E-liquid exposure induces bladder cancer cells to release extracellular vesicles that promote non-malignant urothelial cell transformation.

The vaping of electronic cigarettes (E-cigarettes) has recently emerged as a popular alternative to traditional cigarette smoking, but its association with bladder cancer (BC) risk remains to be established. BC patients exhibit high rates of recurrent disease, possibly as a consequence of the field cancerization effect. We have shown that BC-derived extracellular vesicles (BCEVs) can permanently alter recipient urothelial cells in predisposed fields such that they become fully transformed malignant cells. To model the role that BCEVs may play in this potentially oncogenic setting, we treated TCCSUP BC cells with cigarette smoke extract, unflavored E-liquid, or menthol flavored E-liquid. Those treated BCEVs were then tested for their tumorigenic potential. We found that these smoking- and E-cigarette-related BCEVs were able to promote oxidative stress, inflammatory signaling, and DNA damage in recipient SV-HUC urothelial cells. Strikingly, menthol E-liquid-induced BCEVs significantly increased rates of malignant urothelial cell transformation. While further in vivo validation of the simultaneous effects of E-liquid and E-liquid-induced BCEVs on field cancerization is needed, these data highlight the possibility that E-cigarettes may compound user risk in a manner that can contribute to higher rates of BC incidence or recurrence.

Prevention of Radiation-Induced Bladder Injury: A Murine Study Using Captopril.

PURPOSE: Pelvic radiation therapy (RT) can cause debilitating bladder toxicities but few clinical interventions exist to prevent injury or alleviate symptoms. From a large genome-wide association study in patients with prostate cancer it was previously reported that SNPs tagging AGT, part of the renin-angiotensin system (RAS), correlated with patient-reported late hematuria, identifying a potential targetable pathway to prevent RT-induced bladder injury. To investigate this association, we performed a preclinical study to determine whether RAS modulation protected the bladder against RT injury. METHODS AND MATERIALS: C57BL/6 male mice were treated with an oral angiotensin converting enzyme inhibitor (ACEi: 0.3g/L captopril) 5 days before focal bladder X-irradiation with either single dose (SD) 30 Gy or 3 fractions of 8 Gy (8 Gy × 3 in 5 days). RT was delivered using XStrahl SARRP Muriplan CT-image guidance with parallel-opposed lateral beams. ACEi was maintained for 20 weeks post RT. Bladder toxicity was assessed using assays to identify local injury that included urinalysis, functional micturition, bladder-released exosomes, and histopathology, as well as an assessment of systemic changes in inflammatory-mediated circulating immune cells. RESULTS: SD and fractionated RT increased urinary frequency and reduced the volume of individual voids at >14 weeks, but not at 4 weeks, compared with nonirradiated animals. Urothelial layer width was positively correlated with mean volume of individual voids (P = .0428) and negatively correlated with number of voids (P = .028), relating urothelial thinning to changes in RT-mediated bladder dysfunction. These chronic RT-induced changes in micturition patterns were prevented by captopril treatment. Focal bladder irradiation significantly increased the mean particle count of urine extracellular vesicles and the monocyte and neutrophil chemokines CCL2 and MIP-2, and the proportions of circulating inflammatory-mediated neutrophils and monocytes, which was also prevented by captopril. Exploratory transcriptomic analysis of bladder tissue implicated inflammatory and erythropoietic pathways. CONCLUSIONS: This study demonstrated that systemic modulation of the RAS protected against and alleviated RT-induced late bladder injury but larger confirmatory studies are needed.

Bladder Cancer Extracellular Vesicles Elicit a CD8 T Cell-Mediated Antitumor Immunity.

Tumor-derived extracellular vesicles (TEVs) play crucial roles in mediating immune responses, as they carry and present functional MHC-peptide complexes that enable them to modulate antigen-specific CD8+ T-cell responses. However, the therapeutic potential and immunogenicity of TEV-based therapies against bladder cancer (BC) have not yet been tested. Here, we demonstrated that priming with immunogenic Extracellular Vesicles (EVs) derived from murine MB49 BC cells was sufficient to prevent MB49 tumor growth in mice. Importantly, antibody-mediated CD8+ T-cell depletion diminished the protective effect of MB49 EVs, suggesting that MB49 EVs elicit cytotoxic CD8+ T-cell-mediated protection against MB49 tumor growth. Such antitumor activity may be augmented by TEV-enhanced immune cell infiltration into the tumors. Interestingly, MB49 EV priming was unable to completely prevent, but significantly delayed, unrelated syngeneic murine colon MC-38 tumor growth. Cytokine array analyses revealed that MB49 EVs were enriched with pro-inflammatory factors that might contribute to increasing tumor-infiltrating immune cells in EV-primed MC-38 tumors. These results support the potential application of TEVs in personalized medicine, and open new avenues for the development of adjuvant therapies based on patient-derived EVs aimed at preventing disease progression.

