Leupaxin, a Novel Coactivator of the Androgen Receptor, Is Expressed in Prostate Cancer and Plays a Role in Adhesion and Invasion of Prostate Carcinoma Cells

Abstract In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

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Regulation of prostate-specific antigen by activin A in prostate cancer LNCaP cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00443.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. E927-E931 ◽

Cited By ~ 22

Author(s):

Yasuhisa Fujii ◽

Satoru Kawakami ◽

Yohei Okada ◽

Yukio Kageyama ◽

Kazunori Kihara

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Androgen Receptor ◽

Cell Growth ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Activin A ◽

Prostate Tissue ◽

Dependent Manner ◽

Lncap Cells

Activins are multifunctional growth and differentiation factors and stimulate FSH-β gene expression and FSH secretion by the pituitary gonadotropes. Follistatins bind activin, resulting in the neutralization of activin bioactivity. The activin/follistatin system is present in the prostate tissue. Prostate-specific antigen (PSA) plays an important role in male reproductive physiology as well as being very important as a tumor marker for prostate cancer. Thus the regulation of PSA has important clinical implications. Previous studies showed that PSA is primarily regulated by androgens. In the present study, we evaluated the direct effects of activin A on the proliferation and PSA production of prostate cancer LNCaP cells, which express functional activin receptors and androgen receptor and PSA. LNCaP cells were treated with activin A and 5α-dihydrotestosterone (DHT) with or without their antagonists (follistatin or the nonsteroidal anti-androgen bicalutamide). Activin A decreased cell growth of LNCaP cells in a dose-dependent manner, whereas DHT increased it in a biphasic manner. In contrast to their opposing actions on cell growth, both activin A and DHT upregulated PSA gene expression and increased PSA secretion by LNCaP cells. The effects of activin A and DHT to increase PSA production were synergistic or additive. Follistatin or bicalutamide was without effect on cell growth or PSA production. The effects of activin A on LNCaP cells were blocked by follistatin, not by bicalutamide, whereas effects of DHT were prevented by bicalutamide, not by follistatin. Activin A upregulates PSA production, and the effect is through an androgen receptor-independent pathway. The activin/follistatin system can be a physiological modulator of PSA gene transcription and secretion in the prostate tissue, and activins may cooperate with androgen to upregulate PSA in vivo.

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Role of Testosterone Levels on the Combinatorial Effect of Boswellia serrata Extract and Enzalutamide on Androgen Dependent LNCaP Cells and in Patient Derived Cells

Integrative Cancer Therapies ◽

10.1177/1534735421996824 ◽

2021 ◽

Vol 20 ◽

pp. 153473542199682

Author(s):

Prathesha Pillai ◽

Ginil Kumar Pooleri ◽

Shantikumar V. Nair

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Early Stage ◽

Therapeutic Option ◽

Specific Antigen ◽

Cell Killing ◽

Receptor Expression ◽

Lncap Cells ◽

Boswellia Serrata ◽

Physiological Serum

Co-therapy with herbal extracts along with current clinical drugs is being increasingly recognized as a useful complementary treatment for cancer. The anti-cancer property of the phyto-derivative acetyl-11 keto β boswellic acid (AKBA) has been studied in many cancers, including prostate cancer. However, the whole extract of the gum resin Boswellia serrata (BS) and anti-androgen enzalutamide has not been explored in prostate cancer to date. We hypothesized that the BS extract containing 30% (AKBA) with enzalutamide acted synergistically in the early phase of cancer, especially in LNCaP cells, by inhibiting androgen receptor (AR) and by reducing cell proliferation, and further, that the extract would be superior to the action of the active ingredient AKBA when used alone or in combination with enzalutamide. To test our hypothesis, we treated LNCaP cells with BS extract or AKBA and enzalutamide both individually and in combination to analyze cell viability under different levels of dihydrotestosterone (DHT). The inhibition of androgen receptor (AR) followed by the expression of prostate-specific antigen (PSA) and the efflux mechanism of the cells were analyzed to determine the effect of the combination on the cellular mechanism. Cells derived from prostate cancer patients were also tested with the combination. Only 6 µM enzalutamide along with BS in the range of 4.1 µg/ml to 16.4 µg/ml gave the best synergistic results with nearly 50% cell killing even though standard enzalutamide doses were as high as 48 µM. Cell killing was most effective at intermediate DHT concentrations of approximately 1 nM, which corresponds to normal physiological serum levels of DHT. The Pgp expression level and the androgen receptor expression levels were reduced under the combination treatment; the former helping to minimize drug efflux and the latter by reducing the sensitivity to hormonal changes. Furthermore, the combination reduced the PSA level secreted by the cells. In contrast, AKBA could not achieve the needed synergism for adequate cell killing at equivalent concentrations. The combination of enzalutamide and BS extract containing 30% AKBA because of their synergistic interaction is an attractive therapeutic option for treating early stage (hormone-dependent) prostate cancer and is superior to the use of AKBA alone.

