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Background Markers of stromal activation at future metastatic sites may have prognostic value and may allow clinicians to identify and abolish the pre-metastatic niche to prevent metastasis. In this study, we evaluate tenascin-C as a marker of pre-metastatic niche formation in bladder cancer patient lymph nodes. Methods Tenascin-C expression in benign lymph nodes was compared between metastatic (n = 20) and non-metastatic (n = 27) patients with muscle-invasive bladder cancer. Urinary extracellular vesicle (EV) cytokine levels were measured with an antibody array to examine potential correlation with lymph node inflammation. The ability of bladder cancer EVs to activate primary bladder fibroblasts was assessed in vitro. Results Lymph node tenascin-C expression was elevated in metastatic patients vs. non-metastatic patients, and high expression was associated with worse survival. Urinary EVs contained four cytokines that were positively correlated with lymph node tenascin-C expression. Bladder cancer EVs induced tenascin-C expression in fibroblasts in an NF-κB-dependent manner. Conclusions Tenascin-C expression in regional lymph nodes may be a good predictor of bladder cancer metastasis and an appropriate imaging target. It may be possible to interrupt pre-metastatic niche formation by targeting EV-borne tumour cytokines or by targeting tenascin-C directly.

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.

INTRODUCTION AND OBJECTIVE:Extracellular vesicles (EV) comprise a diverse group of small vesicles secreted by almost all types of cells into the extracellular space. EV have been shown to play cruc...

INTRODUCTION AND OBJECTIVE:Cigarette smoking is the most well-established risk factor for bladder cancer (BC). E-cigarette (E-cig) use is steadily increasing, and there is preclinical evidence that...

INTRODUCTION AND OBJECTIVE:Non-muscle invasive bladder cancer (NMIBC) exhibits a high recurrence rate and tumor multifocality, placing significant financial and psychological burden on patients. Ex...

Recent data suggest that patients with a basal/stem-like bladder cancer (BC) subtype tend to have metastatic disease, but this is unconfirmed. Here we report the identification of murine MB49 cell line sub-clones with stem-like characteristics in culture. Subcutaneous implantation of S2 and S4 MB49 sub-clones into immunocompetent mice resulted in lung metastases in 50% and 80% of mice respectively, whereas none of the mice implanted with the parental cells developed metastasis. Gene profiling of cells cultured from S2 and S4 primary and metastatic tumors revealed that a panel of genes with basal/stem-like/EMT properties is amplified during metastatic progression. Among them, ITGB1, TWIST1 and KRT6B are consistently up-regulated in metastatic tumors of both MB49 sub-clones. To evaluate clinical relevance, we examined these genes in a human public dataset and found that ITGB1 and KRT6B expression in BC patient tumor samples are positively correlated with tumor grade. Likewise, the expression levels of these three genes are correlated with worse clinical outcomes. This MB49 BC metastatic pre-clinical model provides a unique opportunity to validate and recapitulate results discovered in patient studies and to pursue future mechanistic therapeutic interventions for BC metastasis.

