As prostate cancer (PCa) progresses to the lethal castration resistant and metastatic form, genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment contribute to the emergence of unique biological characteristics under the selective pressure of external stresses. These stresses include the therapies applied in the clinic or laboratory and the exposures of cancers to hormonal, paracrine, or autocrine stimuli in the context of the tumor micro- and macro-environment. The androgen receptor (AR) is a key gene involved in PCa etiology and oncogenesis, including disease development, progression, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. Alterations in the AR signaling pathway have been observed in certain selection contexts and contribute to the resistance to agents that target hormonal regulation of the AR, including standard androgen deprivation therapy, antiandrogens such as enzalutamide, and androgen synthesis inhibition with abiraterone acetate. One such resistance mechanism is the synthesis of constitutively active AR variants lacking the canonical ligand-binding domain. This review focuses on the etiology, characterization, biological properties, and emerging data contributing to the clinical characteristics of AR variants, and suggests approaches to full-length AR and AR variant biomarker validation, assessment, and systemic targeting in the clinic.
Kathryn E Ware, Mariano A Garcia-Blanco, Andrew J Armstrong and Scott M Dehm
Jaesung (Peter) Choi, Reena Desai, Yu Zheng, Mu Yao, Qihan Dong, Geoff Watson, David J Handelsman and Ulla Simanainen
Haploinsufficient inactivating phosphatase and tensin homolog (Pten) mutations cause Cowden syndrome, an autosomal dominant risk genotype for hormone dependent reproductive cancers. As androgen actions mediated via the androgen receptor (AR) supports uterine growth and may modify uterine cancer risk, we hypothesized that a functional AR may increase PTEN inactivation induced uterine cancer. To test the hypothesis, we compared the PTEN knockout (PTENKO) induced uterine pathology in heterozygous PTENKO and combined heterozygous PTEN and complete AR knockout (PTENARKO) female mice. PTENKO induced uterine pathology was significantly reduced by AR inactivation with severe macroscopic uterine pathology present in 21% of PTENARKO vs 46% of PTENKO at a median age of 45 weeks. This could be due to reduced stroma ERα expression in PTENARKO compared to PTENKO uterus, while AR inactivation did not modify PTEN or P-AKT levels. Unexpectedly, while progesterone (P4) is assumed protective in uterine cancers, serum P4 was significantly higher in PTENKO females compared to WT, ARKO, and PTENARKO females consistent with more corpora lutea in PTENKO ovaries. Serum testosterone and ovarian estradiol were similar between all females. Hence, our results demonstrated AR inactivation mediated protection against PTENKO induced uterine pathology and suggests a potential role for antiandrogens in uterine cancer prevention and treatment.
W Yue, R J Santen, J P Wang, C J Hamilton and L M Demers
In situ aromatization and enhanced uptake of estradiol from plasma are two potential mechanisms for maintenance of high concentrations of estradiol found in breast tumors of postmenopausal patients. To test the relative importance of these two mechanisms, a nude mouse model was established by inoculating aromatase (A+) and/or sham (A-) transfected MCF-7 cells into ovariectomized mice. Postmenopausal hormonal status was simulated by providing estradiol Silastic implants which clamped plasma estradiol levels at 5-20 pg/ml. We demonstrated that in situ aromatization rather than the uptake mechanism is the key determinant of tumor estradiol levels and tumor growth rate under conditions reflecting the postmenopausal state. The importance of intratumoral aromatase was also suggested by the findings that long-term estrogen deprivation increases sensitivity to estradiol and enhances aromatase activity in MCF-7 cells. The results of our in vivo and in vitro studies suggest that complete blockade of in situ aromatization in the breast would provide added benefit to postmenopausal breast cancer patients, especially those who relapse from antiestrogen therapy.
