Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In this study, the influence of rooibos on the catalytic activity of enzymes 17β -hydroxysteroid dehydrogenase type 3 (17βHSD3), 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), 5α-reductase type 1 (SRD5A1) and 5α-reductase type 2 (SRD5A2), which catalyse prostate androgen metabolism, was investigated. The activities of both 17βHSD3 and AKR1C3 heterologously expressed in CHO-K1 and HEK293 cells were inhibited significantly by rooibos, with rooibos reducing the conversion of androstenedione (A4) and 11keto-androstenedione (11KA4) to testosterone (T) and 11ketotestosterone (11KT), respectively. The catalytic activity of 17βHSD2 towards T, 11hydroxytestosterone (11OHT) and 11KT was also significantly inhibited by rooibos in transiently transfected HEK293 cells. In transiently transfected HEK293 cells rooibos did not inhibit SRD5A1 while the rate of T conversion to dihydrotestosterone (DHT) by SRD5A2 was decreased. Analysis of steroid metabolism in PNT2 cells also suggests that rooibos does not modulate the catalytic activity of endogenously expressed SRD5A towards A4, however, the conversion of T to DHT was reduced. In addition, reductive 17βHSD activity towards A4 was inhibited in the presence of rooibos in both PNT2 and BPH-1 cells. In contrast, the conversion of 11KA4 to 11KT was inhibited in BPH-1, PC-3 and LNCaP cells, with negligible conversion of 11KA4 in PNT2 cells. Interestingly, data suggests inhibition of 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) activity in the production of androsterone (AST) from 5α–androstenedione (5α-dione), as well as the dehydrogenase reaction of T to A4 in PNT2 cells by rooibos. Androgen metabolism pathways were subsequently investigated in LNCaP cells to determine androgen metabolism by endogenous enzymes. Rooibos resulted in the reduced conversion of A4 in LNCaP cells to the same extent as indomethacin, a known AKR1C3 inhibitor. Rooibos also modulated T, DHT and AST metabolism in LNCaP cells. Furthermore, uridine diphosphate glucuronosyltransferase (UGT) activity in LNCaP cells was inhibited by rooibos, decreasing T-, DHT– and AST-glucuronide formation. These data prompted subsequent investigations into the influence of rooibos at cellular level, and prostatespecific antigen (PSA) levels were assayed in the presence of rooibos. PSA was significantly inhibited by rooibos in the absence and presence of DHT, suggesting possible interaction of rooibos with the mutated androgen receptor (AR) or estrogen receptor-β (ERβ) expressed in LNCaP cells. Taken together, rooibos inhibited the catalytic activity of key enzymes that catalyse the activation of androgens in the prostate, as well as inhibiting enzymes involved in the conjugation of androgens. At cellular level, PSA levels were also decreased by rooibos, possibly through AR or ERβ interactions – clearly indicating a modulatory role for rooibos in active androgen production. / AFRIKAANSE OPSOMMING: In hierdie studie was die invloed van rooibos ten opsigte van die katalitiese aktiwiteite van die ensieme 17β-hidroksi-steroïed-dehidrogenase tipe 2, tipe 3 en tipe 5 (17βHSD2, 17βHSD3, AKR1C3), asook 5α-reduktase tipe 1 en tipe 2 (SRD5A1, SRD5A2) ondersoek. Hierdie ensieme is betrokke in die produksie van androgene in die prostaat. Rooibos het die katalitiese aktiwiteit van 17βHSD3 en AKR1C3 in CHO-K1 en HEK293 selle beïnvloed en het vermindere omskakeling van androstenedioon (A4) en 11keto-androstenedioon (11KA4) na testosteroon (T) en 11-ketotestosteroon (11KT), afsonderlik, veroorsaak. Die katalitiese aktiwiteit van 17βHSD2 teenoor T, 11-hidroksie-testosteroon (11OHT) en 11KT was ook beïnvloed in die teenwoordigheid van rooibos in HEK293 selle. Die katalitiese aktiwiteit van SRD5A1 teenoor A4 en T is nie beïnvloed deur rooibos nie, alhoewel dit voorkom asof rooibos die omsettingstempo van T na dihidrotestosteroon (DHT) deur SRD5A2, getransfekteer in HEK293 selle, verminder het. Verdere ondersoeke is in normale prostaat epiteel selle, in die teenwoordigheid van rooibos uitgevoer. Rooibos het geen invloed op die katalitiese aktiwiteit van SRD5A