Production of a new triterpenoid disaccharide saponin from sequential glycosylation of ganoderic acid A by 2 Bacillus glycosyltransferases.

Ganoderic acid A (GAA) is a lanostane-type triterpenoid, isolated from medicinal fungus Ganoderma lucidum, and possesses multiple bioactivities. In the present study, GAA was sequentially biotransformed by 2 recently discovered Bacillus glycosyltransferases (GT), BtGT_16345 and BsGT110, and the final product was purified and identified as a new compound, GAA-15,26-O-ß-diglucoside, which showed 1024-fold aqueous solubility than GAA.

Active vitamin D induces gene-specific hypomethylation in prostate cancer cells developing vitamin D resistance.

Prostate cancer (PCa) is a leading cause of cancer death in men. Despite the antiproliferative effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on PCa, accumulating evidence indicates that 1,25(OH)2D3 promotes cancer progression by increasing genome plasticity. Our investigation of epigenetic changes associated with vitamin D insensitivity found that 1,25(OH)2D3 treatment reduced the expression levels and activities of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively). In silico analysis and reporter assay confirmed that 1,25(OH)2D3 downregulated transcriptional activation of the DNMT3B promoter and upregulated microRNAs targeting the 3'-untranslated regions of DNMT3B. We then profiled DNA methylation in the vitamin D-resistant PC-3 cells and a resistant PCa cell model generated by long-term 1,25(OH)2D3 exposure. Several candidate genes were found to be hypomethylated and overexpressed in vitamin D-resistant PCa cells compared with vitamin D-sensitive cells. Most of the identified genes were associated with mammalian target of rapamycin (mTOR) signaling activation, which is known to promote cancer progression. Among them, we found that inhibition of ribosomal protein S6 kinase A1 (RPS6KA1) promoted vitamin D sensitivity in PC-3 cells. Furthermore, The Cancer Genome Atlas (TCGA) prostate cancer data set demonstrated that midline 1 (MID1) expression is positively correlated with tumor stage. Overall, our study reveals an inhibitory mechanism of 1,25(OH)2D3 on DNMT3B, which may contribute to vitamin D resistance in PCa.

Identification of microRNA-98 as a therapeutic target inhibiting prostate cancer growth and a biomarker induced by vitamin D.

The anti-tumor effect of vitamin D has been well recognized but its translational application is hindered by side effects induced by supra-physiological concentration of vitamin D required for cancer treatment. Thus, exploring the vitamin D tumor suppressive functional mechanism can facilitate improvement of its clinical application. We screened miRNA profiles in response to vitamin D and found that a tumor suppressive miRNA, miR-98, is transcriptionally induced by 1α,25-dihydroxyvitamin D(3) (1,25-VD) in LNCaP. Mechanistic dissection revealed that 1,25-VD-induced miR-98 is mediated through both a direct mechanism, enhancing the VDR binding response element in the promoter region of miR-98, and an indirect mechanism, down-regulating LIN-28 expression. Knockdown of miR-98 led to a reduction of 1,25-VD anti-growth effect and overexpression of miR-98 suppressed the LNCaP cells growth via inducing G2/M arrest. And CCNJ, a protein controlling cell mitosis, is down-regulated by miR-98 via targeting 3'-untranslated region of CCNJ. Interestingly, miR-98 levels in blood are increased upon 1,25-VD treatment in mice suggesting the biomarker potential of miR-98 in predicting 1,25-VD response. Together, the finding that growth inhibitive miR-98 is induced by 1,25-VD provides a potential therapeutic target for prostate cancer and a potential biomarker for 1,25-VD anti-tumor action.

Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression.

PURPOSE: High grade bladder cancer is an extremely aggressive malignancy associated with high rates of morbidity and mortality. Understanding how exosomes may affect bladder cancer progression could reveal novel therapeutic targets. MATERIALS AND METHODS: Exosomes derived from human bladder cancer cell lines and the urine of patients with high grade bladder cancer were assessed for the ability to promote cancer progression in standard assays. Exosomes purified from the high grade bladder cancer cell line TCC-SUP and the nonmalignant urothelial cell line SV-HUC were submitted for mass spectrometry analysis. EDIL-3 was identified and selected for further analysis. Western blot was done to determine EDIL-3 levels in urinary exosomes from patients with high grade bladder cancer. shRNA gene knockdown and recombinant EDIL-3 were applied to study EDIL-3 function. RESULTS: Exosomes isolated from high grade bladder cancer cells and the urine of patients with high grade bladder cancer promoted angiogenesis and migration of bladder cancer cells and endothelial cells. We silenced EDIL-3 expression and found that shEDIL-3 exosomes did not facilitate angiogenesis, and urothelial and endothelial cell migration. Moreover, exosomes purified from the urine of patients with high grade bladder cancer contained significantly higher EDIL-3 levels than exosomes from the urine of healthy controls. EDIL-3 activated epidermal growth factor receptor signaling while blockade of epidermal growth factor receptor signaling abrogated this EDIL-3 induced bladder cell migration. CONCLUSIONS: Exosomes derived from the urine of patients with bladder cancer contains bioactive molecules such as EDIL-3. Identifying these components and their associated oncogenic pathways could lead to novel therapeutic targets and treatment strategies.