Donatella Dondi, Margherita Piccolella, Andrea Biserni, Sara Della Torre, Balaji Ramachandran, Alessia Locatelli, Paola Rusmini, Daniela Sau, Donatella Caruso, Adriana Maggi, Paolo Ciana and Angelo Poletti
Prostate cancer (PC) develops in response to an abnormal activation of androgen receptor induced by circulating androgens and, in its initial stages, is pharmacologically controlled by androgen blockade. However, androgen ablation therapy often allows androgen-independent PC development, generally characterized by increased invasiveness. We previously reported that 5α-androstane-3β,17β-diol (3β-Adiol) inhibits the migration of PC cell lines via the estrogen receptor β (ERβ) activation. Here, by combining in vitro assays and in vivo imaging approaches, we analyzed the effects of 3β-Adiol on PC proliferation, migration, invasiveness, and metastasis in cultured cells and in xenografts using luciferase-labeled PC3 (PC3-Luc) cells. We found that 3β-Adiol not only inhibits PC3-Luc cell migratory properties, but also induces a broader anti-tumor phenotype by decreasing the proliferation rate, increasing cell adhesion, and reducing invasive capabilities in vitro. All these 3β-Adiol activities are mediated by ERβ and cannot be reproduced by the physiological estrogen, 17β-estradiol, suggesting the existence of different pathways activated by the two ERβ ligands in PC3-Luc cells. In vivo, continuous administration of 3β-Adiol reduces growth of established tumors and counteracts metastasis formation when PC3-Luc cells are engrafted s.c. in nude mice or are orthotopically injected into the prostate. Since 3β-Adiol has no androgenic activity, and cannot be converted to androgenic compounds, the effects here described entail a novel potential application of this agent against human PC.
Margaret M Centenera, Sarah L Carter, Joanna L Gillis, Deborah L Marrocco-Tallarigo, Randall H Grose, Wayne D Tilley and Lisa M Butler
Persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) underpins the urgent need for therapeutic strategies that better target this pathway. Combining classes of agents that target different components of AR signaling has the potential to delay resistance and improve patient outcomes. Many oncoproteins, including the AR, rely on the molecular chaperone heat shock protein 90 (Hsp90) for functional maturation and stability. In this study, enhanced anti-proliferative activity of the Hsp90 inhibitors 17-allylamino-demethoxygeldanamycin (17-AAG) and AUY922 in androgen-sensitive and CRPC cells was achieved when the agents were used in combination with AR antagonists bicalutamide or enzalutamide. Moreover, significant caspase-dependent cell death was achieved using sub-optimal agent doses that individually have no effect. Expression profiling demonstrated regulation of a broadened set of AR target genes with combined 17-AAG and bicalutamide compared with the respective single agent treatments. This enhanced inhibition of AR signaling was accompanied by impaired chromatin binding and nuclear localization of the AR. Importantly, expression of the AR variant AR-V7 that is implicated in resistance to AR antagonists was not induced by combination treatment. Likewise, the heat shock response that is typically elicited with therapeutic doses of Hsp90 inhibitors, and is a potential mediator of resistance to these agents, was significantly reduced by combination treatment. In summary, the co-targeting strategy in this study more effectively inhibits AR signaling than targeting AR or HSP90 alone and prevents induction of key resistance mechanisms in prostate cancer cells. These findings merit further evaluation of this therapeutic strategy to prevent CRPC growth.
