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Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers. Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.

Background Testosterone (T) deficiency is associated with erectile dysfunction (ED). The relaxant response of T on the corporal smooth muscle through a non-genomic pathway has been reported; however, the in vitro modulating effects of T on human corpus cavernosum (HCC) have not been studied. Objectives To compare the effects of various concentrations of T on nitric oxide (NO)-dependent and nitric oxide-independent relaxation in organ bath studies and elucidate its mode of action, specifically targeting the cavernous NO/cyclic guanosine monophosphate (cGMP) pathway. Materials and methods Human corpus cavernosum (HCC) samples were obtained from men undergoing penile prosthesis implantation (n = 9). After phenylephrine (Phe) precontraction, the effects of various relaxant drugs of HCC strips were performed using organ bath at low (150 ng/dL), eugonadal (400 ng/dL), and hypergonadal (600 ng/dL) T concentrations. The penile tissue measurements of endothelial nitric oxide synthase (eNOS), neuronal (n)NOS, and phosphodiesterase type 5 (PDE5) were evaluated via immunostaining, Western blot, cGMP and nitrite/nitrate (NOx) assays. Results Relaxation responses to ACh and EFS in isolated HCC strips were significantly increased at all T levels compared with untreated tissues. The sildenafil-induced relaxant response was significantly increased at both eugonadal and hypergonadal T levels. Normal and high levels of T are accompanied by increased eNOS, nNOS, cGMP, and NOx levels, along with reduced PDE5 protein expression. Conclusion This study reveals an important role of short-term and modulatory effects of different concentrations of T in HCC. T positively regulates functional activities, inhibition of PDE5 expression, and formation of cGMP and NOx in HCC. These results demonstrate that T indirectly contributes to HCC relaxation via downstream effects on nNOS, eNOS, and cGMP and by inhibiting PDE5. This action provides a rationale for normalizing T levels in hypogonadal men with ED, especially when PDE5 inhibitors are ineffective. T replacement therapy may improve erectile function by modulating endothelial function in hypogonadal men.

Background Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. Methods C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4+ T cells were isolated from young (16-20 weeks old) and aged (96-104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or quantitative reverse transcription polymerase chain reaction. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and nuclear factor kappa B (NF-κB) signaling. Results Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naive CD4+ T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4+ T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4+ T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion but also activated the NF-κB signaling in PCa cells compared to young mice. Conclusions These results indicate that age-related CD4+ T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

Although well-differentiated papillary thyroid cancer may remain indolent, lymph node metastases and the recurrence rates are approximately 50% and 20%, respectively. No current biomarkers are able to predict metastatic lymphadenopathy and recurrence in early stage papillary thyroid cancer. Hence, identifying prognostic biomarkers predicting cervical lymph-node metastases would prove very helpful in determining treatment.The database of the Cancer Genome Atlas included 495 papillary thyroid cancer samples. Using this database, we developed a machine learning model to define a gene signature that could predict lymph-node metastasis (N0 or N1). Kruskal-Wallis tests, univariate and multivariate logistic and Cox regression models, and Kaplan-Meier analyses were performed to correlate the gene signature with clinical outcomes.We identified a panel of 25 genes and constructed a risk score that can differentiate N0 and N1 papillary thyroid cancer samples (P.001) with a sensitivity of 86%, a specificity of 62%, a positive predictive value of 93%, and a negative predictive value of 42%. This panel represents an independent biomarker to predict metastatic lymphadenopathy (OR = 8.06, P.001) specifically in patients with T1 lesions (OR = 7.65, P = .002) and disease-free survival (HR = 2.64, P = .043).This novel 25-gene panel may be used as a potential prognostic marker for accurately predicting lymph-node metastasis and disease-free survival in patients with early-stage papillary thyroid cancer.