Biotransformation-guided purification of a novel glycoside derived from the extracts of Chinese herb Baizhi.

Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (DgAS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with DgAS. Structural elucidation confirmed the compound as byakangelicin-7″-O-α-glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of DgAS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds.

Testicular nuclear receptor 4 (TR4) regulates UV light-induced responses via Cockayne syndrome B protein-mediated transcription-coupled DNA repair.

UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.

Suppression of prostate cancer cell rolling and adhesion to endothelium by 1α,25-dihydroxyvitamin D3.

Adhesion of circulating prostate cancer (PCa) cells to the microvascular endothelium is a critical step during cancer metastasis. To study PCa cell rolling and adhesion behavior, we developed a dynamic flow-based microtube system to mimic the microvascular environment. We found that PCa cell rolling capacity is mediated by E-selectin and can be enhanced by stromal cell-derived factor-1 under different wall shear stresses. Using this device, we tested if the chemopreventive agent, vitamin D, could interfere with PCa cell adhesion. We found that 1α,25-dihydroxyvitamin D(3) (1,25-VD), the bioactive form of vitamin D, reduced PCa cell rolling numbers and increased rolling velocities resulting in a significant decreased number of PCa cells adhering to the microtube. The inhibitory effects of 1,25-VD on PCa cell heterotypic adhesion were further confirmed using microvascular endothelial cells in a static condition. Furthermore, we demonstrated that 1,25-VD can increase E-cadherin expression in PCa cells and promote the homotypic cell-cell aggregation, which can then hinder PCa cell adhesion to the endothelium. Blocking E-cadherin with a neutralizing antibody can reverse 1,25-VD-mediated suppression of PCa cell adhesion to the endothelium. Taken together, our data revealed that 1,25-VD promoted PCa cell aggregation by increasing E-cadherin expression, thus interfering with circulating PCa cell adhesion to microvascular endothelial cells and potentially reducing their metastatic potential.

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation.

The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.

Premature aging with impaired oxidative stress defense in mice lacking TR4.

Early studies suggest that TR4 nuclear receptor is a key transcriptional factor regulating various biological activities, including reproduction, cerebella development, and metabolism. Here we report that mice lacking TR4 (TR4(-/-)) exhibited increasing genome instability and defective oxidative stress defense, which are associated with premature aging phenotypes. At the cellular level, we observed rapid cellular growth arrest and less resistance to oxidative stress and DNA damage in TR4(-/-) mouse embryonic fibroblasts (MEFs) in vitro. Restoring TR4 or supplying the antioxidant N-acetyl-l-cysteine (NAC) to TR4(-/-) MEFs reduced the DNA damage and slowed down cellular growth arrest. Focused qPCR array revealed alteration of gene profiles in the DNA damage response (DDR) and anti-reactive oxygen species (ROS) pathways in TR4(-/-) MEFs, which further supports the hypothesis that the premature aging in TR4(-/-) mice might stem from oxidative DNA damage caused by increased oxidative stress or compromised genome integrity. Together, our finding identifies a novel role of TR4 in mediating the interplay between oxidative stress defense and aging.

The roles of testicular nuclear receptor 4 (TR4) in male fertility-priapism and sexual behavior defects in TR4 knockout mice.