INTRODUCTION AND OBJECTIVE: High recurrence rates found in non-muscle invasive bladder cancer (NMIBC) pose a financial and psychological burden for patients. We demonstrated that bladder cancer (BC) cells release protein disulfide isomerase (PDI), a redox-regulated ER-resident protein, via extracellular vesicles (EVs) when experiencing high levels of ER stress for their own survival. Chronic exposure to PDI-enriched BC EVs induced malignant transformation in recipient non-transformed cells. We hypothesized that the dynamic redox state in BC cells determines PDI cellular localization and tumors with elevated PDI preferentially incorporate PDI into EVs for removal. Furthermore, PDI expression levels and subcellular localization in tumors from NMIBC patients may predict NMIBC recurrence. METHODS: Retrospective chart review identified 122 patients with newly diagnosed low-grade NMIBC classified into 2 groups: recurrent (N=55) and non-recurrent (N=67) during follow-up period. Formalin fixed paraffin embedded tumor specimens from index transurethral resection of bladder tumor (TURBT) were obtained and a tissue microarray (TMA) was constructed. Immunofluorescent staining of TMA sections was conducted using antibodies against PDI and BiP, an ER stress marker. Stained tissues were photographed and tumor regions in each field were manually masked and confirmed by a genitourinary pathologist. Within each tumor region, total antibody labeling was determined by measuring the mean pixel intensity values with NIH ImageJ/Fiji. Manders coefficient was used to assess the colocalization of epitopes using JACoP plugin in ImageJ. RESULTS: Tumor tissues from patients that recurred expressed significantly higher levels of PDI and BiP. Moreover, high PDI and BiP expression predicted worse recurrence-free survival. These data suggest that tissues under higher levels of ER stress—as indicated by higher levels of PDI and BiP expression—have a higher risk of recurrence. Significantly higher levels of PDI were detected in non-ER cytosol in recurrent cohort tumors. CONCLUSIONS: This study provides a novel mechanism for BC recurrence and valuable clinical predictive biomarkers for NMIBC recurrence and progression. Future clinical applications targeting this pathway may be an avenue to prevent NMIBC recurrence. Citation Format: Kit L. Yuen, Chia-Hao Wu, Christopher R. Silvers, Alexis R. Steinmetz, Hiroshi Miyamoto, Edward M. Messing, Yi-Fen Lee. Protein disulfide isomerase as a predictive biomarker for non-muscle invasive bladder cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 517.

INTRODUCTION AND OBJECTIVE:Increasing evidence suggests that inflammation and stromal activation at pre-metastatic sites constitute a niche that supports the colonization and outgrowth of dissemina...

// Christopher R. Silvers 1 , Hiroshi Miyamoto 1, 2, 3 , Edward M. Messing 1 , George J. Netto 3 and Yi-Fen Lee 1, 2 1 Department of Urology, University of Rochester Medical Center, Rochester, NY, USA 2 Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA 3 Departments of Pathology and Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Yi-Fen Lee, email: yifen_lee@urmc.rochester.edu Keywords: extracellular vesicles, exosomes, bladder cancer, proteomic, transaldolase Received: January 10, 2017 Accepted: July 26, 2017 Published: August 08, 2017 ABSTRACT The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers ( n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples ( n = 51). While cytoplasmic transaldolase was found in 89–90% of both normal urothelium ( n = 79) and non-muscle-invasive samples ( n = 71), the rate falls to 39% in MIBC samples ( P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients ( P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation.

// Christopher R. Silvers 1 , Yu-Ru Liu 1 , Chia-Hao Wu 1 , Hiroshi Miyamoto 2 , Edward M. Messing 1 , Yi-Fen Lee 1 1 Department of Urology, University of Rochester Medical Center, Rochester, NY, USA 2 Departments of Pathology and Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Yi-Fen Lee, e-mail: yifen_lee@urmc.rochester.edu Keywords: periostin, extracellular vesicle, exosome, muscle-invasive bladder cancer and biomarker Received: August 24, 2015 Accepted: February 25, 2016 Published: March 10, 2016 ABSTRACT Muscle-invasive bladder cancer (MIBC) is an aggressive malignancy with high mortality, and heterogeneity in MIBC results in variable clinical outcomes, posing challenges for clinical management. Extracellular vesicles (EVs) derived from MIBC have been shown to promote cancer progression. EVs derived from bladder cell lines were subjected to proteomic analysis, and periostin was chosen for further characterization due to its stage-specific gene expression profile. Knockdown of periostin by RNA interference reduces invasiveness in vitro and produces a rounder morphology. Importantly, treating low grade BC cells with periostin-rich EVs promotes cell aggressiveness and activates ERK oncogenic signals, and periostin suppression reverses these effects. These data suggest that MIBC might transfer periostin in an EV-mediated paracrine manner to promote the disease. To determine the potential of periostin as a bladder cancer indicator, patient urinary EVs were examined and found to have markedly higher levels of periostin than controls. In addition, immunohistochemical staining of a bladder cancer tissue microarray revealed that the presence of periostin in MIBC cells is correlated with worse prognosis. In conclusion, periostin is a component of bladder cancer cells associated with poor clinical outcome, and EVs can transfer oncogenic molecules such as periostin to affect the tumor environment and promote cancer progression.