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The role of the androgen receptor as a driver and mitigator of cellular stress

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0057 ◽

2020 ◽

Vol 65 (2) ◽

pp. R19-R33

Author(s):

Dimitrios Doultsinos ◽

Ian Mills

Keyword(s):

Prostate Cancer ◽

Protein Synthesis ◽

Androgen Receptor ◽

Cancer Progression ◽

Prostate Gland ◽

Specific Antigen ◽

Nuclear Hormone Receptor ◽

Biological Processes ◽

Normal Prostate ◽

Mtor Activity

Prostate cancer is a high-incidence male cancer, which is dependent on the activity of a nuclear hormone receptor, the androgen receptor (AR). Since the AR is required for both normal prostate gland development and for prostate cancer progression, it is possible that prostate cancer evolves from perturbations in AR-dependent biological processes that sustain specialist glandular functions. The archetypal example of course is the use of prostate specific antigen (PSA), an organ-type specific component of the normal prostate secretome, as a biomarker of prostate cancer. Furthermore, localised prostate cancer is characterised by a low proliferative index and a heterogenous array of somatic mutations aligned to a multifocal disease pattern. We and others have identified a number of biological processes that are AR dependent and represent aberrations in significant glandular processes. Glands are characterised by high rates of metabolic activity including protein synthesis supported by co-dependent processes such as glycosylation, organelle biogenesis and vesicle trafficking. Impairments in anabolic metabolism and in protein folding/processing will inevitably impose proteotoxic and oxidative stress on glandular cells and, in particular, luminal epithelial cells for which secretion is their primary function. As cancer develops there is also significant metabolic dysregulation including impaired negative feedback effects on glycolytic and anabolic activity under conditions of hypoxia and heightened protein synthesis due to dysregulated PI 3-kinase/mTOR activity. In this review we will focus on the components of the AR regulome that support cancer development as well as glandular functions focussing on the unfolded protein response and on regulators of mTOR activity.

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CR6-Interacting Factor 1 Represses the Transactivation of Androgen Receptor by Direct Interaction

Molecular Endocrinology ◽

10.1210/me.2007-0194 ◽

2008 ◽

Vol 22 (1) ◽

pp. 33-46 ◽

Cited By ~ 19

Author(s):

Ji Ho Suh ◽

Minho Shong ◽

Hueng-Sik Choi ◽

Keesook Lee

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Prostate Specific Antigen ◽

Transcriptional Activation ◽

Mrna Level ◽

Specific Antigen ◽

Negative Regulator ◽

Dependent Manner ◽

Target Promoters

Abstract CR6-interacting factor 1 (CRIF1) was previously identified as a nuclear protein that interacts with members of the Gadd45 family and plays a role as a negative regulator in cell growth. However, the nuclear function of CRIF1 remains largely unknown. In this study, we demonstrate that CRIF1 acts as a novel corepressor of the androgen receptor (AR) in prostatic cells. Transient transfection studies show that CRIF1 specifically represses AR transcriptional activation of target promoters in a dose-dependent manner. Additionally, CRIF1 is recruited with AR to the endogenous AR target promoters. In vivo and in vitro protein interaction assays reveal that CRIF1 directly interacts with AR via the activation function-1 domain of AR. Interestingly, both the N-terminal and C-terminal half-regions of CRIF1 are independently capable of interacting with and repressing the transactivation of AR. CRIF1 represses AR transactivation through competition with AR coactivators. In addition, the CRIF1-mediated inhibition of AR transactivation involves the recruitment of histone deacetylase 4. Down-regulation of CRIF1 by small interfering RNA increases the transactivation of AR and the mRNA level of the AR target gene prostate-specific antigen, whereas the overexpression of CRIF1 decreases the prostate-specific antigen mRNA level. Finally, the overexpression of CRIF1 inhibits the androgen-induced proliferation and cell cycle progression of prostate cancer cells. Taken together, these results suggest that CRIF1 acts as an AR corepressor and may play an important role in the regulation of AR-positive growth of prostate cancer.

