Chung S. Song

Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction

Abstract: Androgens are C‐19 steroids secreted primarily from the testes and adrenals that play a critical role in reproduction. Reproductive functions of androgens are mediated through coordination of diverse physiological processes ranging from brain functions to specific cell proliferation and apoptosis. At the molecular level, most of these regulatory influences are exerted by altered expression of appropriate genes by the androgen receptor (AR), a member of the nuclear receptor (NR) superfamily. The unliganded AR is a cytoplasmic protein and, upon ligand binding, it translocates into the nucleus. Thereafter, in conjunction with other transcription factors and coactivators, the AR influences transcription of target genes through a multistep process that includes its clustering in a subnuclear compartment. Here, we describe the genomic organization of the AR, the role of individual structural domains in specific AR function, and the influence of agonistic/antagonistic ligands in the intracellular movement of the receptor. We also show that the AR is capable of undergoing multiple rounds of nucleocytoplasmic recycling after ligand binding and dissociation. Xenobiotic ligands, considered as selective androgen receptor modulators (SARMs), can modulate AR activity by inhibiting either its nuclear translocation or its subnuclear clustering and subsequent transactivation function.

Tissue-specific and Androgen-repressible Regulation of the Rat Dehydroepiandrosterone Sulfotransferase Gene Promoter

Dehydroepiandrosterone sulfotransferase (Std) catalyzes sulfonation of androgenic steroids and certain aromatic procarcinogens. In rats, this enzyme is selectively expressed in the liver, and its expression is strongly repressed by androgens. DNase I footprinting and electrophoretic mobility shift analyses revealed two hepatocyte nuclear factor-1 (HNF1), three CCAAT/enhancer-binding protein (C/EBP), and one consensus palindromic thyroid hormone response elements within the first 215 base pairs (bp) of the promoter sequence of rat Std. This promoter is normally inactive in fibroblast-derived NIH 3T3 cells. However, overexpression of HNF1 and C/EBP resulted in synergistic activation of the Std promoter in this cell type, indicating essential roles of these two trans-regulators in liver-selective expression of the rat Std gene. On the other hand, point mutations at any one of five cis elements proximal to the −215 bp region markedly reduced reporter gene expression, suggesting that all of these sites are important for overall promoter function. Androgenic repression of the Std gene in rat liver can be recapitulated in androgen receptor (AR)-negative HepG2 hepatoma cells after cotransfection with an AR expression plasmid. Functional assay of a nested set of 5′-deleted promoters mapped the negative androgen response region between positions −235 and −310. Antibody supershift and oligonucleotide competition identified three OCT-1 and two C/EBP elements between bp −231 and −292. An additional OCT-1 site was found to overlap with a C/EBP element at the −262/−252 position. Mutational inactivation of any one of five cis elements within the −231/−292 region abolished negative androgen response. However, none of these cis elements showed DNase I protection by recombinant AR in footprinting assay, suggesting the absence of a direct AR-DNA interaction. Thus, these studies on rat Std promoter function indicate that (i) HNF1 and C/EBP are responsible for liver specificity of the ratStd gene; (ii) androgenic repression of the gene requires the presence of all of the OCT-1 and C/EBP elements between positions −231 and −292; and (iii) AR may exert its negative regulatory effect indirectly through transcriptional interference of OCT-1 and C/EBP rather than through a direct DNA-AR interaction.

Interplay of reactive oxygen species, intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca  i2+ surge, increased mitochondrial membrane potential (MMP, ΔΨm), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca  i2+ surge, on the other hand the Ca2+ chelator BAPTA inhibited the Ca  i2+ overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca2+ flux. This suggested that both ROS and Ca  i2+ signaling play roles in the increased MMP via Ca  i2+ ‐dependent and/or ‐independent mechanisms, since ΔΨm elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca  i2+ ‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca2+ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013.

SULT2B1b Sulfotransferase: Induction by Vitamin D Receptor and Reduced Expression in Prostate Cancer

An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3β-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house-developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α-bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease progression.

Dual targeting of androgen receptor and mTORC1 by salinomycin in prostate cancer

Androgen receptor (AR) and PI3K/AKT/mTORC1 are major survival signals that drive prostate cancer to a lethal disease. Reciprocal activation of these oncogenic pathways from negative cross talks contributes to low/limited success of pathway-selective inhibitors in curbing prostate cancer progression. We report that the antibiotic salinomycin, a cancer stem cell blocker, is a dual-acting AR and mTORC1 inhibitor, inhibiting PTEN-deficient castration-sensitive and castration-resistant prostate cancer in culture and xenograft tumors. AR expression, its transcriptional activity, and androgen biosynthesis regulating enzymes CYP17A1, HSD3β1 were reduced by sub-micro molar salinomycin. Estrogen receptor-α expression was unchanged. Loss of phosphorylated AR at serine-81, which is an index for nuclear AR activity, preceded total AR reduction. Rapamycin enhanced the AR protein level without altering phosphoAR-Ser81 and CYP17A1. Inactivation of mTORC1, evident from reduced phosphorylation of mTOR and downstream effectors, as well as AMPK activation led to robust autophagy induction. Apoptosis increased modestly, albeit significantly, by sub-micro molar salinomycin. Enhanced stimulatory TSC2 phosphorylation at Ser-1387 by AMPK, and reduced inhibitory TSC2 phosphorylation at Ser-939/Thr-1462 catalyzed by AKT augmented TSC2/TSC1 activity, which led to mTORC1 inhibition. AMPK-mediated raptor phosphorylation further reduced mTORs kinase function and mTORC1 activity. Our novel finding on dual inhibition of AR and mTORC1 suggests that salinomycin is potentially active as monotherapy against advanced prostate cancer.