teenoor A4 gehad nie, alhoewel vermindere omskakeling van T na DHT aangetoon kon word. Rooibos het ook die omskakeling van A4 na T in beide PNT2 en BPH-1 selle tot „n mate geïnhibeer. Die omskakeling van 11KA4 na 11KT was ook verminder in BPH-1, PC-3 en LNCaP selle. Die omskakeling van 11KA4 na 11KT was beduidend laer in PNT2 selle en kon die invloed van rooibos nie aangetoon word nie. Bykomende data toon dat rooibos ook die omskakeling van 5α-androstenedioon (5α-dione) na androsteroon (AST), gekataliseer deur 3α-hidroksi-dehidrogenase tipe 3 (AKR1C2), verminder, gesamentlik met die vermindere omskakeling van T na A4, deur 17βHSD2, in PNT2 selle. Hierdie studie het ook ondersoek ingestel, na die metabolisme van androgene in LNCaP selle. Vermindere A4 metabolisme is in die teenwoordigheid van rooibos asook in die teenwoordigheid van indometasien, „n bekende AKR1C3 inhibitor, gevind. Rooibos verminder dus die aktiwiteit van reduktiewe 17βHSD in LNCaP selle. Verandering in die metabolisme van T, DHT en AST in LNCaP selle, in die teenwoordigheid van rooibos, is ook gevind. Verdere ondersoek in LNCaP selle het gewys dat rooibos „n vermindering in die produksie van gekonjugeerde T, DHT en AST veroorsaak. Die studie het die invloed van rooibos op prostaat-spesifieke antigeen (PSA) ook ondersoek. Daar is vasgestel dat rooibos die vlakke van PSA verminder in die afwesigheid en teenwoordigheid van DHT in LNCaP selle. Hierdie resultaat dui op moontlike interaksie van rooibos met die androgeen (AR) of estrogeen-reseptor-β (ERβ), teenwoordig in LNCaP selle. Rooibos het die katalitiese aktiwiteit van ensieme, wat bydra tot androgeen produksie, geïnhibeer, asook die konjugasie van androgene. Op „n sellulêre vlak, het rooibos die vlakke van PSA-sekresie verminder, wat moontlike interaksie met die AR en ERβ aandui. Hierdie bevindings dui daarop dat rooibos wel n rol het om te speel in die modulasie van aktiewe androgene in die prostaat.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

For the past few years there has been increasing concern surrounding a group of chemicals known as phthalate esters. In mammals, phthalates are known antiandrogens, interfering with the production or activity of testosterone. Phthalates are ubiquitous in the aquatic environment. With recent findings suggesting that antiandrogens may be responsible for much of the endocrine disruption found in wild fish populations, the study of phthalate esters has become integral to determining whether or not these anti-androgenic chemicals are of concern. I investigated whether di-n-butyl phthalate (DBP) was able to cause antiandrogenic endocrine disruption in fish under controlled laboratory conditions. Three experiments were undertaken. In the first study, two generations of fathead minnows were exposed to nominal concentrations of 6 to 100 μg/L for 21 and 150 days, respectively. The second experiment examined the effects of early life-stage exposure to DBP (50, 100 and 200 μg DBP/L) on three-spined sticklebacks. The final experiment examined the effects of DBP on adult male three-spined sticklebacks in a 21-day nesting study (15 and 35 μg DBP/L). DBP had no effect on the fecundity, survival, growth, sex ratio, or gonadal histology of the exposed fish in any of the experiments. Further, it failed to alter the expression of two steroidogenic genes in adult male sticklebacks. In contrast, DBP was often found to significantly alter plasma androgen concentrations in both species, and spiggin concentrations in the three-spined stickleback, most notably causing significantly reduced spiggin concentrations in the adult males exposed to DBP. Ultimately, DBP-exposure did not disrupt the ability of the fish to reproduce successfully, and did not appear to alter reproductive behaviours or the expression of secondary sexual characteristics. In conclusion, while DBP did appear to have some capacity for endocrine disruption in fish, it was unable to interfere with the ability of the fish to develop normally and reproduce successfully. Thus, environmentallyrelevant concentrations of phthalate esters are likely not of particular concern to fish populations.

