Hong-Chiang Chang

Cloning and Characterization of Androgen Receptor Coactivator, ARA55, in Human Prostate*

Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors. We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system. ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-β1-inducible gene. Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a ligand-dependent manner. Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nmdihydrotestosterone or its antagonists such as 100 nm17β-estradiol or 1 μm hydroxyflutamide. Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR. Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells. Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation.

Tocopherol-Associated Protein Suppresses Prostate Cancer Cell Growth by Inhibition of the Phosphoinositide 3-Kinase Pathway

Epidemiologic studies suggested that vitamin E has a protective effect against prostate cancer. We showed here that tocopherol-associated protein (TAP), a vitamin E-binding protein, promoted vitamin E uptake and facilitated vitamin E antiproliferation effect in prostate cancer cells. Interestingly, without vitamin E treatment, overexpression of TAP in prostate cancer cells significantly suppressed cell growth; knockdown of endogenous TAP by TAP small interfering RNA (siRNA) in nonmalignant prostate HPr-1 cells increased cell growth. Further mechanism dissection studies suggested that the tumor suppressor function of TAP was via down-regulation of phosphoinositide 3-kinase (PI3K)/Akt signaling, but not by modulating cell cycle arrest or androgen receptor signaling. Immunoprecipitation results indicated that TAP inhibited the interaction of PI3K subunits, p110 with p85, and subsequently reduced Akt activity. Constitutively active Akt could negate the TAP-suppressive activity on prostate cancer cell growth. Moreover, stable transfection of TAP in LNCaP cells suppressed LNCaP tumor incidence and growth rate in nude mice. Furthermore, TAP mRNA and protein expression levels were significantly down-regulated in human prostate cancer tissue samples compared with benign prostate tissues as measured by reverse transcription-PCR, in situ hybridization, and immunohistochemistry. Together, our data suggest that TAP not only mediates vitamin E absorption to facilitate vitamin E antiproliferation effect in prostate cancer cells, but also functions like a tumor suppressor gene to control cancer cell viability through a non-vitamin E manner. Therefore, TAP may represent a new prognostic marker for prostate cancer progression.

Differential induction of androgen receptor transactivation by different androgen receptor coactivators in human prostate cancer DU145 cells.

Recently identified androgen receptor (AR) coactivators were used in this study to determine whether the specificity of sex hormones and antiandrogens could be modulated at the coactivator level. We found that ARA 70 is the best coactivator to confer the androgenic activity on 17β-estradiol. Only ARA 70 and ARA 55 could increase significantly the androgenic activity of hydroxyflutamide, a widely used antiandrogen for the treatment of prostate cancer. None of the AR coactivators we tested could significantly confer androgenic activity on progesterone and glucocorticoid at their physiological concentrations (1–10 nM). We also found that ARA70, ARA55, and ARA 54, but not steroid receptor coactivator-1 (SRC-1) and Rb, could significantly enhance the Δ5-androstenediol-mediated AR transactivation. Furthermore, in comparing the relative specificity of these coactivators to AR in DU 145 cells, our results suggested that ARA70 has a relatively higher specificity and that SRC-1 can enhance almost equally well many other steroid receptors. Finally, our data demonstrated that AR itself and some select AR coactivators such as ARA70 or ARA54 could, respectively, interact with CBP and p300/CBP-associated factors that have histone acetyl-transferase activity for assisting chromatin remodeling. Together, our data suggest that the specificity of sex hormones and antiandrogens can be modulated by some selective AR coactivators. These findings may not only help us to better understand the specificity of the sex hormones and antiandrogens, but also facilitate the development of better antiandrogens to fight the androgen-related diseases, such as prostate cancer.

A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth.

The ligand-bound androgen receptor (AR) regulates target genes via a mechanism involving coregulators such as androgen receptor-associated 54 (ARA54). We investigated whether the interruption of the AR coregulator function could lead to down-regulation of AR activity. Using in vitro mutagenesis and a yeast two-hybrid screening assay, we have isolated a mutant ARA54 (mt-ARA54) carrying a point mutation at amino acid 472 changing a glutamic acid to lysine, which acts as a dominant-negative inhibitor of AR transactivation. In transient transfection assays of prostate cancer cell lines, the mt-ARA54 suppressed endogenous mutated AR-mediated and exogenous wild-type AR-mediated transactivation in LNCaP and PC-3 cells, respectively. In DU145 cells, the mt-ARA54 suppressed exogenous ARA54 but not other coregulators, such as ARA55-enhanced or SRC-1-enhanced AR transactivation. In the LNCaP cells stably transfected with the plasmids encoding the mt-ARA54 under the doxycycline inducible system, the overexpression of the mt-ARA54 inhibited cell growth and endogenous expression of prostate-specific antigen. Mammalian two-hybrid assays further demonstrated that the mt-ARA54 can disrupt the interaction between wild-type ARA54 molecules, suggesting that ARA54 dimerization or oligomerization may play an essential role in the enhancement of AR transactivation. Together, our results demonstrate that a dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells. Further studies may provide a new therapeutic approach for blocking AR-mediated prostate cancer growth.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF‐1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear.