Non-stem bladder cancer cell-derived extracellular vesicles promote cancer stem cell survival in response to chemotherapy.

BACKGROUND: Chemosenstive non-stem cancer cells (NSCCs) constitute the bulk of tumors and are considered as part of the cancer stem cell (CSC) niche in the tumor microenvironment (TME). Tumor-derived extracellular vesicles (EVs) mediate the communication between tumors and the TME. In this study, we sought to investigate the impacts of EVs released by NSCCs on the maintenance of CSC properties and chemoresistance. METHODS: We employed murine MB49 bladder cancer (BC) sub-lines representing CSCs and NSCCs as a model system. Chemotherapy drugs were used to treat NSCCs in order to collect conditioned EVs. The impacts of NSCC-derived EVs on CSC progression were evaluated through sphere formation, cytotoxicity, migration, and invasion assays, and by analyzing surface marker expression on these BC cells. Differential proteomic analyses were conducted to identify cargo protein candidates involved in the EV-mediated communication between NSCCs and CSCs. RESULTS: NSCC-derived EVs contained cargo proteins enriched in proteostasis-related functions, and significantly altered the development of CSCs such that they were more intrinsically chemoresistant, aggressive, and better able to undergo self-renewal. CONCLUSIONS: We thus identified a novel communication mechanism whereby NSCC-EVs can alter the relative fitness of CSCs to promote disease progression and the acquisition of chemoresistance.

Tenascin-C expression in the lymph node pre-metastatic niche in muscle-invasive bladder cancer.

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.

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.

Enhanced metastatic potential in the MB49 urothelial carcinoma model.

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.

Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells.

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 UPRER signaling activated the survival branch of the UPRER pathway, 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.

Extracellular Vesicles in Bladder Cancer: Biomarkers and Beyond.

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.

Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer.

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.

Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer.

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.

Cell-to-cell communication in cancer: workshop report.

Recent advances in cancer biology and the development of new research tools have enabled interrogations of single cells and cell-cell interactions. Emerging technologies are capable of revealing data on the physical characteristics of cells, differences in the genome and proteome between cancerous and healthy cells, and variations in distinct cell subpopulations. Dynamic measurements enable studies that can reveal the evolution of cell characteristics. Cells can also be assembled in vitro or ex vivo into two- and three-dimensional cell environments, allowing for studies of cell-cell interactions and cell signaling. The Memorial Sloan Kettering Cancer Center, in collaboration with the Breast Cancer Research Foundation and the National Cancer Institute, co-organized a workshop as an opportunity for leading researchers in their respective fields to present and discuss scientific research highlights relevant to the utilization of techniques and technologies for studying cell-to-cell communications in cancer. Avenues of future development and the potential for clinical utility were primary features of these discussions. The scientific presentations and extensive ensuing discussions resulted in the identification of a number of research opportunities, which are summarized in this report.

E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis.

Circulating prostate cancer (PCa) cells preferentially roll and adhere on bone marrow vascular endothelial cells, where abundant E-selectin and stromal cell-derived factor 1 (SDF-1) are expressed, subsequently initiating a cascade of activation events that eventually lead to the development of metastases. To elucidate the roles of circulating PCa cells' rolling and adhesion behaviors in cancer metastases, we applied a dynamic cylindrical flow-based microchannel device that is coated with E-selectin and SDF-1, mimicking capillary endothelium. Using this device we captured a small fraction of rolling PCa cells. These rolling cells display higher static adhesion ability, more aggressive cancer phenotypes and stem-like properties. Importantly, mice received rolling PCa cells, but not floating PCa cells, developed cancer metastases. Genes coding for E-selectin ligands and genes associated with cancer stem cells and metastasis were elevated in rolling PCa cells. Knock down of E-selectin ligand 1(ESL-1), significantly impaired PCa cells' rolling capacity and reduced cancer aggressiveness. Moreover, ESL-1 activates RAS and MAP kinase signal cascade, consequently inducing the downstream targets. In summary, circulating PCa cells' rolling capacity contributes to PCa metastasis, and that is in part controlled by ESL-1.

TR4 nuclear receptor functions as a tumor suppressor for prostate tumorigenesis via modulation of DNA damage/repair system.

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.

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.

1 alpha, 25-dihydroxylvitamin D3 promotes Bacillus Calmette-Guérin immunotherapy of bladder cancer.

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1 alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.

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.

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

BACKGROUND: Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. METHODS: We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. RESULTS: We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. CONCLUSIONS: Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis.

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.