Espécies silvestres com grande potencial zootécnico devem ser exploradas de forma racional a fim de se evitar a extinção das mesmas. Assim se dá a importância de pesquisas voltadas à reprodução daquelas criadas em cativeiro, como a cutia (Dasyprocta sp.). Este animal é um mamífero e roedor vivente, em sua maioria, na Caatinga brasileira. A ultraestrutura é a base para determinar os estágios celulares e, assim, facilitar as comparações dos processos entre cutias e roedores silvestres ou outros mamíferos. As enzimas P450 aromatase e P450c17 são responsáveis pela regulagem da produção de estrógenos e andrógenos, respectivamente. Considerando a hipótese de que o comportamento de expressão das enzimas do complexo citocromo P540 permanece o mesmo no testículo e na via espermática de cutias durante as fases de desenvolvimento sexual, objetivou-se observar a atuação das enzimas P450 aromatase e P450c17 (17-α-hidroxilase/17,20-liase) nas diferentes fases do desenvolvimento sexual, detalhar a ultraestrutura dos componentes desta via e constatar o desenvolvimento do processo espermatogênico. Segmentos do ducto deferente, epidídimo e testículo de 28 cutias machos em diferentes idades (um dia, 2-14 meses) foram fixados em paraformoldeído e glutaraldeído. O material foi coletado no Centro de Multiplicação da Universidade Federal Rural do Semiárido, Mossoró, RN (Autorização IBAMA nº 2028236/2008). Foram feitos: histologia, seguindo o protocolo padrão para hematoxilina e eosina; processamento para corte semifino (azul de toluidina); microscopia eletrônica de transmissão e varredura; e imunohistoquímica. Este trabalho foi pioneiro ao observar que o epidídimo de cutias é composto por células basais, células principais, células haloides e, quando impúbere, por células \"limpas\", e por células apicais, quando a partir da puberdade. O ducto deferente de cutias antes da puberdade era caracterizado por duas camadas musculares, possivelmente devido à falta de trânsito espermático. No epitélio germinativo foram encontradas, em sua maioria, células em prófase I, principalmente em paquíteno. A espermiogênese é completa quando na pré-puberdade, entretanto, a espermiação ocorre a partir dos 9 meses de idade. A expressão da enzima P450 aromatase variou ao longo do desenvolvimento sexual, sendo na puberdade seu pico de atividade. A P450c17 não mostrou nenhuma ação em qualquer fase sexual. Pode-se concluir que o epitélio germinativo testicular e intersticial, bem como o epitélio pseudoestratificado estereociliado do epidídimo e do ducto deferente de cutias criadas em cativeiro sofrem mudanças morfológicas e funcionais ao longo do desenvolvimento sexual. As atividades androgênicas preponderantes em cutias criadas em cativeiro ocorrem no período da puberdade. / Wild species with great potential livestock should be explored rationally in order to prevent the extinction of the same. Thus is the importance of research aimed at reproducing those bred in captivity, such as agouti (Dasyprocta sp.). This animal is a mammal and rodent living mostly in the Brazilian Caatinga. The ultrastructure is the basis for determining the stages and thus facilitates comparisons of cases between agouti and wild rodents or other mammals. The enzymes P450 aromatase and P450c17 are responsible for regulating the production of estrogens and androgens, respectively. On the assumption that the behavior of expression of the enzymes of complex cytochrome P540 remains the same in the testis and excurrent canals of the agouti during the stages of sexual development, aimed to observe the activity of the enzymes P450 aromatase and P450c17 (17-α- hidroxilase/17,20-lyase) in different stages of sexual development, detail the ultrastructure of the components of this pathway and observe the development of spermatogenesis. Segments of the vas deferens, epididymis and testis of 28 agouti males at different ages (1 day, 2-14 months) were fixed in glutaraldehyde and paraformoldehyde. The material was collected on Center of Multiplication of Federal Rural University of the Semi-arid, Natal, RN (IBAMA Authorization No. 2028236/2008). Were made: histology following the standard protocol for hematoxylin and eosin; processing to semithin (blue toluidine), electron microscopy of transmission and scanning; and immunohistochemistry. This work was pioneered by observing that the epididymis is composed by basal cells, principal cells, haloids cells; and for clean cells when impubertal and apical cells after puberty in agoutis. The vas deferens before puberty was characterized by two muscle layers, possibly due to the lack of sperm transit. In the germinal epithelium were found mostly cells in prophase I, mainly in pachytene. Spermiogenesis is complete when prepubertal phase; however, spermiation takes place from 9 months of age. The expression of enzymes of the cytochrome complex varied over sexual development and peak activity of P450 aromatase was at puberty. The P450c17 showed no action at any stage of sexual development. It can be concluded that the testicular germinal epithelium and interstitial epithelium as well as the pseudostratified estereociliated epithelium of the epididymis and vas deferens undergo morphological and functional changes during the sexual development. Androgenic activities prevalent in agoutis kept in captivity occur during puberty.