C J Fabian, B F Kimler, M S Mayo and S A Khan
Breast tissue and duct fluid provide a rich source of biomarkers to both aid in the assessment of short-term risk of developing breast cancer and predict and assess responses to prevention interventions. There are three methods currently being utilized to sample breast tissue in asymptomatic women for risk assessment: nipple-aspirate fluid (NAF), random periareolar fine-needle aspiration (RPFNA) and ductal lavage. Prospective single-institution trials have shown that the presence of atypical cells in NAF fluid or RPFNA specimens is associated with an increased risk of breast cancer. Furthermore, RPFNA-detected atypia has been observed to further stratify risk based on the commonly used Gail risk-assessment model. A prospective trial evaluating risk prediction on the basis of atypical cells in ductal-lavage fluid is ongoing. The ability of other established non-genetic biomarkers (mammographic breast density; serum levels of bioavailable estradiol, testosterone, insulin-like growth factor-1 and its insulin like growth factor binding protein-3) to stratify risk based on the Gail model is as yet incompletely defined. Modulation of breast intra-epithelial neoplasia (i.e. hyperplasia with or without atypia) with or without associated breast-tissue molecular markers, such as proliferation, is currently being used to evaluate response in Phase II chemoprevention trials. RPFNA has been the method most frequently used for Phase II studies of 6–12 months duration. However, ductal lavage, RPFNA and random and directed core needle biopsies are all being utilized in ongoing multi-institutional Phase II studies. The strengths and weaknesses of each method are reviewed.
S A Khan, D Bhandare and R T Chatterton Jr
Recent developments in breast epithelial sampling techniques (nipple fluid aspiration, ductal lavage, and random fine needle aspiration) provide new opportunities for the acquisition of hormonal and cellular biomarker data in asymptomatic women, and thereby the possibility of developing a unified vision of how the hormonal environment of the breast may interact with the cellular expression of proteins, and with other evolving candidate markers of breast cancer risk. The purpose of this review is to integrate available information regarding cellular and breast fluid biomarkers of hormone action on the breast, to identify candidate biomarkers for studies of breast cancer risk and prevention. These include the estrogen receptors α andβ, markers of proliferative and apoptotic response, and protein markers of estrogen action in breast cells and nipple fluid. Studies of breast hormone levels in nipple aspiration fluid (NAF) show that estrone sulphate is present in large quantities in the normal breast, while the differences in serum ovarian steroids that are seen in pre- and postmenopausal women are blunted in NAF. The variability of several estradiol precursors in NAF over time is relatively small, a useful attribute of potential biomarkers of breast cancer risk, particularly if they are reversible with intervention in Phase 2 prevention trials. These studies are already providing new insights into the hormonal etiology of breast cancer, and should lead to the identification of robust, reversible biomarkers for use in breast cancer prevention studies.
Kiyoshi Takagi, Yasuhiro Miki, Shuji Nagasaki, Hisashi Hirakawa, Yoshiaki Onodera, Jun-ichi Akahira, Takanori Ishida, Mika Watanabe, Izo Kimijima, Shin-ichi Hayashi, Hironobu Sasano and Takashi Suzuki
Sex steroids play important roles in the development of many human breast carcinomas, and aromatase inhibitors are used for the anti-estrogen therapy. Recent studies have demonstrated that aromatase suppressed 5α-dihydrotestosterone (DHT) synthesis in breast carcinoma cells, but intratumoral concentration of androgens and its significance have not been reported in the breast carcinoma patients treated with aromatase inhibitors. Therefore, we examined androgen concentrations in breast carcinoma tissues treated with exemestane, and further performed in vitro studies to characterize the significance of androgen actions. Intratumoral DHT concentration was significantly higher in breast carcinoma tissues following exemestane treatment (n=9) than those without the therapy (n=7), and 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) status was significantly altered to be positive after the treatment. Following in vitro studies showed that 17βHSD2 expression was dose dependently induced by both DHT and exemestane in T-47D breast carcinoma cells, but these inductions were not additive. DHT-mediated induction of 17βHSD2 expression was markedly suppressed by estradiol (E2) in T-47D cells. E2-mediated cell proliferation was significantly inhibited by DHT in T-47D cells, associated with an increment of 17βHSD2 expression level. These findings suggest that intratumoral androgen actions are increased during exemestane treatment. 17 β HSD2 is a potent DHT-induced gene in human breast carcinoma, and may not only be involved in anti-proliferative effects of DHT on breast carcinoma cells but also serve as a potential marker for response to aromatase inhibitor in the breast carcinoma patients.
