Androgen receptor-regulated circFNTA activates KRAS signaling to promote bladder cancer invasion.

The androgen receptor (AR) has been linked to bladder cancer (BCa) progression, but if this involves circular RNAs (circRNAs) remains unclear. Here, we find that AR alters the levels of circRNA-FNTA (circFNTA) to increase BCa cell invasion and chemo-resistance. Mechanistically, AR represses the RNA editing gene ADAR2 via direct binding to its 5' promoter region to increase circFNTA levels, which then sponges the microRNA miR-370-3p to increase the expression of its host gene FNTA. This AR-mediated ADAR2/circFNTA/miR-370-3p/FNTA pathway then activates KRAS signaling to alter BCa cell invasion and chemo-sensitivity to cisplatin. A clinical BCa sample survey shows that circFNTA expression is elevated in BCa tissues, and results from a BCa mouse model indicate that depletion of circFNTA leads to the suppression of BCa metastases and increased cisplatin chemo-sensitivity. Together, based on our results using multiple BCa cell lines and an in vivo mouse model we suggest that targeting this newly identified AR/ADAR2/circFNTA/miR-370-3p/FNTA/KRAS axis may lead to the development of therapies to suppress BCa metastasis and to increase its chemo-sensitivity.

Olaparib and enzalutamide synergistically suppress HCC progression via the AR-mediated miR-146a-5p/BRCA1 signaling.

Hepatocellular carcinoma (HCC) is one of most common cancers worldwide, however, the treatment for advanced HCC remains unsatisfactory. We focused on the function of the androgen receptor (AR) in HCC and tried to find new treatment strategy based on antiandrogen enzalutamide (Enz). Here, we found that olaparib, a FDA-approved PARP inhibitor, could enhance the cytotoxicity in HCC cells with a lower BRCA1 expression, and suppressing the AR with either Enz or AR-shRNA could further increase the olaparib sensitivity to better suppress the HCC cell growth via a synergistic mechanism that may involve suppressing the expression of BRCA1 and other DNA damage response (DDR) genes. Mechanism studies revealed that Enz/AR signaling might transcriptionally regulate the miR-146a-5p expression via binding to the Androgen Response Elements on its 5' promoter region, which could then lead to suppress the homologous recombination-related BRCA1 expression via direct binding to the mRNA 3'UTR. Preclinical studies using an in vivo mouse model also demonstrated that combining Enz plus olaparib led to better suppression of the HCC progression. Together, these in vitro/in vivo data suggest that combining Enz and olaparib may help in the development of a novel therapy to better suppress the HCC progression.

LncRNA PCAT1 activates AKT and NF-κB signaling in castration-resistant prostate cancer by regulating the PHLPP/FKBP51/IKKα complex.

In PTEN-deficient prostate cancers, AKT signaling may be activated upon suppression of androgen receptor signaling. Activation of AKT as well as NF-κB signaling involves a key regulatory protein complex containing PHLPP, FKBP51 and IKKα. Here, we report a critical role of lncRNA PCAT1 in regulating the PHLPP/FKBP51/IKKα complex and progression of castration-resistant prostate cancer (CRPC). Using database queries, bioinformatic analyses, as well as RIP and RNA pull-down assays, we discovered and validated that the lncRNA-PCAT1 perturbs the PHLPP/FKBP51/IKKα complex and activates AKT and NF-κB signaling. Expression of lncRNA-PCAT1 is positively linked to CRPC progression. PCAT1 binds directly to FKBP51, displacing PHLPP from the PHLPP/FKBP51/IKKα complex, leading to activation of AKT and NF-κB signaling. Targeting PCAT1 restores PHLPP binding to FKBP1 leading to suppression of AKT signaling. Preclinical study in a mouse model of CRPC suggests therapeutic potential by targeting lncRNA PCAT1 to suppress CRPC progression. Together, the newly identified PCAT1/FKBP51/IKKα complex provides mechanistic insight in the interplay between AKT, NF-κB and AR signaling in CRPC, and the preclinical studies suggest that a novel role for PCAT1 as a therapeutic target.

Correction to: Targeting TR4 nuclear receptor suppresses prostate cancer invasion via reduction of infiltrating acrophages with alteration of the TIMP-1/MMP2/MMP9 signals.

An amendment to this paper has been published and can be accessed via the original article.

Preclinical studies using miR-32-5p to suppress clear cell renal cell carcinoma metastasis via altering the miR-32-5p/TR4/HGF/Met signaling.