The androgenic glands of the Pacific crayfish, Pacifastacus leniusculus trowbridgii Stimpson, 1859, were studied experimentally and by light and electron microscopy. The androgenic glands proliferate in the spring of the year concurrently with an increase in mitotic activity in the testes. Degeneration of a major portion of the glands occurs in the fall, when spermatogenic activity in the testes is near completion. Cellular development appears to progress from small undifferentiated cells through stages of vacuolization and hypertrophy and terminates with degeneration or lysis. The fine structure of the vacuolated gland cells is indicative of increased metabolic activity and probable hormone secretion. However, experimental manipulation of the glands of P. leniusculus gave no evidence of their presumed endocrine function. A discussion of the similarities of the androgenic glands with other known arthropod endocrine glands is also included.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility and the most common endocrinopathy of women of reproductive age. Genome-wide association studies (GWAS) identified a number of loci associated PCOS in different ethnic populations, including women with Asian and European ancestry. Replication studies have confirmed some of these associations. Among the loci identified are those located near the LH receptor gene (LHCGR), a clathrin-binding protein gene (DENND1A) that also functions as a guanine nucleotide exchange factor, and the gene encoding RAB5B, a GTPase and protein involved in vesicular trafficking. The functional significance of one of these GWAS candidates (DENND1A) was supported by our discovery that a truncated protein splice variant of DENND1A termed DENND1A.V2, is elevated in PCOS theca cells, and that forced expression of DENND1A.V2 in normal theca cells increased CYP11A1 and CYP17A1 expression and androgen synthesis, a hallmark of PCOS. We previously proposed that the PCOS GWAS loci could be assembled into a functional network that contributes to altered gene expression in ovarian theca cells, resulting in increased androgen synthesis. Here we demonstrate the localization of LHCGR, DENND1AV.2 and RAB5B proteins in various cellular compartments in normal and PCOS theca cells. hCG and forskolin stimulation affects the distribution and co-localization of DENND1A.V2 and RAB5B in various cellular compartments This cytological evidence supports our PCOS gene network concept, and raises the intriguing possibility that LHCGR activation, via a cAMP-mediated process, promotes the translocation of DENND1A.V2 and RAB5B-containing vesicles from the PCOS theca cell cytoplasm into the nucleus, resulting in increased transcription of genes involved in androgen synthesis.

No description available.

[Truncated abstract] Androgens inhibit the growth of human breast tumours and have been successfully used to treat breast cancer in women. Expression of the androgen receptor (AR), which mediates androgen action, is upregulated in breast cancer cells and the AR is the most frequently expressed steroid hormone receptor in breast tumours. AR levels and activity are modulated by the activity of other signalling pathways, however interactions between the AR and signalling pathways and the consequent alterations to the androgen responsiveness of breast cancer cells are largely uncharacterised. The extracellular signal regulated kinase (ERK1/2) pathway is hyperactivated in ~30% of breast tumours and these tumours are often associated with low oestrogen receptor-a (ERa) levels, reduced responsiveness to antioestrogen therapies and an overall poorer prognosis. In this thesis, the MCF-7 human breast cancer cell line which expresses ERa, progesterone receptor (PR) and the AR, was used to investigate ERK1/2-mediated regulation of the AR and the androgen responsiveness of cells. Inhibition of ERK1/2 signalling was achieved by treatment of cells with U0126, an inhibitor of MEK1/2, the upstream activator of ERK1/2. Hyperactivation of ERK1/2 signalling was achieved by stably transfecting cells with a plasmid encoding a constitutively active form of the MEK1 protein (¿MEK1), resulting in the isolation of two clonal cell populations stably expressing ¿MEK1, ¿C3 and ¿6B, and a monoclonal cell line stably expressing the empty vector, MT3-1. Steady state AR mRNA levels, quantitated using real-time RT-PCR, were increased following U0126 treatment of MCF-7, MT3-1 and ¿6B cells. Conversely, treatment of cells with 10-8M 5a-dihydrotestosterone (DHT) for up to 72 hours decreased AR mRNA levels, indicating that ERK1/2 hyperactivation did not alter the androgenresponsiveness of AR mRNA. '...' Overall levels of AR phosphorylation were enhanced in ¿6B cells in the absence and presence of ligand, indicating that ERK1/2 hyperactivation either directly or indirectly induced receptor phosphorylation. The AR is localised in the cytoplasm in the absence of ligand and was more rapidly translocated to the nucleus in the presence of DHT in ¿C3 cells, an effect that was abrogated in the presence of U0126, thereby indicating an ERK1/2-specific mechanism. AR transcriptional activity, measured using androgen responsive reporter plasmids was not significantly altered in ¿6B cells in either the absence or presence of DHT, although the trend towards enhanced AR activity may be confirmed in future studies using optimised reporter assays. Consistent with the cell cycle regulatory functions of ERK1/2 signalling, proliferation of ¿C3 cells and ¿6B cells was increased in comparison to that of MT3-1 and MCF-7 cells. Treatment of ¿C3 cells and MCF-7 cells with 10-10 – 10-8M DHT produced similar inhibition of proliferation (~40%) during 8 days of culture, with no evidence of cytotoxicity. The results obtained in this thesis demonstrate that while ERK1/2 signalling regulates AR phosphorylation, processing and intracellular localisation, ERK1/2 hyperactivation in breast cancer cells does not inhibit the anti-proliferative effects of androgens. These findings support the development of tissue-specific androgenic treatments for breast tumours including poor prognosis tumours exhibiting ERK1/2 hyperactivation.