BACKGROUND: Successful reproductive efforts require the establishment of a situation favorable for reproduction that requires integration of both behavior and internal physiological events. TR4 nuclear receptor is known to be involved in male fertility via controlling spermatogenesis, yet its roles in regulating other biological events related to reproduction have not been completely revealed. METHODS: Male TR4 knockout (TR4 -/-) and wild type mice were used for the sexual behavior and penile dysfunction studies. Mice were sacrificed for histological examination and corresponding genes profiles were analyzed by quantitative RT-PCR. Reporter gene assays were performed. RESULTS: We describe an unexpected finding of priapism in TR4 -/- mice. As a transcriptional factor, we demonstrated that TR4 transcriptionally modulates a key enzyme regulating penis erection and neuronal nitric oxide synthese NOS (nNOS). Thereby, elimination of TR4 results in nNOS reduction in both mRNA and protein levels, consequently may lead to erectile dysfunction. In addition, male TR4 -/- mice display defects in sexual and social behavior, with increased fear or anxiety, as well as reduced mounting, intromission, and ejaculation. Reduction of ER alpha, ER beta, and oxytocin in the hypothalamus may contribute to defects in sexual behavior and stress response. CONCLUSIONS: Together, these results provide in vivo evidence of important TR4 roles in penile physiology, as well as in male sexual behavior. In conjunction with previous finding, TR4 represents a key factor that controls male fertility via regulating behavior and internal physiological events.

Improving Aqueous Solubility of Natural Antioxidant Mangiferin through Glycosylation by Maltogenic Amylase from Parageobacillus galactosidasius DSM 18751.

Mangiferin is a natural antioxidant C-glucosidic xanthone originally isolated from the Mangifera indica (mango) plant. Mangiferin exhibits a wide range of pharmaceutical activities. However, mangiferin's poor solubility limits its applications. To resolve this limitation of mangiferin, enzymatic glycosylation of mangiferin to produce more soluble mangiferin glucosides was evaluated. Herein, the recombinant maltogenic amylase (MA; E.C. 3.2.1.133) from a thermophile Parageobacillus galactosidasius DSM 18751T (PgMA) was cloned into Escherichia coli BL21 (DE3) via the expression plasmid pET-Duet-1. The recombinant PgMA was purified via Ni2+ affinity chromatography. To evaluate its transglycosylation activity, 17 molecules, including mangiferin (as sugar acceptors), belonging to triterpenoids, saponins, flavonoids, and polyphenol glycosides, were assayed with ß-CD (as the sugar donor). The results showed that puerarin and mangiferin are suitable sugar acceptors in the transglycosylation reaction. The glycosylation products from mangiferin by PgMA were isolated using preparative high-performance liquid chromatography. Their chemical structures were glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin, determined by mass and nucleic magnetic resonance spectral analysis. The newly identified maltosyl-α-(1→6)-mangiferin showed 5500-fold higher aqueous solubility than that of mangiferin, and both mangiferin glucosides exhibited similar 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activities compared to mangiferin. PgMA is the first MA with glycosylation activity toward mangiferin, meaning mangiferin glucosides have potential future applications.

Biotransformation of celastrol to a novel, well-soluble, low-toxic and anti-oxidative celastrol-29-O-ß-glucoside by Bacillus glycosyltransferases.

Celastrol is a quinone-methide triterpenoid isolated from the root extracts of Tripterygium wilfordii (Thunder god vine). Although celastrol possesses multiple bioactivities, the potent toxicity and rare solubility in water hinder its clinical application. Biotransformation of celastrol using either whole cells or purified enzymes to form less toxic and more soluble derivatives has been proven difficult due to its potent antibiotic and enzyme-conjugation property. The present study evaluated biotransformation of celastrol by four glycosyltransferases from Bacillus species and found one glycosyltransferase (BsGT110) from Bacillus subtilis with significant activity toward celastrol. The biotransformation metabolite was purified and identified as celastrol-29-O-ß-glucoside by mass and nuclear magnetic resonance spectroscopy. Celastrol-29-O-ß-glucoside showed over 53-fold higher water solubility than celastrol, while maintained 50% of the free radical scavenging activity of celastrol. When using zebrafish as the in vivo animal model, celastrol-29-O-ß-glucoside exhibited 50-fold less toxicity than celastrol. To our knowledge, the present study is not only the first report describing the biotransformation of celastrol, but also the first one detailing a new compound, celastrol-29-O-ß-glucoside, that is generated in the biotransformation process. Moreover, celastrol-29-O-ß-glucoside may serve as a potential candidate in the future medicine application due to its higher water solubility and lower toxicity.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Protective role of 1 alpha, 25-dihydroxyvitamin D3 against oxidative stress in nonmalignant human prostate epithelial cells.