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet the mechanisms of this effect remain unknown. In this work, using cell biology, flow cytometry, and qPCR analyses, along with a xenograft mouse tumor model, we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) results in the neoplastic transformation of nonmalignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented this transformation. Transformed cells not only possessed several oncogenic properties, such as increased genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology and cell structures, such as an enlarged cytoplasm with disrupted endoplasmic reticulum (ER) alignment and the accumulation of smaller mitochondria. Exposure of SV-HUC cells to TEVs provoked the unfolded protein response in the endoplasmic reticulum (UPRER). Prolonged induction of UPRERsignaling activated the survival branch of the UPRERpathway, in which cells had elevated expression of inositol-requiring enzyme 1 (IRE1), NF-κB, and the inflammatory cytokine leptin, and incurred loss of the pro-apoptotic protein C/EBP homologous protein (CHOP). More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides detailed insight into the mechanisms underlying the bladder cancer field effect and tumor recurrence.

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet its mechanisms remain unknown. In this work we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) resulted in the neoplastic transformation of non-malignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented transformation. Transformed cells not only possessed several oncogenic properties, such as genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology where cells show enlarged cytoplasm with disrupted ER alignment and the accumulation of smaller mitochondria. Treatment of SV-HUC cells with TEVs provoked the unfolded protein response of endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling led to the activation of the survival branch of the UPRER pathway, where cells had elevated expression of the IRE1, NFi«B and the inflammatory cytokine leptin, and loss of CHOP, a pro-apoptotic protein. More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides insight into the mechanisms of the bladder cancer field effect and tumor recurrence. Funding Statement: This work is supported by NCI R01 CA173986 (YF Lee, PI). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The study was approved by the University of Rochester Committee on Animal Resources, and the mice were kept in a specific pathogen-free environment.

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.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.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.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.

Testicular nuclear receptor 4 (TR4), a member of the nuclear receptor superfamily, plays important roles in metabolism, fertility and aging. The linkage of TR4 functions in cancer progression, however, remains unclear. Using three different mouse models, we found TR4 could prevent or delay prostate cancer (PCa)/prostatic intraepithelial neoplasia development. Knocking down TR4 in human RWPE1 and mouse mPrE normal prostate cells promoted tumorigenesis under carcinogen challenge, suggesting TR4 may play a suppressor role in PCa initiation. Mechanism dissection in both in vitro cell lines and in vivo mice studies found that knocking down TR4 led to increased DNA damage with altered DNA repair system that involved the modulation of ATM expression at the transcriptional level, and addition of ATM partially interrupted the TR4 small interfering RNA-induced tumorigenesis in cell transformation assays. Immunohistochemical staining in human PCa tissue microarrays revealed ATM expression is highly correlated with TR4 expression. Together, these results suggest TR4 may function as a tumor suppressor to prevent or delay prostate tumorigenesis via regulating ATM expression at the transcriptional level.

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.