Prostate cancer (PCa) is associated with advanced age, but how age contributes to prostate carcinogenesis remains unknown. The prostate-specific Pten conditional knockout mouse model closely imitates human PCa initiation and progression. To better understand how age impacts PCa in an experimental model, we have generated a spatially and temporally controlled Pten-null PCa murine model at different ages (aged vs. non-aged) of adult mice. Here, we present a protocol to inject the Cre-expressing adenovirus with luciferin tag, intraductally, into the prostate anterior lobes of Pten-floxed mice; Pten-loss will be triggered post-Cre expression at different ages. In vivo imaging of luciferin signal following viral infection confirmed successful delivery of the virus and Cre activity. Immunohistochemical staining confirmed prostate epithelial-specific expression of Cre recombinase and the loss of Pten and activation of P-Akt, P-S6, and P-4E-BP1. The Cre-expression, Pten ablation, and activated PI3K/AKT/mTOR pathways were limited to the prostate epithelium. All mice developed prostatic epithelial hyperplasia within 4 weeks after Pten ablation and prostatic intraepithelial neoplasia (PIN) within 8 weeks post-Pten ablation. Some PINs had progressed to invasive adenocarcinoma at 8–16 weeks post-Pten ablation. Aged mice exhibited significantly accelerated PI3K/AKT/mTOR signaling and increased PCa onset and progression compared to young mice. The viral infection success rate is ∼80%. This model will be beneficial for investigations of cancer-related to aging.

INTRODUCTION AND OBJECTIVE:Renal cell carcinoma remains an aggressive malignancy with known substantial cross talk between cells. Exosomes (exo) are small extracellular vesicles (EVs) secreted from...

Androgen receptor (AR) signaling is fundamental to prostate cancer (PC) progression, and hence, androgen deprivation therapy (ADT) remains a mainstay of treatment. However, augmented AR signaling via both full length AR (AR-FL) and constitutively active AR splice variants, especially AR-V7, is associated with the recurrence of castration resistant prostate cancer (CRPC). Oxidative stress also plays a crucial role in anti-androgen resistance and CRPC outgrowth. We examined whether a triterpenoid antioxidant drug, Bardoxolone-methyl, known as CDDO-Me or RTA 402, can decrease AR-FL and AR-V7 expression in PC cells. Nanomolar (nM) concentrations of CDDO-Me rapidly downregulated AR-FL in LNCaP and C4-2B cells, and both AR-FL and AR-V7 in CWR22Rv1 (22Rv1) cells. The AR-suppressive effect of CDDO-Me was evident at both the mRNA and protein levels. Mechanistically, acute exposure (2 h) to CDDO-Me increased and long-term exposure (24 h) decreased reactive oxygen species (ROS) levels in cells. This was concomitant with an increase in the anti-oxidant transcription factor, Nrf2. The anti-oxidant N-acetyl cysteine (NAC) could overcome this AR-suppressive effect of CDDO-Me. Co-exposure of PC cells to CDDO-Me enhanced the efficacy of a clinically approved anti-androgen, enzalutamide (ENZ), as evident by decreased cell-viability along with migration and colony forming ability of PC cells. Thus, CDDO-Me which is in several late-stage clinical trials, may be used as an adjunct to ADT in PC patients.

MicroRNAs (miRNAs) are a small functional non-coding RNAs that post-transcriptionally regulate gene expression through mRNA degradation or translational repression. miRNAs are key regulatory components of various cellular networks. Current evidence support that multiple mammalian genome-encoded miRNAs impact the cellular biology, including proliferation, apoptosis, differentiation, and tumorigenesis, by targeting specific subsets of mRNAs. This minireview is focused on the current themes underlying the interactions between miRNAs and their mRNA targets and pathways in prostate tumorigenesis and progression, and their potential clinical utility as biomarkers for prostate cancer. Impact statement The primary goal of this article was to review recent literature on miRNA biogenesis and further elaborate on the identity of newly discovered miRNAs and their potential functional significance in the complex biological network associated with prostate tumorigenesis and disease progression and as biomarkers for prostate cancer.

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression.

The increased expression of phosphatase of regenerating liver-3 (PRL‑3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL‑3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP‑SF) revealed the constitutive cytoplasmic expression of PRL‑3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL‑3. The AR-negative cell lines exhibited negligible PRL‑3 expression, and the ectopic overexpression of PRL‑3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL‑3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL‑3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL‑3 expression was significantly higher in the malignant cores, as compared to the non‑malignant areas. Increases in both total and nuclear PRL‑3 levels were also associated with a higher GS and GG. Metastatic tumors (T4‑stage) had lower cytoplasmic, but higher nuclear PRL‑3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL‑3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL‑3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL‑3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.