Overproduction of reactive oxygen species (ROS), through either endogenous or exogenous sources, could induce DNA damage, and accumulation of DNA damage might lead to multistep carcinogenesis. The antioxidative effects of vitamin D have been suggested by epidemiological and many in vitro and in vivo laboratory studies. While exploring the antioxidative effects of vitamin D in prostate cells, we found that the active form of vitamin D, 1 alpha, 25-dihydroxyvitamin D(3) (1,25-VD), can protect nonmalignant human prostate epithelial cell lines, BPH-1 and RWPE-1, but not malignant human prostate epithelial cells, CWR22R and DU 145, from oxidative stress-induced cell death. Glucose-6-phosphate dehydrogenase (G6PD), a key antioxidant enzyme, was dose- and time-dependently induced by 1,25-VD. Mechanistic studies using chromatin immunoprecipitation (ChIP) assay revealed that a direct repeat-3 (DR3) vitamin D response element located in the first intron of the G6PD genome can be bound by liganded vitamin D receptor, thereby regulating G6PD gene expression. Increasing G6PD activity and glutathione level by 1,25-VD can scavenge cellular ROS. Moreover, the protective effects of 1,25-VD were abolished by dehydroepiandrosterone, a noncompetitive inhibitor of G6PD activity. Together, our results showed that 1,25-VD can protect nonmalignant prostate cells from oxidative stress-induced cell death by elimination of ROS-induced cellular injuries through transcriptional activation of G6PD activity. The antioxidative effect of vitamin D strengthens its roles in cancer chemoprevention and adds to a growing list of beneficial effects of vitamin D against cancer.

Increased expression of corepressors in aggressive androgen-independent prostate cancer cells results in loss of 1alpha,25-dihydroxyvitamin D3 responsiveness.

Vitamin D has antiproliferative activity in prostate cancer; however, resistance to vitamin D-mediated growth inhibition occurs. To investigate the mechanisms of vitamin D resistance, we screened two prostate cancer sublines of CWR22rv1, CWR22R-1, and CWR22R-2, with differential sensitivity to vitamin D. CWR22R-2 showed less response to the antiproliferative effect of vitamin D than CWR22R-1. The vitamin D receptor (VDR)-mediated transcriptional activity was also decreased in CWR22R-2. We further showed that the DNA-binding ability of VDR was decreased and the amount of NCoR in VDR response element was increased in CWR22R-2. Analysis of VDR-associated protein profiles found higher expression of the corepressors, NCoR1 and SMRT, in CWR22R-2 cells. Treatment with the histone deacetylase inhibitor, trichostatin A, increased vitamin D/VDR transcriptional activity and promoted the antiproliferative effect of vitamin D in CWR22R-2 cells. Targeted down-regulation of NCoR1 and SMRT by small interference RNA was able to restore CWR22R-2 response to vitamin D. Together, we showed that increased NCoR1 and SMRT expression in CWR22R-2 cells resulted in reduced VDR-mediated transcriptional activity and attenuated antiproliferative response to vitamin D. Our data suggest that the integrity of the vitamin D/VDR-mediated signaling pathway is crucial in predicting vitamin D responsiveness and thus provide a rational design to improve vitamin D-based treatment efficacy based on molecular profiles of patients.

Actin associated proteins function as androgen receptor coregulators: an implication of androgen receptor's roles in skeletal muscle.

This review of androgen receptor (AR) coregulators, which also function as actin-binding proteins, intends to establish the connection between actin cytoskeletal components and androgen signaling, especially in skeletal muscle. In cellular and animal models, androgen activated AR modulates myoblasts proliferation, promotes sexual dimorphic muscle development, and alters muscle fiber type. In the clinical setting, administration of anabolic androgens can decrease cachexia and speed wound healing. During myogenesis and regeneration of skeletal muscle in embryo and adult, the membrane of myoblasts fuse and the actin cytoskeleton is rearranged to form an alignment with myosin to form myotubes then ultimately the myofibrils. Contraction of skeletal muscle promotes the growth of myocytes by coordinating signals from the neuromuscular junction to intra-myofibrils through costameres, the functional structure comprised of signal proteins closely associated with actin filaments and involved in muscular dystrophy. Therefore, the discovery of actin-binding proteins functioning as AR coregulators implies that androgen signaling is tightly regulated during the process of the development and regeneration of skeletal muscle. The search for selective androgen receptor modulators (SARM) that act precisely in skeletal muscle instead of other tissues could target the engineering of a SARM-AR complex that selectively recruits these coregulators.

Docetaxel-induced growth inhibition and apoptosis in androgen independent prostate cancer cells are enhanced by 1alpha,25-dihydroxyvitamin D3.