Abstract 1917: Loss of steroid sulfotransferase (SULT) in human prostate cancer: Potential implications for SULT as a biomarker and therapeutic target.

Purpose: Reactivated androgen receptor, fueled by elevated tumor tissue 5α-dihydrotestosterone (DHT), plays a key role in the reemergence of prostate cancer. Primary prostate cancer almost always regresses in response to androgen deprivation therapy. The disease, however, recurs and progresses to therapy resistance and death in ∼30% patients. New-generation drugs that inhibit androgen receptor (MDV3100™) or enzymes involved in androgen biosynthesis (Zytiga™, TAK-700™, Dutasteride™) extend survival of post-chemotherapy patients for a limited period following which therapy resistance emerges– hence the challenge to develop alternate means for reducing intra-tumor androgen signaling. Design and Results: 3β-sulfation of dehydroepiandrosterone (DHEA), mediated by the prostate-expressed sulfotransferase SULT2B1b (hereon SULT), is likely to reduce intra-prostate androgen levels since sulfated DHEA cannot be converted to androstenedione and thus to testosterone and DHT. We tested whether enzyme-catalyzed sulfation of DHEA, the obligate precursor steroid for androgen synthesis, is targetable to inhibit prostate cancer. Results show that SULT silencing increased prostate cancer cell proliferation. Clinical prostate cancer specimens showed markedly reduced SULT expression, revealed by immunohistochemistry, western blotting and mRNA quantification. 1,25-dihydroxy vitamin D3 (D3) and sterols enhanced SULT expression due to induction of the SULT2B-encoding gene by D3-activated vitamin D receptor (VDR) and sterol-activated liver X receptor (LXRα). Conclusion: Tumor SULT levels may be a marker for predicting prostate cancer recurrence and progression. We also anticipate that enhancement of SULT activity in tumors by activating VDR and LXRα through combined therapeutic intervention with D3 and sterols would limit the tumor androgen pool and inhibit recurrent, therapy-resistant prostate cancer. Citation Format: Nooshin Mirkheshti, Chung S. Song, Bandana Chatterjee. Loss of steroid sulfotransferase (SULT) in human prostate cancer: Potential implications for SULT as a biomarker and therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1917. doi:10.1158/1538-7445.AM2013-1917

Abstract 1252: Interacting role of the androgen receptor and the corepressor Alien in the proliferation inhibition of prostate cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Androgen receptor (AR) plays a central role in normal prostate growth and in prostate cancer development. The goal of this study was to identify negative regulatory factor(s) that contribute to proliferation inhibition of prostate cancer cells. The biphasic proliferation pattern of LNCaP human prostate cancer cells at low versus high androgen concentrations was utilized to examine regulatory factors involved in AR-mediated regulation of E2F1 gene transcription. E2F1 is a key cell cycle regulatory transcription factor and an important proliferation stimulator of prostate cancer cells. LNCaP cells are known to be stimulated by ≤ 1 nM androgen, while the cells are arrested at G1/S at high androgen (≥10 nM). Quantitative RT-PCR revealed increased expression of E2F1 and E2F1 target genes (such as cyclin A and the antiapoptotic protein Bcl2) in LNCaP cells treated with 0.1nM R1881 (a synthetic androgen). This up regulation was markedly reduced at the 50 nM R1881 treatment condition. The E2F1 promoter activity showed a similar biphasic regulation by low versus high androgen. Functional analysis of deleted E2F1 promoter fragments identified an androgen responsive region (ARR) in the E2F1 upstream promoter. Chromatin immunoprecipitation (ChIP) of LNCaP cells treated with 0.1nM R1881 showed co-recruitment of AR and the p160 coactivator SRC3 to the ARR in parallel to E2F1 gene induction and increased cell proliferation. AR and SRC3 occupancy at ARR was abrogated by 50 nM R1881 treatment, in parallel to de-induction of E2F1 gene expression. Proliferation inhibition at 50 nM R1881 and loss of AR occupancy at ARR was prevented by SRC3 over expression. Reciprocally, proliferation stimulation by 0.1nM R1881 was lost in SRC3-silenced cells. In further novel findings, we show that the corepressor protein Alien (also known as TRIP15) strongly interacted with AR at 50 nM R1881, while interaction of AR with Alien was much weaker at 0.1 nM R1881. Androgen dose-dependent differential interaction between AR and Alien was demonstrated by co-immunoprecipitation and GST pull down assays of LNCaP cell nuclear extracts. Alien was absent constitutively from ARR in the E2F1 promoter at all conditions (i.e. LNCaP cells treated with vehicle, 0.1 nM R1881 or 50 nM R1881). This suggests that Alien interacts with AR in the nucleoplasm, not in a chromatin environment of the E2F1 promoter. Stable knockdown of Alien prevented proliferation inhibition of LNCaP cells at 50 nM R1881. We conclude that the corepressor Alien/TRIP15 negatively regulates proliferation of prostate cancer cells by sequestering AR in the nucleoplasm, which in turn leads to interference with E2F1 gene induction by androgen-activated AR. In ongoing studies we are utilizing a xenograft tumor model in immune-compromised mice to validate the inhibitory role of the corepressor Alien/TRIP15 in the proliferation of human prostate cancer cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1252.