Prostate cancer (PCa) is associated with advanced age. To better understand how age impacts PCa, it is critical to use PCa animal models generated at different ages (aged vs. non-aged). The PB-Cre4 driven phosphatase and tensin homolog (Pten) conditional knockout mouse model, which closely imitates human PCa initiation and progression. However, the Pten deletion is triggered in a 2-week-old prostate, when comparing the extent of PCa between aged and non-aged mice, it is difficult to distinguish the extent to which the onset and progression of PCa are due to the acceleration of the normal aging process or due to the manifestation of PCa pathologies over time. We present here a protocol to inject Cre-expressing adenovirus with luciferin tag intraductally into the prostate anterior lobes of Pten floxed mice, thus, the Pten-loss will be triggered at different ages post-Cre expression. The in vivo imaging of luciferin signals following viral infection was conducted to confirm the Cre expression and activity. Immunohistochemical staining was performed to confirm the Cre expression, Pten loss, and p-Akt and p-S6 activation. Prostate weight and histopathology were compared between aged and non-aged mice. The results showed that the virus infection was limited in the prostate glands and aged mice had significantly increased PCa onset and progression compared to young mice. Although technical skill is required to carry out this procedure and the success rate of viral infection is about 80%, this model of PCa is of great use to all investigators in the aging and cancer research field.

PubMedID: 31741421 Objective: To evaluate the effect of the If channel inhibitor, ivabradine on human corpus cavernosum (HCC) smooth muscle tone. Methods: HCC samples were obtained from erectile dysfunction(ED) patients (n = 12) undergoing penile prosthesis surgery. Concentration–response curves for ivabradine were exposed to various inhibitory and stimulatory agents. The relaxant and contractile responses to electrical field stimulation (EFS, 10 Hz and 80 Hz) were examined in the presence or absence of ivabradine (10 µM). HCN3 and HCN4 channel expression and localization were determined by Western blot and immunohistochemical analyses of HCC tissues. Results: Increasing ivabradine concentrations dependently reduced the maximal contractile responses of isolated HCC strips induced by KCl (59.5 ± 2.5%) and phenylephrine (84.0 ± 9.8%), which was not affected by nitric oxide synthase and soluble guanylyl cyclase inhibitors after phenylephrine-induced contraction. Nifedipine and tetraethylammonium inhibited the maximum relaxation to ivabradine by 75% and 39.3%, respectively. Fasudil and sildenafil increased the relaxation response to ivabradine without altering the maximum response. Pre-incubation with ivabradine significantly increased relaxant responses to EFS (p < 0.01) and reduced the contractile tension evoked by EFS (72.3%) (p < 0.001). Ivabradine incubation did not affect the expression and localization of HCN3 and HCN4 channels in the HCC smooth muscle cells. Conclusions: Ivabradine exhibits a relaxant effect on HCC tissues, which is likely to be attributed to the blocking of L-type Ca2+ channels and the opening of K+ channels, independent of changes in the activation of the nitric oxide/cyclic guanosine monophosphate system. Inhibition of HCN channels localized in cavernosal smooth muscle cells may offer pharmacological benefits for patients with cardiovascular risk factors. © 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group.

340 Background: Over the last decade, medical treatment for metastatic renal cell carcinoma has made significant advances through the development of tyrosine kinase inhibitors (TKI) like sunitinib. However, of patients initiated on TKI therapy, 70% respond well while 30% are believed to be primarily resistant to treatment. Additionally, 30% of patients who initially respond to treatment gain secondary drug resistance and present with increased tumor burden. In this study, we seek to develop a combination therapy of Tipifarnib + Sunitinib to target exosome conferred drug resistance. Methods: 786-O, 786-0 Sunitinib Resistant (SR), and 293-T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 1% penicillin/streptomycin (P/S). Exosomes were collected from conditioned media treated with Tipifarnib and isolated using differential ultracentrifugation. Exosomes were analyzed using the qNano IZON system. Colony forming units assay and Immunoblot analysis were used to further characterize or samples. Results: Exosomes collected from 786-O, 786-0 SR, and 293-T cells treated with 0.5 uM of Tipifarnib were compared using our qNANO IZON system. Exosome concentrations of all cell lines showed a decrease after Tipifarnib treatment. However, our 293-T cells showed a 16% decrease in exosome concentration while our 786-O and 786-0 SR lines displayed a 66% and 75% decrease respectively. To assess the pathway Tipifarnib used to decrease exosome concentrations, immunoblot assay was used after treating cells with 0, 0.1, 0.25, 0.5, 1 uM of tipifarnib. 293-T cells showed a dose dependent increase in ESCRT-dependent marker Alix and no change in either ESCRT-independent marker nSMase or trafficking marker Rab27a. Conversely, our 786-O and 786-0 SR cell lines showed a decrease in all 3 markers: Alix, nSMase, and Rab27a. Furthermore, a colony forming units assay was used to assess the drug combination of tipifarnib + sunitinib ability to alter cell growth. After 48hr, 293-T cell showed no decrease in colony forming units when compared to DMSO control. Our drug combination showed a synergistic ability to decrease colony forming units in the RCC 786-O cell line. 786-O SR cells were resistant to sunitinib treatment, showing comparable CFUs to DMSO control. When treated with the combination of sunitinib and tipifarnib, CTUs of 786-0 SR cells dropped significantly when compared to unaccompanied sunitinib and tipifarnib treatments. Conclusions: Tipifarnib has the ability to attenuate both the exosome ESCRT–dependent and –independent pathways. Our study also showed that when used injunction with sunitinib, tipifarnib is effective at decreasing cell proliferation. This drug combination is also pre-clinically useful in sunitinib resistance cancer cells. We believe this drug combination to be efficacious at decreasing tumor burden through blocking exosome biogenesis and secretion.