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Prostate cancer stromal cells and LNCaP cells coordinately activate the androgen receptor through synthesis of testosterone and dihydrotestosterone from dehydroepiandrosterone

Endocrine Related Cancer ◽

10.1677/erc-09-0070 ◽

2009 ◽

Vol 16 (4) ◽

pp. 1139-1155 ◽

Cited By ~ 41

Author(s):

Atsushi Mizokami ◽

Eitetsu Koh ◽

Kouji Izumi ◽

Kazutaka Narimoto ◽

Masashi Takeda ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Stromal Cells ◽

Promoter Activity ◽

Reductase Inhibitor ◽

Specific Antigen ◽

Receptor Activation ◽

Lncap Cells ◽

Steroidogenic Enzymes ◽

Adrenal Androgen

One of the mechanisms through which advanced prostate cancer (PCa) usually relapses after androgen deprivation therapy (ADT) is the adaptation to residual androgens in PCa tissue. It has been observed that androgen biosynthesis in PCa tissue plays an important role in this adaptation. In the present study, we investigated how stromal cells affect adrenal androgen dehydroepiandrosterone (DHEA) metabolism in androgen-sensitive PCa LNCaP cells. DHEA alone had little effect on prostate-specific antigen (PSA) promoter activity and the proliferation of LNCaP cells. However, the addition of prostate stromal cells or PCa-derived stromal cells (PCaSC) increased DHEA-induced PSA promoter activity via androgen receptor activation in the LNCaP cells. Moreover, PCaSC stimulated the proliferation of LNCaP cells under physiological concentrations of DHEA. Biosynthesis of testosterone or dihydrotestosterone from DHEA in stromal cells and LNCaP cells was involved in this stimulation of LNCaP cell proliferation. Androgen biosynthesis from DHEA depended upon the activity of various steroidogenic enzymes present in stromal cells. Finally, the dual 5α-reductase inhibitor dutasteride appears to function not only as a 5α-reductase inhibitor but also as a 3β-hydroxysteroid dehydrogenase inhibitor in LNCaP cells. Taken together, this coculture assay system provides new insights of coordinate androgen biosynthesis under the microenvironment of PCa cells before and after ADT, and offers a model system for the identification of important steroidogenic enzymes involved in PCa progression and for the development of the corresponding inhibitors of androgen biosynthesis.

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Suppression of Androgen Receptor Transactivation and Prostate Cancer Cell Growth by Heterogeneous Nuclear Ribonucleoprotein A1 via Interaction with Androgen Receptor Coregulator ARA54

Endocrinology ◽

10.1210/en.2006-0716 ◽

2007 ◽

Vol 148 (3) ◽

pp. 1340-1349 ◽

Cited By ~ 12

Author(s):

Zhiming Yang ◽

Yu-Jia Chang ◽

Hiroshi Miyamoto ◽

Shuyuan Yeh ◽

Jorge L. Yao ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Cell Growth ◽

Specific Antigen ◽

Suppressive Effect ◽

Hnrnp A1 ◽

Lncap Cells ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein ◽

Interfering Rna

The androgen receptor (AR) requires coregulators for its optimal transactivation. Whether AR coregulators also need interacting proteins to modulate their function remains unclear. Here we describe heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as an associated negative modulator for the AR coregulator ARA54. hnRNP A1 selectively suppressed ARA54-enhanced wild-type and mutant AR transactivation via interruption of AR-ARA54 interaction and ARA54 homodimerization. Stable transfection of hnRNP A1 in the LNCaP cells suppressed AR-mediated cell growth and the expression of prostate-specific antigen, and this suppressive effect was abolished by the addition of ARA54-small interfering RNA. Small interfering RNA knockdown of endogenous hnRNP A1 enhanced cell growth and prostate-specific antigen expression in LNCaP cells. These results not only suggest that the loss of hnRNP A1 expression might activate the ARA54-enhanced cell growth and contribute to the prostate cancer progression, but also demonstrate the dual functional roles for ARA54 as an AR coregulator directly and as a mediator for the suppressive effect of hnRNP A1 indirectly. The novel finding that a protein can modulate AR function without direct interaction with AR might provide a new therapeutic approach to battle prostate cancer by targeting AR indirectly with fewer side effects.