While testicular nuclear receptor 4 (TR4) may promote prostate cancer (PCa) metastasis, its role in the clear cell renal cell carcinoma (ccRCC) remains unclear. Here we found a higher expression of TR4 in ccRCC tumors from patients with distant metastases than those from metastasis-free patients, suggesting TR4 may play positive roles in the ccRCC metastasis. Results from multiple in vitro ccRCC cell lines also confirmed TR4's positive roles in promoting ccRCC cell invasion/migration via altering the microRNA (miR-32-5p)/TR4/HGF/Met/MMP2-MMP9 signaling. Mechanism dissection revealed that miR-32-5p could suppress TR4 protein expression levels via direct binding to the 3'UTR of TR4 mRNA, and TR4 might then alter the HGF/Met signaling at the transcriptional level via direct binding to the TR4-response-elements (TR4RE) on the HGF promoter. Then the in vitro data also demonstrated the efficacy of Sunitinib, a currently used drug to treat ccRCC, could be increased after targeting this newly identified miR-32-5p/TR4/HGF/Met signaling. The preclinical study using the in vivo mouse model with xenografted ccRCC cells confirmed the in vitro cell lines data. Together, these findings suggest that TR4 is a key player to promote ccRCC metastasis and targeting this miR-32-5p/TR4/HGF/Met signaling with small molecules including TR4-shRNA or miR-32-5p may help to develop a new therapy to better suppress the ccRCC metastasis.

Sorafenib with ASC-J9® synergistically suppresses the HCC progression via altering the pSTAT3-CCL2/Bcl2 signals.

Sorafenib is currently used as a standard treatment to suppress the progression of hepatocellular carcinoma (HCC), especially in advanced stages. However, patients who receive Sorafenib treatment eventually develop resistance without clear mechanisms. There is a great need for better efficacy of Sorafenib treatment in combination with other therapies. Here, we demonstrated that the treatment combining Sorafenib with ASC-J9® could synergistically suppress HCC progression via altering cell-cycle regulation, apoptosis and invasion. Mechanism dissection suggests that while Sorafenib impacts little or even slightly increases the activated/phosphorylated STAT3 (p-STAT3), a key stimulator to promote the HCC progression, adding ASC-J9® significantly suppresses the p-STAT3 expression and its downstream genes including CCL2 and Bcl2. Interrupting these signals via constitutively active STAT3 partially reverses the synergistic suppression of Sorafenib-ASC-J9® combination on HCC progression. In vivo studies further confirmed the synergistic effect of Sorafenib-ASC-J9® combination. Together, these results suggest the newly developed Sorafenib-ASC-J9® combination is a novel therapy to better suppress HCC progression.

Targeting fatty acid synthase with ASC-J9 suppresses proliferation and invasion of prostate cancer cells.

Fatty acid synthase (FASN) is the key enzyme for the control of fatty acid synthesis that contributes significantly to the prostate cancer (PCa) progression. It was reported that androgens were able to induce FASN expression in PCa, and addition of the anti-androgen Casodex might suppress the androgen-induced FASN expression. However, here we found androgen-deprivation-therapy (ADT) with anti-androgens Bicalutamide (Casodex) or Enzalutamide (MDV3100) had little effect to suppress FASN expression and FASN-mediated cell growth and invasion during the castration resistant stage when the androgen concentration is 1 nM DHT (dihydrotestosterone). In contrast, the newly developed androgen receptor (AR) degradation enhancer ASC-J9® suppressed FASN expression and FASN-mediated cell growth and invasion in various PCa cell lines at 1 nM DHT. Mechanism dissection found ASC-J9® could suppress significantly the FASN expression and FASN-mediated PCa progression via the AR-dependent pathway involving AR→SREBP-1→FASN signaling in AR-positive C4-2 and LNCaP cells and via the AR-independent pathway involving the modulation of PI3K/AKT→SREBP-1→FASN signaling in AR-negative PC-3 and DU145 cells. Together, these results suggest that FASN is one of the important mechanism why the current ADT eventually fails. ASC-J9® might represent a new potential therapeutic approach to suppress FASN-mediated PCa progression via both AR-dependent and AR-independent pathways during the castration resistant stage of PCa. © 2016 Wiley Periodicals, Inc.

Stromal androgen receptor roles in the development of normal prostate, benign prostate hyperplasia, and prostate cancer.