Both epidemiologic and laboratory studies have shown the chemopreventive effects of 1α,25-dihydroxyvitamin D3 (1,25-VD) in tumorigenesis. However, understanding of the molecular mechanism by which 1,25-VD prevents tumorigenesis remains incomplete. In this study, we used an established mouse model of chemical carcinogenesis to investigate how 1,25-VD prevents malignant transformation. In this model, 1,25-VD promoted expression of the DNA repair genes RAD50 and ATM, both of which are critical for mediating the signaling responses to DNA damage. Correspondingly, 1,25-VD protected cells from genotoxic stress and growth inhibition by promoting double-strand break DNA repair. Depletion of the vitamin D receptor (VDR) reduced these genoprotective effects and drove malignant transformation that could not be prevented by 1,25-VD, defining an essential role for VDR in mediating the anticancer effects of 1,25-VD. Notably, genotoxic stress activated ATM and VDR through phosphorylation of VDR. Mutations in VDR at putative ATM phosphorylation sites impaired the ability of ATM to enhance VDR transactivation activity, diminishing 1,25-VD–mediated induction of ATM and RAD50 expression. Together, our findings identify a novel vitamin D–mediated chemopreventive mechanism involving a positive feedback loop between the DNA repair proteins ATM and VDR. Cancer Res; 72(4); 958–68. ©2011 AACR.

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.

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.

OBJECTIVE TR4 is a nuclear receptor without clear pathophysiological roles. We investigated the roles of hepatic TR4 in the regulation of lipogenesis and insulin sensitivity in vivo and in vitro. RESEARCH DESIGN AND METHODS TR4 activity and phosphorylation assays were carried out using hepatocytes and various TR4 wild-type and mutant constructs. Liver tissues from TR4 knockout, C57BL/6, and db/db mice were examined to investigate TR4 target gene stearoyl-CoA desaturase (SCD) 1 regulation. RESULTS TR4 transactivation is inhibited via phosphorylation by metformin-induced AMP-activated protein kinase (AMPK) at the amino acid serine 351, which results in the suppression of SCD1 gene expression. Additional mechanistic dissection finds TR4-transactivated SCD1 promoter activity via direct binding to the TR4-responsive element located at −243 to −255 on the promoter region. The pathophysiological consequences of the metformin→AMPK→TR4→SCD1 pathway are examined via TR4 knockout mice and primary hepatocytes with either knockdown or overexpression of TR4. The results show that the suppression of SCD1 via loss of TR4 resulted in reduced fat mass and increased insulin sensitivity with increased β-oxidation and decreased lipogenic gene expression. CONCLUSIONS The pathway from metformin→AMPK→TR4→SCD1→insulin sensitivity suggests that TR4 may function as an important modulator to control lipid metabolism, which sheds light on the use of small molecules to modulate TR4 activity as a new alternative approach to battle the metabolic syndrome.

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.

Tumor-derived extracellular vesicles (TEVs) are membrane-bound, nanosized vesicles released by cancer cells and taken up by cells in the tumor microenvironment to modulate the molecular makeup and behavior of recipient cells. In this report, we summarize the pivotal roles of TEVs involved in bladder cancer (BC) development, progression and treatment resistance through transferring their bioactive cargos, including proteins and nucleic acids. We also report on the molecular profiling of TEV cargos derived from urine and blood of BC patients as non-invasive disease biomarkers. The current hurdles in EV research and plausible solutions are discussed.

Vitamin D anti-tumor effect is often found reduced in the late stages of cancer. To uncover vitamin D resistance mechanism, we established a vitamin D-resistant human prostate cancer LNCaP cell line, LNCaP-R, by chronic exposure of cells to 1alpha,25-dihydroxyvitamin D(3) (1,25-VD). The vitamin D receptor (VDR)-mediated transcriptional activity was reduced in LNCaP-R, whereas VDR expression level and DNA-binding capacity were similar compared to parental cells (LNCaP-P). The expressions of the key factors involved in VDR transactivity, including CYP24A1 and VDR-associated proteins are all increased in LNCaP-R cells, and yet treatment with ketoconazole, P450 enzymes inhibitor, as well as trichostatin A (TSA), a histone deacetylase inhibitor, did not sensitize LNCaP-R cells response to vitamin D, suggesting that neither a local 1,25-VD availability, nor VDR-associated proteins are responsible for the vitamin D resistance. Interestingly, nuclear factor-kappaB (NF-kappaB) signaling, which is critical for 1,25-VD/VDR activity was found reduced in LNCaP-R cells, thereby treatment with NF-kappaB activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), can sensitize LNCaP-R vitamin D response. Together, we conclude that NF-kappaB signaling is critical for vitamin D sensitivity, and dysregulation of this pathway would result in vitamin D resistance and disease progression.