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Nutlin3a Contributes to the Cytoplasmic Retention of Androgen Receptor

Proceedings ◽

10.3390/proceedings2251580 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1580

Author(s):

Gulseren Ozduman ◽

Bilge Debelec Butuner ◽

Kemal Sami Korkmaz

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Cellular Localization ◽

Nuclear Translocation ◽

Intracellular Localization ◽

Critical Role ◽

Cancer Therapeutics ◽

Luciferase Reporter ◽

Dependent Manner ◽

Normal Prostate

Prostate cancer cells need androgens to grow and maintain like normal prostate cells, both utilize that Androgen Receptor (AR) function. Androgen receptor (AR) is expressed throughout the prostate cancer progression plays a critical role as a transcription factor in castration-dependent stages of disease. AR also interacts to many cellular proteins, including p53, to regulate apoptosis. Further, as the stabilization of p53 protein triggers apoptosis, p53 interacting small molecules such as Nutlin3a, are interpreted as cancer therapeutics. In this study, to find out how Nutlin3a-mediated p53 stabilization effect on AR signaling. Here, we investigated the dynamics of p53 binding to transcriptional targets of AR, and further investigated the variations of AR intracellular localization as well as transactivation in the presence of Nutlin3a. To do this, the changes in AR transactivation were investigated via luciferase reporter assay, which was performed by treating LNCaPs with different doses of Nutlin3a and resulted that transactivation was suppressed by Nutlin3a in a dose dependent manner. AR transactivation and sub-cellular localization were also studied by immunofluorescence assay and found that cytoplasmic-nuclear fractionation-coupled western blot analysis showed that Nutlin3a inhibits AR phosphorylation and nuclear translocation regardless of androgens.

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Loss of Fibroblast-Dependent Androgen Receptor Activation in Prostate Cancer Cells is Involved in the Mechanism of Acquired Resistance to Castration

Journal of Clinical Medicine ◽

10.3390/jcm8091379 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1379

Author(s):

Kenichiro Ishii ◽

Izumi Matsuoka ◽

Takeshi Sasaki ◽

Kohei Nishikawa ◽

Hideki Kanda ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Acquired Resistance ◽

Specific Antigen ◽

Receptor Activation ◽

Androgen Deprivation ◽

Mitogen Activated Protein Kinase ◽

Castration Resistant Prostate Cancer ◽

Lncap Cells

Loss of androgen receptor (AR) dependency in prostate cancer (PCa) cells is associated with progression to castration-resistant prostate cancer (CRPC). The tumor stroma is enriched in fibroblasts that secrete AR-activating factors. To investigate the roles of fibroblasts in AR activation under androgen deprivation, we used three sublines of androgen-sensitive LNCaP cells (E9 and F10 cells: low androgen sensitivity; and AIDL cells: androgen insensitivity) and original fibroblasts derived from patients with PCa. We performed in vivo experiments using three sublines of LNCaP cells and original fibroblasts to form homotypic tumors. The volume of tumors derived from E9 cells plus fibroblasts was reduced following androgen deprivation therapy (ADT), whereas that of F10 or AIDL cells plus fibroblasts was increased even after ADT. In tumors derived from E9 cells plus fibroblasts, serum prostate-specific antigen (PSA) decreased rapidly after ADT, but was still detectable. In contrast, serum PSA was increased even in F10 cells inoculated alone. In indirect cocultures with fibroblasts, PSA production was increased in E9 cells. Epidermal growth factor treatment stimulated Akt and p44/42 mitogen-activated protein kinase phosphorylation in E9 cells. Notably, AR splice variant 7 was detected in F10 cells. Overall, we found that fibroblast-secreted AR-activating factors modulated AR signaling in E9 cells after ADT and loss of fibroblast-dependent AR activation in F10 cells may be responsible for CRPC progression.