The prostate is an androgen-sensitive organ that needs proper androgen/androgen receptor (AR) signals for normal development. The progression of prostate diseases, including benign prostate hyperplasia (BPH) and prostate cancer (PCa), also needs proper androgen/AR signals. Tissue recombination studies report that stromal, but not epithelial, AR plays more critical roles via the mesenchymal-epithelial interactions to influence the early process of prostate development. However, in BPH and PCa, much more attention has been focused on epithelial AR roles. However, accumulating evidence indicates that stromal AR is also irreplaceable and plays critical roles in prostate disease progression. Herein, we summarize the roles of stromal AR in the development of normal prostate, BPH, and PCa, with evidence from the recent results of in vitro cell line studies, tissue recombination experiments, and AR knockout animal models. Current evidence suggests that stromal AR may play positive roles to promote BPH and PCa progression, and targeting stromal AR selectively with AR degradation enhancer, ASC-J9, may allow development of better therapies with fewer adverse effects to battle BPH and PCa.

Targeting TR4 nuclear receptor suppresses prostate cancer invasion via reduction of infiltrating macrophages with alteration of the TIMP-1/MMP2/MMP9 signals.

BACKGROUND: TR4 nuclear receptor 4 (TR4) plays an important role in macrophages-associated foam cell formation of cardiovascular diseases and infiltrating macrophages are critical for prostate cancer (PCa) progression. However, the linkage of macrophages and TR4 and their impacts on PCa metastasis remains unclear. RESULTS: Knocking-down TR4 in human PCa cells (C4-2, CWR22Rv1), but not in human macrophages cells (THP-1), led to suppress the macrophages infiltration to PCa cells. The consequences of such suppression of the recruitment of macrophages toward PCa then resulted in suppressing the PCa cell invasion. Mechanism dissection found that knocking-down TR4 in PCa cells suppressed metastasis-related genes including MMP2, with induction of TIMP-1. Interruption assays using TIMP-1 neutralizing antibody could then reverse TR4-macrophage-mediated PCa invasion. IHC staining showed higher TR4 level, more macrophage infiltration, lower TIMP-1 and stronger MMP2/MMP9 in tumor tissues of the Gleason score 5 + 4 patients compared with the Gleason score 3 + 3 patients. CONCLUSION: Targeting TR4 in prostate tumor microenvironment might represent a potential new therapeutic approach to better battle PCa metastasis.

TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

Testicular nuclear receptor 4 (TR4) plays protective roles against oxidative stress and DNA damage and might contribute to aging. Our recent clinical tumor tissue staining results showed higher expression of TR4 in prostate cancer (PCa) patients with high Gleason scores compared to the tissues with the low Gleason scores. In vitro migration/invasion assays after manipulation of the TR4 expression in PCa cells showed that TR4 promoted PCa cells migration/invasion. Mechanism dissection found that the CCL2/CCR2 signal plays the key role in the mediation of TR4-promoted PCa cells migration/invasion. Chromatin immunoprecipitation and Luciferase assays further confirmed TR4 modulation of CCL2 at the transcriptional level and addition of the CCR2 antagonist led to interruption of the TR4-enhanced PCa cells migration/invasion. Finally, the orthotopic xenografted mice studies using the luciferase expressing CWR22Rv1 cells found that TR4 enhanced PCa metastasis and this increased metastasis was reversed when the CCR2 antagonist was injected into the mice. Together, these in vitro and in vivo results revealed a positive role of TR4 in PCa metastasis and demonstrated CCL2/CCR2 signaling as an important mediator in exerting TR4 action. This finding suggests that TR4 may represent a biomarker related to PCa metastasis and targeting the TR4-CCL2/CCR2 axis may become a new therapeutic approach to battle PCa metastasis.

Testicular orphan nuclear receptor 4 is associated with the radio-sensitivity of prostate cancer.

