Calmodulin protects androgen receptor from calpain-mediated breakdown in prostate cancer cells

Although inactivation of the androgen receptor (AR) by androgen-ablation or anti-androgen treatment has been frontline therapy for disseminated prostate cancer for over 60 years, it is not curative because castration-resistant prostate cancer cells retain AR activity. Therefore, curative strategy should include targeted elimination of AR protein. Since AR binds to calmodulin (CaM), and since CaM-binding proteins are targets of calpain (Cpn)-mediated proteolysis, we studied the role of CaM and Cpn in AR breakdown in prostate cancer cells. Whereas the treatment of prostate cancer cells individually with anti-CaM drug or calcimycin, which increases intracellular Ca(++) and activates Cpn, led to minimal AR breakdown, combined treatment led to a precipitous decrease in AR protein levels. This decrease in AR protein occurred without noticeable changes in AR mRNA levels, suggesting an increase in AR protein turnover rather than inhibition of AR mRNA expression. Thus, CaM inactivation seems to sensitize AR to Cpn-mediated breakdown in prostate cancer cells. Consistent with this possibility, purified recombinant human AR (rhAR) underwent proteolysis in the presence of purified Cpn, and the addition of purified CaM to the incubation blocked rhAR proteolysis. Together, these observations demonstrate that AR is a Cpn target and AR-bound CaM plays an important role in protecting AR from Cpn-mediated breakdown in prostate cancer cells. These observations raise an intriguing possibility that anti-CaM drugs in combination with Cpn-activating agents may offer a curative strategy for the treatment of prostate cancer, which relies on AR for growth and survival.     

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.     

Calpain-mediated androgen receptor breakdown in apoptotic prostate cancer cells

Since androgen receptor (AR) plays an important role in prostate cancer development and progression, androgen-ablation has been the frontline therapy for treatment of advanced prostate cancer even though it is rarely curative. A curative strategy should involve functional and structural elimination of AR from prostate cancer cells. We have previously reported that apoptosis induced by medicinal proteasome-inhibitory compound celastrol is associated with a decrease in AR protein levels. However celastrol-stimulated events contributing to this AR decrease have not been elucidated. Here, we report that a variety of chemotherapeutic agents, including proteasome inhibitors, a topoisomerase inhibitor, DNA-damaging agents and docetaxel that cause cell death, decrease AR levels in LNCaP prostate cancer cells. This decrease in AR protein levels was not due to the suppression of AR mRNA expression in these cells. We observed that a proteolytic activity residing in cytosol of prostate cancer cells is responsible for AR breakdown and that this proteolytic activity was stimulated upon induction of apoptosis. Interestingly, proteasome inhibitor celastrol- and chemotherapeutic drug VP-16-stimulated AR breakdown was attenuated by calpain inhibitors calpastatin and N-acetyl-L-leucyl-L-leucyl-L-methioninal. Furthermore, AR proteolytic activity pulled down by calmodulin-agarose beads from celastrol-treated PC-3 cells showed immunoreactivity to a calpain antibody. Taken together, these results demonstrate calpain involvement in proteasome inhibitor-induced AR breakdown, and suggest that AR degradation is intrinsic to the induction of apoptosis in prostate cancer cells.     

Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation

Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor beta with Src, activates the Src/Raf-1/Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar findings for oestradiol receptor alpha were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK(-) abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor alpha or beta is detected using glutathione S:-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor alpha and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor alpha with Src and consequent activation of Src in intact Cos cells.     

Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.     

Identification and characterization of androgen receptor associated coregulators in prostate cancer cells

The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily that mediates the effects of androgens on target tissues. Over the last decade, it has become apparent that NRs require accessory factors for optimal activation of target gene expression. Numerous NR coregulators have been identified, with diverse structures and potential mechanisms of coregulation, creating an increasingly complicated picture of NR action. Due to the expanding complexity of the coregulator field, this review will focus on the AR ligand-binding domain (LBD) and N-terminal interacting proteins identified by our lab. The LBD-interacting proteins ARA70, ARA55 and ARA54 were first characterized and ARA70 was found to have a relatively higher specificity for the AR in human prostate cancer DU145 cells. Characterization of the functional relationship between the AR and these coregulators indicated that ARA70 and ARA55 could enhance the androgenic effects of 17beta-estradiol (E2) and hydroxyflutamide (HF), an antiandrogen commonly used in the treatment of prostate cancer. ARA160, an AR N-terminal interacting protein also known as TATA element modulatory factor (TMF), was subsequently shown to cooperate with ARA70 in enhancing AR activity. Another AR N-terminal interacting protein, ARA24, interacted with the poly-Q tract, a region within the N-terminus of the AR linked to Kennedy's disease (X-linked spinal and bulbar muscular atrophy). More recently, our lab has identified ARA267, a SET domain containing protein, and supervillin, an F-actin binding protein, as AR coregulators. Collectively, the data from these studies indicate that these coregulators are necessary for optimal AR transactivation. Interruption of the interaction between AR and these proteins may serve as a new therapeutic target in the treatment of prostate cancer.     

The androgen receptor antagonist enzalutamide induces apoptosis,dysregulates the heat shock protein system,and diminishes the androgen receptor and estrogen receptor β1 expression in prostate cancer cells

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.     

Alterations of androgen receptor in prostate cancer

The significance of androgens in the development of prostate cancer has been known for more than half century. During the last decade, a lot of effort has been put to study the significance of the specific nuclear receptor of the hormone, androgen receptor (AR). It has been suggested that polymorphisms, especially the length of CAG repeat in exon 1 of the gene, are associated with the risk of prostate cancer. However, not all studies have confirmed the association. Most surprisingly, it has now become clear that prostate carcinomas emerging during the androgen withdrawal therapy (i.e. hormone-refractory tumors) are capable of reactivating the AR-mediated signalling despite of the low levels of androgens. In addition, it has been shown that AR gene itself is genetically targeted. One-third of the hormone-refractory prostate carcinomas contains amplification of the gene. In addition, 10-30% of prostate carcinomas treated by antiandrogens acquire point mutation in the AR gene. The genetic alterations in AR indicate that receptor should be considered as putative treatment target. Evidently, the currently available antiandrogens are not capable to abolish the AR-mediated signalling efficiently enough.     

Effects of androgen receptor and androgen on gene expression in prostate stromal fibroblasts and paracrine signaling to prostate cancer cells

The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-β, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.     

Suppression of androgen receptor signaling in prostate cancer cells by an inhibitory receptor variant

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.     

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.     

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.