Martin K Bakht, Iulian Derecichei, Yinan Li, Rosa-Maria Ferraiuolo, Mark Dunning, So Won Oh, Abdulkadir Hussein, Hyewon Youn, Keith F Stringer, Chang Wook Jeong, Gi Jeong Cheon, Cheol Kwak, Keon Wook Kang, Alastair D Lamb, Yuzhuo Wang, Xuesen Dong and Lisa A Porter
Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate adenocarcinoma (AdPC) cells and acts as a target for molecular imaging. However, some case reports indicate that PSMA-targeted imaging could be ineffectual for delineation of neuroendocrine (NE) prostate cancer (NEPC) lesions due to the suppression of the PSMA gene (FOLH1). These same reports suggest that targeting somatostatin receptor type 2 (SSTR2) could be an alternative diagnostic target for NEPC patients. This study evaluates the correlation between expression of FOLH1, NEPC marker genes and SSTR2. We evaluated the transcript abundance for FOLH1 and SSTR2 genes as well as NE markers across 909 tumors. A significant suppression of FOLH1 in NEPC patient samples and AdPC samples with high expression of NE marker genes was observed. We also investigated protein alterations of PSMA and SSTR2 in an NE-induced cell line derived by hormone depletion and lineage plasticity by loss of p53. PSMA is suppressed following NE induction and cellular plasticity in p53-deficient NEPC model. The PSMA-suppressed cells have more colony formation ability and resistance to enzalutamide treatment. Conversely, SSTR2 was only elevated following hormone depletion. In 18 NEPC patient-derived xenograft (PDX) models we find a significant suppression of FOLH1 and amplification of SSTR2 expression. Due to the observed FOLH1-supressed signature of NEPC, this study cautions on the reliability of using PMSA as a target for molecular imaging of NEPC. The observed elevation of SSTR2 in NEPC supports the possible ability of SSTR2-targeted imaging for follow-up imaging of low PSMA patients and monitoring for NEPC development.
Päivi Järvensivu, Taija Heinosalo, Janne Hakkarainen, Pauliina Kronqvist, Niina Saarinen and Matti Poutanen
Hydroxysteroid (17-beta) dehydrogenase type 1 (HSD17B1) converts low-active estrogen estrone to highly active estradiol. Estradiol is necessary for normal postpubertal mammary gland development; however, elevated estradiol levels increase mammary tumorigenesis. To investigate the significance of the human HSD17B1 enzyme in the mammary gland, transgenic mice universally overexpressing human HSD17B1 were used (HSD17B1TG mice). Mammary glands obtained from HSD17B1TG females at different ages were investigated for morphology and histology, and HSD17B1 activity and estrogen receptor activation in mammary gland tissue were assessed. To study the significance of HSD17B1 enzyme expression locally in mammary gland tissue, HSD17B1-expressing mammary epithelium was transplanted into cleared mammary fat pads of wild-type females, and the effects on mammary gland estradiol production, epithelial cells and the myoepithelium were investigated. HSD17B1TG females showed increased estrone to estradiol conversion and estrogen-response element-driven estrogen receptor signaling in mammary gland tissue, and they showed extensive lobuloalveolar development that was further enhanced by age along with an increase in serum prolactin concentrations. At old age, HSD17B1TG females developed mammary cancers. Mammary-restricted HSD17B1 expression induced lesions at the sites of ducts and alveoli, accompanied by peri- and intraductal inflammation and disruption of the myoepithelial cell layer. The lesions were shown to be estrogen dependent, as treatment with an antiestrogen, ICI 182,780, starting when lesions were already established reversed the phenotype. These data elucidate the ability of human HSD17B1 to enhance estrogen action in the mammary gland in vivo and indicate that HSD17B1 is a factor inducing phenotypic alterations associated with mammary tumorigenesis.