Long terminal repeats act as androgen-responsive enhancers for the PSA-Kallikrein locus

The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units

Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades (P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins (P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade (P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue (P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

Analysis of androgen and anti-androgen regulation of KLK-related peptidase 2, 3, and 4 alternative transcripts in prostate cancer

We assessed whether alternative transcripts (using KLK2, KLK3 and KLK4 as models) are differentially regulated by androgens and anti-androgens as an indicator of prostate cancers as they acquire treatment resistance. Using RNAseq of LNCaP cells treated with dihydrotestosterone, bicalutamide and enzalutamide, we show that the expression of variant KLK transcripts is markedly different to other variant transcripts at those loci. We also reveal that KLK variants are also over 2-fold more highly expressed in prostate cancers compared to their corresponding normal prostate. We propose that androgens and anti-androgens can activate specific variant transcripts of critical prostate cancer genes during treatment resistance

An androgen receptor mutation in the MDA-MB-453 cell line model of molecular apocrine breast cancer compromises receptor activity

Recent evidence indicates that the estrogen receptor-a-negative, androgen receptor (AR)- positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as 5a-dihydrotestosterone (DHT) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDAMB- 453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to DHT and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that DHT and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that DHT- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to DHT and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype. Endocrine-Related Cancer (2012) 19 599–613

Differential Regulation of Clusterin Isoforms by the Androgen Receptor

Clusterin (CLU) was initially reported as an androgen-repressed gene which is now shown to be an androgen-regulated ATP-independent cytoprotective molecular chaperone. CLU binds to a wide variety of client proteins to potently inhibit stress-induced protein aggregation and chaperone or stabilise conformations of proteins at times of cell stress. CLU is an enigmatic protein, being ascribed both pro- and anti-apoptotic roles. Recent evidence has shown that both secreted (sCLU) and nuclear (nCLU) isoforms can be produced, and that protein function is dependent on the sub-cellular localisation. We and others have shown that sCLU is cytoprotective, while nCLU is pro-apoptotic. It now seems likely that the apparently dichotomous functions of CLU result from the expression of different but related CLU isoforms and splice variants, and that cell survival depends in part on the relative expression of pro- versus anti-apoptotic CLU proteins. In cancer cells, increased sCLU expression is associated with increased resistance to apoptotic triggers and treatment resistance. CLU is a stress-induced protein upregulated after apoptotic triggers like androgen ablation and chemotherapy. Treatment strategies targeting stress-associated increases in sCLU expression enhance treatment-induced apoptosis and delay the emergence of androgen independence. Differential regulation of CLU isoforms and splice variants by androgens may be a pathway whereby cancer cells develop treatment resistance and evade apoptosis.

Molecular and structural basis of androgen receptor responses to dihydrotestosterone, medroxyprogesterone acetate and Δ4-tibolone

Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3β-OH-tibolone, and a delta-4 isomer (Δ4-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ4-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ4-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ4-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

BM18 : a novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis

BACKGROUND Androgen-dependent prostate cancer (PrCa) xenograft models are required to study PrCa biology in the clinically relevant in vivo environment. METHODS Human PrCa tissue from a femoral bone metastasis biopsy (BM18) was grown and passaged subcutaneously through male severe combined immune-deficient (SCID) mice. Human mitochondria (hMt), prostate specific antigen (PSA), androgen receptor (AR), cytokeratin-18 (CK-18), pan-cytokeratin, and high molecular weight-cytokeratin (HMW-CK) were assessed using immunohistochemistry (IHC). Surgical castration was performed to examine androgen dependence. Serum was collected pre- and post-castration for monitoring of PSA levels. RESULTS: BM18 stained positively for hMt, PSA, AR, CK-18, pan keratin, and negatively for HMW-CK, consistent with the staining observed in the original patient material. Androgen-deprivation induced tumor regression in 10/10 castrated male SCID mice. Serum PSA levels positively correlated with BM18 tumor size. CONCLUSIONS BM18 expresses PSA and AR, and rapidly regresses in response to androgen withdrawal. This provides a new clinically significant PrCa model for the study of androgen-dependent growth.

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.

Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer : an opportunity for intervention

Androgens regulate biological pathways to promote proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen receptor (AR) targeted therapies exploit this dependence and are used in advanced prostate cancer to control disease progression. Contemporary treatment regimens involve sequential use of inhibitors of androgen synthesis or AR function. Although targeting the androgen axis has clear therapeutic benefit, its effectiveness is temporary, as prostate tumor cells adapt to survive and grow. The removal of androgens (androgen deprivation) has been shown to activate both epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD) programs. EMT has established roles in promoting biological phenotypes associated with tumor progression (migration/invasion, tumor cell survival, cancer stem cell-like properties, resistance to radiation and chemotherapy) in multiple human cancer types. NEtD in prostate cancer is associated with resistance to therapy, visceral metastasis, and aggressive disease. Thus, activation of these programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression. Brachyury, Axl, MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications. Understanding tumor cell plasticity will be important in further defining the rational use of androgen-targeted therapies clinically and provides an opportunity for intervention to prolong survival of men with metastatic prostate cancer.

Adverse effects of androgen-deprivation therapy in prostate cancer and their management

Objective To provide an up-to-date summary of current literature on the management of adverse effects of androgen-deprivation therapy (ADT). Patients and Methods All relevant medical literature on men with prostate cancer treated with ADT from 2005 to 2014, and older relevant papers, were reviewed. Recent health advisory statements from the Australian government, societies and advocacy groups have been incorporated to the document. Results There are numerous adverse effects of ADT that require pro-active prevention and treatment. Ranging from cardiovascular disease, diabetes and osteoporosis, to depression, cognitive decline and sexual dysfunction, the range of adverse effects is wide. Baseline assessment, monitoring, prevention and consultation from a multidisciplinary team are important in minimising the harm from ADT. Conclusions This review provides a series of practical recommendations to assist with managing the adverse effects of ADT.