The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.
SUMMARY Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These “double-negative” PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo. Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.
We have shown that a polypeptide of M(r) 60,000 (60K) that shares N-terminal homology with a calcium-binding protein, calreticulin, can bind to an amino-acid sequence motif, KXGFFKR, found in the cytoplasmic domains of all integrin alpha-subunits. The homologous amino-acid sequence, KXFFKR (where X is either G, A or V), is also present in the DNA-binding domain of all known members of the steroid hormone receptor family; amino acids in this sequence make direct contact with nucleotides in their DNA-responsive elements and are crucial for DNA binding. Here we show that both the 60K protein (p60), purified on a KLGFFKR-Sepharose affinity matrix, and recombinant calreticulin can inhibit the binding of androgen receptor to its hormone-responsive DNA element in a KXFFKR-sequence-specific manner. Calreticulin can also inhibit androgen receptor and retinoic acid receptor transcriptional activities in vivo, as well as retinoic acid-induced neuronal differentiation. Our results indicate that calreticulin can act as an important modulator of the regulation of gene transcription by nuclear hormone receptors.
Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser 78 and Ser 82
Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.
Background. Since postcastration progression of tumors to an androgen‐independent state appears to be linked to the cessation of androgen‐induced differentiation of tumorigenic stem cells, the authors hypothesized that the replacement of androgens at the end of a period of apoptotic regression might result in the regeneration of differentiated tumor cells with further apoptotic potential. Methods and Results. To determine the effect of intermittent exposure of androgens on the androgen‐dependent Shionogi carcinoma, the tumor was transplanted into a succession of male mice, each of which was castrated when the estimated tumor weight became about 3 g. After the tumor had regressed to 30% of the original weight, it was transplanted into the next noncastrated male. This cycle of transplantation and castration‐induced apoptosis was repeated successfully four times before growth became androgen‐independent during the fifth cycle. In four of Stage C and three of Stage D patients with prostate cancer, androgen withdrawal was initiated with cyproterone acetate (100 mg/d) and diethylstilbestrol (0.1 mg/d) and then maintained with cyproterone acetate in combination with the luteinizing hormone‐releasing hormone agonist, goserelin acetate (3.6 mg/month). After 6 or more months of suppression of serum prostate‐specific antigen (PSA) into the normal range, treatment was interrupted for 2 to 11 months. After recovery of testicular function, androgen‐withdrawal therapy was resumed when serum PSA increased to a level of about 20 μg/l. This cycle was repeated sequentially to a total of two to four times over treatment periods of 21 to 47 months with no loss of androgen dependence. Conclusions. These results demonstrate that intermittent androgen suppression can be used to induce multiple apoptotic regressions of a tumor; they also suggest that the cyclic effects of such treatment on prostate cancer can be followed by the sequential measurement of serum PSA levels.
BackgroundThe androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.ResultsWe previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.ConclusionsThis work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users.
We provide evidence that the androgen receptor (AR) can promote nuclear translocation of -catenin in LNCaP and PC3 prostate cancer cells. Using AR-expressing cells (LNCaP) and non-AR-expressing cells (PC3) we showed by time course cell fractionation that the AR can shuttle -catenin into the nucleus when exposed to exogenous androgen. Cells exposed to the synthetic androgen, R1881, show distinct, punctate, nuclear co-localization of the AR and -catenin. We further showed that the AR does not interact with adenomatous polyposis coli or glycogen synthase kinase-3 and, therefore, conclude that androgenmediated transport of -catenin occurs through a distinct pathway. The minimal necessary components of the AR and -catenin required for binding nuclear accumulation of -catenin nuclear import appears to be the DNA/ligand binding regions and the Armadillo repeats of -catenin. We also employed a novel DNA binding assay to illustrate that -catenin has the capacity to bind to the probasin promoter in an AR-dependent manner. The physiological relevance of AR-mediated transport of -catenin and binding to an AR promoter appeared to be a substantial increase in AR transcriptional reporter activity. ARmediated import represents a novel mode of nuclear accumulation of -catenin. The androgen receptor (AR)1 has a fundamental role in development and differentiation of androgen-sensitive tissue but also has an important role in prostate cancer (1). Proliferation of prostatic epithelium is dependent on the uptake of androgens from the serum through the cell membrane, binding to the cognate steroid receptors, and translocation to the nucleus leading to activation of transcription (2, 3) of downstream genes (4). Structurally, the AR belongs to a superfamily of ligand-activated transcription factors composed of a highly conserved DNA binding domain (AR DBD ) and a moderately conserved ligand binding domain (AR LBD ), while containing an N-terminal domain (AR Nt ), which is least conserved (5-7). The AR Nt contains a ligand-independent transcriptional activating function whereas the AR Ct contains one that is ligand-dependent (8). The ligand binding domain of nuclear receptors interact with a variety of other proteins following ligand binding (9), which has the potential to augment or modulate transcriptional response. The transcriptional activity of the AR is largely determined by the presence or absence of other co-factors, including co-activators, which enhance AR activity, and corepressors, which repress AR activity. Examples of previously identified co-activating molecules of the AR include CBP, SRC1, and TIF-2 (10 -12).There is strong documentation to suggest steroid receptor shuttling upon exposure to the cognate ligand. Such studies have pertained to the AR (13-16), glucocorticoid receptor (GR) (17), estrogen receptor (ER) (19), mineralocorticoid receptor (20), and thyroid receptor (TR) (21). These receptors show a certain degree of trafficking either to or from the nucleus but also in a subnuclear fashion. Those that s...
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