 prostaatkankersellyn toon dat ligand-geaktiveerde AR die uitdrukking van die AR- gereguleerde TMPRSS2-geen afreguleer. Ligandlose PRA, uitgedruk teen dieselfde vlakke as die AR, lig hierdie afname, terwyl ligandlose en ligand-gebonde PRA en PRB ooruitgedruk teenoor die AR, die TMRPSS2 mRNA uitdrukking opreguleer. CTSD mRNA uitdrukking is verhoog in MDA-MB-231 borskankerselle wat met PRA getransfekteer was, wat daarop dui dat PRA transkripsie van 'n ER-gereguleerde geen kan aktiveer. In teenstelling, verminder PRB die uitdrukking van 'n ER-gereguleerde geen, soos aangedui deur die verminderde uitdrukking van CTSD mRNA in MDA-MB-231 selle wat met PRB getransfekteer is. Onder die eksperimentele toestande wat in hierdie studie gebruik was, kon ons nie 'n molekulêre interaksie tussen AR en PRA of PRB in COS-1 of MDA-MB-231 selle toon nie. Gesamentlik ondersteun die qPKR resultate egter vorige bevindings met promotor-rapporteerders wat wisselwerking tussen AR en PR in borskanker toon, en dit dra by tot ons kennis van steroïedreseptor wisselwerkingsmeganismes in borskanker.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/110328
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