In earlier studies, we had suggested that the fasting signal induces TR4 orphan nuclear receptor expression in vivo. The detailed mechanism(s), however, remain unclear. In this study, we found that cAMP/PKA, the mediator of fasting and glucagon signals, could induce TR4 gene expression that in turn modulates gluconeogenesis. Mechanistic dissection by in vitro studies in hepatocytes demonstrated that cAMP/PKA might trigger C/EBP alpha and beta binding to the selective cAMP response element, which is located at the TR4 promoter, thus inducing TR4 transcription. We also demonstrated that the binding activity of C/EBPs to the TR4 promoter is increased in response to cAMP treatment. Together, our data identified a new signaling pathway from the fasting signal --cAMP/PKA --C/EBP alpha and beta --TR4 --gluconeogenesis in hepatocytes; and suggested that TR4 could be an important regulator to control glucose homeostasis. The identification of activator(s)/inhibitor(s) or ligand(s) of TR4 may provide us an alternative way to control gluconeogenesis.

Early studies reveal that testicular orphan nuclear receptor 4 (TR4) modulates signaling pathways that control various cell functions. However, how TR4 activity is regulated without the involvement of specific ligand(s) remains unclear. Here we identify a daf-16 family protein-binding element (DBE; 5′-TGTTTAC-3′) in the TR4 promoter that can be recognized by the forkhead transcriptional factor FOXO3a, a key stress-responsive factor, through which TR4 gene expression is activated. The interaction between DBE and FOXO3a was confirmed using EMSA and chromatin immunoprecipitation assays. Activation of FOXO3a by oxidative stress and phosphatidylinositol 3-kinase inhibitor induced TR4 expression; in contrast, suppression of FOXO3a by small interfering RNA can reduce oxidative stress-induced TR4 expression. The biological consequence of the FOXO3a-induced TR4 by oxidative stress is to protect against stress-induced cell death in which cells with reduced FOXO3a are less resistant to oxidative stress, and addition of functional TR4 can increase stress resistance. These results suggest that this new identified oxidative stress-FOXO3a-TR4 pathway is a fundamentally important mechanism regulating stress resistance and cell survival.

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. (Mol Cancer Res 2007;5(9):967–80)

Testicular orphan nuclear receptor 4 (TR4) has been suggested to play important roles in the development and functioning of the central nervous system (CNS). We find reduced myelination in TR4 knockout (TR4(-/-)) mice, which is particularly obvious in forebrains and in early developmental stages. Further analysis reveals that CC-1-positive (CC-1+) oligodendrocytes are decreased in TR4(-/-) forebrains. The O4+ signals are also reduced in TR4(-/-) forebrains when examined at postnatal d 7. However, the number and proliferation rate of platelet-derived growth factor receptor alpha-positive (PDGFalphaR+) oligodendrocyte precursor cells (OPCs) remain unaffected in these regions, suggesting that loss of TR4 interrupts oligodendrocyte differentiation. This is further supported by the observation that CC-1+ oligodendrocytes derived from 5-bromo-2'-deoxyuridine incorporating OPCs are significantly reduced in TR4(-/-) forebrains. We also find higher Jagged1 expression levels in axon fiber-enriched regions in TR4(-/-) forebrains, suggesting a more activated Notch signaling in these regions that correlates with previous reports showing that Notch activation inhibits oligodendrocyte differentiation. Together, our results suggest that TR4 is required for proper myelination in the CNS and is particularly important for oligodendrocyte differentiation and maturation in the forebrain regions. The altered Jagged1-Notch signaling in TR4(-/-) forebrain underlies a potential mechanism that contributes to the reduced myelination in the forebrain.