Altered prostate epithelial development and IGF-1 signal in mice lacking the androgen receptor in stromal smooth muscle cells †‡§

Androgens and the androgen receptor (AR) play critical roles in the prostate development via mesenchymal–epithelial interactions. Smooth muscle cells (SMC), differentiated from mesenchyme, are one of the basic components of the prostate stroma. However, the roles of smooth muscle AR in prostate development are still obscure.

CCDC62/ERAP75 functions as a coactivator to enhance estrogen receptor beta-mediated transactivation and target gene expression in prostate cancer cells

Human prostate cancer (PCa) and prostate epithelial cells predominantly express estrogen receptor (ER) beta, but not ERalpha. ERbeta might utilize various ER coregulators to mediate the E2-signaling pathway in PCa. Here, we identified coiled-coil domain containing 62 (CCDC62)/ERAP75 as a novel ER coactivator. CCDC62/ERAP75 is widely expressed in PCa cell lines and has low expression in MCF7 cells. Both in vitro and in vivo interaction assays using mammalian two-hybrid, glutathione S-transferase pull-down and coimmunoprecipitation methods proved that ERbeta can interact with the C-terminus of CCDC62/ERAP75 via the ligand-binding domain. The first LXXLL motif within CCDC62/ERAP75 is required for the interaction between ERbeta and CCDC62/ERAP75. Electrophoretic mobility shift assay showed that CCDC62/ERAP75 can be recruited by the estrogen response element-ER complex in the presence of ligand. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of CCDC62/ERAP75 within the promoter of the estrogen-responsive gene cyclin D1. In addition, using silencing RNA (siRNA) against endogeneous CCDC62/ERAP75, we demonstrated that inhibition of endogenous CCDC62/ERAP75 results in the suppression of ERbeta-mediated transactivation as well as target gene expression in LNCaP cells. More importantly, using the tet-on overexpression system, we showed that induced expression of CCDC62/ERAP75 can enhance the E2-regulated cyclin D1 expression and cell growth in LNCaP cells. Together, our results revealed the role of CCDC62/ERAP75 as a novel coactivator in PCa cells that can modulate ERbeta transactivation and receptor function.

Androgen receptor regulates expression of skeletal muscle-specific proteins and muscle cell types.

C2C12 myoblasts expressing the androgen receptor (AR) were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. Marked up-regulation of AR expression was observed in differentiated myotubes. A nuclear run-on transcription assay demonstrated that transcription of the AR gene is increased during skeletal muscle cell differentiation. Regulation of skeletal muscle-specific protein expression by the androgen-AR signaling pathway was further analyzed using quadriceps skeletal muscle from wild-type (WT) and AR knock-out (ARKO) male mice. A histological analysis of quadriceps skeletal muscle indicates no morphological differences between ARKO and WT mice. However, the androgen-AR signaling pathway increases expression of slow-twitch-specific skeletal muscle proteins and downregulates fast-twitch-specific skeletal muscle proteins, resulting in an increase of slow-twitch muscle fiber type cells in quadriceps muscle.

Infiltrating T cells promote prostate cancer metastasis via modulation of FGF11→miRNA-541→androgen receptor (AR)→MMP9 signaling

Early clinical studies suggested infiltrating T cells might be associated with poor outcomes in prostate cancer (PCa) patients. The detailed mechanisms how T cells contribute to PCa progression, however, remained unclear. Here, we found PCa cells have a better capacity to recruit more CD4(+) T cells than the surrounding normal prostate cells via secreting more chemokines‐CXCL9. The consequences of more recruited CD4(+) T cells to PCa might then lead to enhance PCa cell invasion. Mechanism dissection revealed that infiltrating CD4(+) T cells might function through the modulation of FGF11→miRNA‐541 signals to suppress PCa androgen receptor (AR) signals. The suppressed AR signals might then alter the MMP9 signals to promote the PCa cell invasion. Importantly, suppressed AR signals via AR‐siRNA or anti‐androgen Enzalutamide in PCa cells also enhanced the recruitment of T cells and the consequences of this positive feed back regulation could then enhance the PCa cell invasion. Targeting these newly identified signals via FGF11‐siRNA, miRNA‐541 inhibitor or MMP9 inhibitor all led to partially reverse the enhanced PCa cell invasion. Results from in vivo mouse models also confirmed the in vitro cell lines in co‐culture studies. Together, these results concluded that infiltrating CD4(+) T cells could promote PCa metastasis via modulation of FGF11→miRNA‐541→AR→MMP9 signaling. Targeting these newly identified signals may provide us a new potential therapeutic approach to better battle PCa metastasis.

Desipramine-Induced Apoptosis in Human Pc3 Prostate Cancer Cells: Activation of Jnk Kinase and Caspase-3 Pathways and a Protective Role of Ca2+ (I) Elevation

The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+]i) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+]i rises may slow down or alleviate desipramine-induced cytotoxicity.