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Inhibition of MAPK-signaling pathway promotes the interaction of the corepressor SMRT with the human androgen receptor and mediates repression of prostate cancer cell growth in the presence of antiandrogens

Journal of Molecular Endocrinology ◽

10.1677/jme-08-0084 ◽

2009 ◽

Vol 42 (5) ◽

pp. 429-435 ◽

Cited By ~ 15

Author(s):

Michael Eisold ◽

Mohammad Asim ◽

Hanna Eskelinen ◽

Thomas Linke ◽

Aria Baniahmad

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Cell Growth ◽

Signaling Pathway ◽

Specific Inhibitor ◽

Mapk Signaling ◽

Lncap Cells ◽

Cancer Cell Growth ◽

Normal Prostate ◽

Prostate Epithelial Cells

Prostate cancer is one of the most prominent malignancies of elderly males. The growth of normal prostate and prostate cancer (PCa) cells depend on functional androgen receptor (AR), a ligand controlled transcription factor and member of the nuclear receptor superfamily. Binding of agonistic ligand enhances the transactivation function of AR and hence promotes the growth of prostate epithelial cells. We have earlier shown that AR antagonistic ligands such as cyproterone acetate (CPA) promote the recruitment of transcriptional corepressors such as silencing mediator of retinoid and thyroid receptor (SMRT) leading to repression of AR transactivation in non-PCa cells. Unfortunately, however, in LNCaP PCa cells, CPA functions as an agonist and thereby increases AR transactivation function. Here, we show that activated MEK signaling cascade inhibits functional recruitment of corepressor SMRT to CPA-bound AR in PCa cells. Chemical blockade of MEK kinase using a specific inhibitor U0126 increases the interaction and hence repression of AR by the corepressor SMRT in LNCaP PCa cells. This inhibition also results in enhanced antagonistic behavior of CPA as assessed by reporter and cell-growth assays. Moreover, the growth of LNCaP cells stably overexpressing SMRT was more robustly inhibited in the presence of CPA and U1026. In line with this, the growth rate of LNCaP cells was decelerated in the presence of both CPA and U0126. This suggests that activated MEK signaling pathway attenuates the functional recruitment of corepressor SMRT to AR induced by antagonists and thus indicates the important role of corepressors in mediating repression of both AR transactivation and PCa cell growth by antagonists. Furthermore, these findings suggest that combining receptor antagonists with signaling inhibitors could be a beneficial approach for PCa treatment.

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Androgens Suppress EZH2 Expression Via Retinoblastoma (RB) and p130-Dependent Pathways: A Potential Mechanism of Androgen-Refractory Progression of Prostate Cancer

Endocrinology ◽

10.1210/en.2010-0436 ◽

2010 ◽

Vol 151 (11) ◽

pp. 5136-5145 ◽

Cited By ~ 55

Author(s):

Laura R. Bohrer ◽

Shuai Chen ◽

Timothy C. Hallstrom ◽

Haojie Huang

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Target Genes ◽

Retinoblastoma Protein ◽

Lncap Cells ◽

Normal Prostate ◽

Androgen Ablation ◽

Androgen Treatment ◽

Polycomb Protein ◽

Ezh2 Expression

Androgens and the androgen receptor are important for both normal prostate development and progression of prostate cancer (PCa). However, the underlying mechanisms are not fully understood. The Polycomb protein enhancer of zeste homolog 2 (EZH2) functions as an epigenetic gene silencer and plays a role in oncogenesis by promoting cell proliferation and invasion. EZH2 has been implicated in human PCa progression, because its expression is often elevated in hormone-refractory PCa. Here, we demonstrated that expression of EZH2 is lower in androgen-sensitive LNCaP PCa cells compared with Rf and C4-2 cells, two androgen-refractory sublines that are derived from LNCaP cells. Androgen ablation by castration increased the level of EZH2 proteins in LNCaP xenografts in mice. In contrast, treatment of LNCaP cells in culture with the synthetic androgen methyltrieolone (R1881) at doses of 1 nm or higher suppressed EZH2 expression. Moreover, our data suggest that androgen repression of EZH2 requires a functional androgen receptor and this effect is mediated through the retinoblastoma protein and its related protein p130. We further showed that androgen treatment not only increases expression of EZH2 target genes DAB2IP and E-cadherin but also affects LNCaP cell migration. Our results reveal that androgens function as an epigenetic regulator in prostatic cells by repression of EZH2 expression through the retinoblastoma protein and p130-dependent pathways. Our findings also suggest that blockade of EZH2 derepression during androgen deprivation therapy may represent an effective tactic for the treatment of androgen-refractory PCa.

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