BACKGROUND: It is well known that a significant number of prostate cancers (PCa) showed different extents of radio-resistance and the tumor may recur after treatment. Recent studies demonstrated that Testicular orphan nuclear receptor 4 (TR4) could play a critical role in anti-oxidative stress responses and might modulate the DNA damage repair. The objective of this study is to investigate the role of TR4 in the radiotherapy for PCa. METHODS: The TR4 expression in tissue samples from PCa patients treated with brachytherapy was measured by immunohistochemistry (IHC). Cell survival test and colony formation assay were applied to test the radio-sensitivity of PCa cells with modulated TR4 gene expression upon irradiation. RESULTS: PCa patients with biochemical recurrence (BCR) after brachytherapy tend to have higher TR4 expression (80%, n = 30) as compared to those without BCR (36.67%, n = 30). Survival analysis demonstrated a significant higher BCR occurrence in patients with high level of TR4 expression (HR = 3.474, 95%CI 1.678-7.192, P = 0.0008). Multivariate analysis showed that the TR4 staining score on IHC was the only significant variable for predicting the PCa patients' clinical outcomes after radiotherapy (OR = 9.919, 95% CI 2.516-39.101, P = 0.001). Using cell survival test and colony forming assay, we found that the addition of functional TR4 in PC3 cells lead to elevated radio-resistance. In contrast, knocking-down TR4 in LNCaP cells resulted in increased radio-sensitivity. The γH2AX foci kinetic analysis suggested that knocking down TR4 might delay the PCa cell's DNA damage repair which would enhance the radio-sensitivity. CONCLUSION: TR4 could mediate the PCa cells' radio-sensitivity and might become a prognostic indicator for PCa patients received radiotherapy. This study provides a novel approach to manipulate radio-sensitivity of PCa cells, and may bring a promoted therapeutic outcome of radiotherapy to battle PCa in future.

Concise review: androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal, and hematopoietic lineages.

Stem/progenitor (S/P) cells are special types of cells that have the ability to generate tissues throughout their entire lifetime and play key roles in the developmental process. Androgen and the androgen receptor (AR) signals are the critical determinants in male gender development, suggesting that androgen and AR signals might modulate the behavior of S/P cells. In this review, we summarize the AR effects on the behavior of S/P cells, including self-renewal, proliferation, apoptosis, and differentiation in normal S/P cells, as well as proliferation, invasion, and self-renewal in prostate cancer S/P cells. AR plays a protective role in the oxidative stress-induced apoptosis in embryonic stem cells. AR inhibits the self-renewal of embryonic stem cells, bone marrow stromal cells, and prostate S/P cells, but promotes their differentiation except for adipogenesis. However, AR promotes the proliferation of hematopoietic S/P cells and stimulates hematopoietic lineage differentiation. In prostate cancer S/P cells, AR suppresses their self-renewal, metastasis, and invasion. Together, AR differentially influences the characteristics of normal S/P cells and prostate cancer S/P cells, and targeting AR might improve S/P cell transplantation therapy, especially in embryonic stem cells and bone marrow stromal cells.

Proteomic analysis of urethral protein expression in an estrogen receptor α-deficient murine model of stress urinary incontinence.

PURPOSE: The roles of estrogen receptor α (ERα) in stress urinary incontinence (SUI) remain elusive. This study was conducted to understand the molecular mechanism of ERα against SUI. METHODS: Wild-type (ERα(+/+)) and ACTB-cre ERα knockout (ERα(-/-)) female mice were generated. Urethral function and protein expression were measured. Leak point pressures (LPP) and maximum urethral closure pressure (MUCP) were assessed in mice under urethane anesthesia. After the measurements, the urethras were removed for proteomic analysis using the two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry technology. Interaction between these ERα pathway proteins was further analyzed by using MetaCore. Lastly, Western blot and immunochemistry (IHC) were used to confirm the candidate protein expression levels and locations, respectively. RESULTS: Compared with the ERα(+/+) group, the LPP and MUCP values of the ERα(-/-) group were significantly decreased. Additionally, we identified 11 differentially expressed proteins in the urethra of ERα(-/-) female mice; five proteins were down-regulated and six were up-regulated. The majority of the ERα knockout-modified proteins were involved in muscle development, contraction, and regulation, as well as immune response (amphoterin signaling and phagocytosis), proteolysis, and cell adhesion (platelet aggregation and integrin-mediated cell-matrix adhesion). IHC and Western blot confirmed the down-regulation of tropomyosin and up-regulation of myosin in urethra. CONCLUSIONS: This is the first study to estimate protein expression changes in urethras from ERα(-/-) female mice. These changes could be related to the molecular mechanism of ERα in SUI.

Evidence of TGF-ß1 mediated epithelial-mesenchymal transition in immortalized benign prostatic hyperplasia cells.