Pre-treatment with high-dose 1alpha,25-dihydroxyvitamin D(3) (1,25-VD) enhanced the antitumor activity of docetaxel in the androgen-independent prostate cancer cell line, PC-3. The effect manifested as an increasing population of apoptotic cells and amount of pro-apoptotic protein, Bax, under combined treatment compared with single treatment of either 1,25-VD or docetaxel alone. We further demonstrated that pre-treatment with 1,25-VD reduced the expression of multidrug resistance-associated protein-1 at both the mRNA and protein levels. This suggests pre-treatment with 1,25-VD can potentiate cytotoxicity of docetaxel in PC-3 due to 1,25-VD reducing multidrug resistance-associated protein-1 expression.

Modulation of the mRNA-binding protein HuR as a novel reversal mechanism of epirubicin-triggered multidrug resistance in colorectal cancer cells.

HuR (ELAVL1), a RNA-binding protein, plays a key role in posttranscriptional regulation of multidrug resistance (MDR)-related genes. Among various HuR-regulated oncogenic transcripts, the activation of galectin-3/ß-catenin survival pathway is critical to induce transcription of cyclin D1, P-glycoprotein (P-gp) and/or multidrug resistance-associated proteins (MRPs). In this study, we aim to elucidate the HuR-regulating pathways related to epirubicin-mediated resistance in human colorectal carcinoma cells. The effects and mechanisms of epirubicin treatment on the expressions of upstream survival signals (e.g., ß-catenin) and downstream MDR transporters (e.g., P-gp) and anti-apoptotic pathways (e.g., Bcl-2) were assessed with or without HuR knockdown (siHuR) or overexpression (overHuR; ectopic HuR or pcDNA3/HA-HuR). Our results showed that siHuR decreased transcriptional expressions of galectin-3, ß-catenin, cyclin D1, Bcl-2, P-gp, MRP1, and MRP2 in epirubicin-treated colon cancer cells. Consistently, the co-treatment of epirubicin and siHuR diminished the expressions of galectin-3, ß-catenin, c-Myc, P-gp and MRP1. HuR silencing enhanced the intracellular accumulation of epirubicin in colon cancer cells. On the other hand, overHuR abolished such effects. Furthermore, siHuR significantly intensified epirubicin-mediated apoptosis via increasing reactive oxygen species and thus promoted the cytotoxic effect of epirubicin. The combined treatments of siHuR and epirubicin significantly reduced the expression of Bcl-2, but increased the expression of Bax, as well as activity and expression levels of caspase-3 and -9. In contrast, overHuR abrogated these effects. Our findings provide insight into the mechanisms by which siHuR potentiated epirubicin-induced cytotoxicity via inhibiting galectin-3/ß-catenin signaling, suppressing MDR transporters and provoking apoptosis. To our best knowledge, this is an innovative investigation linking the post-transcriptional control by HuR silencing to survival signaling repression, efflux transporter reversal and apoptosis induction. Our study thus provides a powerful regimen for circumventing MDR in colon cancer cells.

Vitamin D receptor-binding site variants affect prostate cancer progression.

Vitamin D is an important modulator of cellular proliferation through the vitamin D receptor (VDR) that binds to DNA in the regulatory sequences of target genes. We hypothesized that single nucleotide polymorphisms (SNPs) in VDR-binding sites might affect target gene expression and influence the progression of prostate cancer. Using a genome-wide prediction database, 62 SNPs in VDR-binding sites were selected for genotyping in 515 prostate cancer patients and the findings were replicated in an independent cohort of 411 patients. Prognostic significance on prostate cancer progression was assessed by Kaplan-Meier analysis and the Cox regression model. According to multivariate analyses adjusted for known predictors, HFE rs9393682 was found to be associated with disease progression for localized prostate cancer, and TUSC3 rs1378033 was associated with progression for advanced prostate cancer in both cohorts. Vitamin D treatment inhibited HFE mRNA expression, and down-regulation of HFE by transfecting small interfering RNA suppressed PC-3 human prostate cancer cell proliferation and wound healing ability. In contrast, vitamin D treatment induced TUSC3 expression, and silencing TUSC3 promoted prostate cancer cell growth and migration. Further analysis of an independent microarray dataset confirmed that low TUSC3 expression correlated with poor patient prognosis. Our results warrant further studies using larger cohorts. This study identifies common variants in VDR-binding sites as prognostic markers of prostate cancer progression and HFE and TUSC3 as plausible susceptibility genes.