Expression of epithelial-mesenchymal transition (EMT) markers has been detected clinically in benign prostatic hyperplasia (BPH) tissues. To understand the molecular basis, we investigated the role of stromal microenvironment in the progression of EMT in BPH cells. First, we used cell culture supernatant from normal prostate stromal WPMY-1 cells to provide supernatant-conditioned medium (WSCM) to culture the BPH-1 cell line. Then, the morphological changes and migratory capacity were detected in BPH-1 cells. The expression of EMT markers was examined in BPH-1 cells by Western blot and immunofluorescent analysis. Finally, to investigate the role of transforming growth factor beta 1 (TGF-ß1) in this process, the WSCM-cultured cells were treated with monoclonal antibody against TGF-ß1 to study its effect on EMT. We found that the morphology of BPH-1 cells changed to a spindle-like shape after cultured in WSCM, and the levels of E-cadherin and cytokeratin 5/8 (CK5/8) were significantly lower than the cells cultured in ordinary medium. These BPH-1 cells were also tested positive for mesenchymal markers vimentin and a-smooth muscle actin (SMA) as well as Snail. We also found WSCM can increase the migratory capacity of BPH-1 cells. In addition, when they were treated with anti-TGF-ß1, upregulation of E-cadherin and CK5/8 levels was observed but no expression of vimentin, alpha-SMA or Snail was detected. Furthermore, phosphorylated-Smad3 expression in WSCM-cultured BPH-1 cells was also suppressed by anti-TGF-ß1 treatment. Our results demonstrated that stromal cell supernatant was able to induce EMT in BPH-1 cells, possibly through secreting TGF-ß1 to activate Smad signaling. Our results suggest novel molecular targets for clinical treatment of BPH by modification of stromal microenvironment through inhibiting TGF-ß1/Smad expression.

Abnormal mitochondrial function and impaired granulosa cell differentiation in androgen receptor knockout mice.

In the ovary, the paracrine interactions between the oocyte and surrounded granulosa cells are critical for optimal oocyte quality and embryonic development. Mice lacking the androgen receptor (AR⁻/⁻) were noted to have reduced fertility with abnormal ovarian function that might involve the promotion of preantral follicle growth and prevention of follicular atresia. However, the detailed mechanism of how AR in granulosa cells exerts its effects on oocyte quality is poorly understood. Comparing in vitro maturation rate of oocytes, we found oocytes collected from AR⁻/⁻ mice have a significantly poor maturating rate with 60% reached metaphase II and 30% remained in germinal vesicle breakdown stage, whereas 95% of wild-type AR (AR⁺/⁺) oocytes had reached metaphase II. Interestingly, we found these AR⁻/⁻ female mice also had an increased frequency of morphological alterations in the mitochondria of granulosa cells with reduced ATP generation (0.18 ± 0.02 vs. 0.29 ± 0.02 µM/mg protein; p < 0.05) and aberrant mitochondrial biogenesis. Mechanism dissection found loss of AR led to a significant decrease in the expression of peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1-ß (PGC1-ß) and its sequential downstream genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), in controlling mitochondrial biogenesis. These results indicate that AR may contribute to maintain oocyte quality and fertility via controlling the signals of PGC1-ß-mediated mitochondrial biogenesis in granulosa cells.

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.

Differential androgen deprivation therapies with anti-androgens casodex/bicalutamide or MDV3100/Enzalutamide versus anti-androgen receptor ASC-J9(R) Lead to promotion versus suppression of prostate cancer metastasis.

Despite the fact that androgen deprivation therapy (ADT) can effectively reduce prostate cancer (PCa) size, its effect on PCa metastasis remains unclear. We examined the existing data on PCa patients treated with ADT plus anti-androgens to analyze ADT effects on primary tumor size, prostate-specific antigen (PSA) values, and metastatic incidence. We found that the current ADT with anti-androgens might lead to primary tumor reduction, with PSA decreased yet metastases increased in some PCa patients. Using in vitro and in vivo metastasis models with four human PCa cell lines, we evaluated the effects of the currently used anti-androgens, Casodex/bicalutamide and MDV3100/enzalutamide, and the newly developed anti-AR compounds, ASC-J9® and cryptotanshinone, on PCa cell growth and invasion. In vitro results showed that 10 µm Casodex or MDV3100 treatments suppressed PCa cell growth and reduced PSA level yet significantly enhanced PCa cell invasion. In vivo mice studies using an orthotopic xenograft mouse model also confirmed these results. In contrast, ASC-J9® led to suppressed PCa cell growth and cell invasion in in vitro and in vivo models. Mechanism dissection indicated these Casodex/MDV3100 treatments enhanced the TGF-ß1/Smad3/MMP9 pathway, but ASC-J9® and cryptotanshinone showed promising anti-invasion effects via down-regulation of MMP9 expression. These findings suggest the potential risks of using anti-androgens and provide a potential new therapy using ASC-J9® to battle PCa metastasis at the castration-resistant stage.