OBJECTIVE To investigate tyrosine kinase inhibitors (TKI) and gold nanorods (AuNRs) paired with photothermal ablation in a human metastatic clear cell renal cell carcinoma (RCC) mouse model. Nanoparticles have been successful as a platform for targeted drug delivery in the treatment of urological cancers. Likewise, the use of nanoparticles in photothermal tumour ablation, although early in its development, has provided promising results. Our previous in vitro studies of nanoparticles loaded with both TKI and AuNRs and activated with photothermal ablation have shown significant synergistic cell kill greater than each individual arm alone. This study is a translation of our initial findings to an in vivo model. MATERIALS AND METHODS Immunologically naive nude mice (athymic nude-Foxn1nu ) were injected subcutaneously bilaterally in both flanks (n = 36) with 2.5 × 106 cells of a human metastatic renal cell carcinoma cell line (RCC 786-O). Subcutaneous xenograft tumours developed into 1-cm palpable nodules. AuNRs encapsulated in human serum albumin protein (HSA) nanoparticles were synthesised with or without a TKI and injected directly into the tumour nodule. Irradiation was administered with an 808-nm light-emitting diode laser for 6 min. Mice were humanely killed 14 days after irradiation; tumours were excised, formalin fixed, paraffin embedded, and evaluated for size and the percentage of necrosis by a genitourinary pathologist. The untreated contralateral flank tumours were used as controls. RESULTS In mice that did not receive irradiation, TKI alone yielded 4.2% tumour necrosis on the injected side and administration of HSA-AuNR-TKI alone yielded 11.1% necrosis. In the laser-ablation models, laser ablation alone yielded 62% necrosis and when paired with HSA-AuNR there was 63.4% necrosis. The combination of laser irradiation and HSA-AuNR-TKI had cell kill rate of 100%. CONCLUSIONS In the absence of laser irradiation, TKI treatment alone or when delivered via nanoparticles produced moderate necrosis. Irradiation with and without gold particles alone also improves tumour necrosis. However, when irradiation is paired with gold particles and drug-loaded nanoparticles, the combined therapy showed the most significant and synergistic complete tumour necrosis of 100% (P < 0.05). This study illustrates the potential of combination nanotechnology as a new approach in the treatment of urological cancers.

// Hogyoung Kim 1 , Amrita Datta 1 , Sudha Talwar 1 , Sarmad N. Saleem 1 , Debasis Mondal 2 and Asim B. Abdel-Mageed 1, 2, 3 1 Department of Urology, Tulane University School of Medicine, New Orleans, Louisiana, 70112, USA 2 Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, 70112, USA 3 Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana, 70112, USA Correspondence to: Asim B. Abdel-Mageed, email: amageed@tulane.edu Keywords: estrogen, ERβ2, TMPRSS2:ETV5, IGF-1R, prostate cancer Received: September 02, 2015 Accepted: July 26, 2016 Published: August 17, 2016 ABSTRACT Estrogen receptor beta (ERβ) splice variants are implicated in prostate cancer (PC) progression; however their underlying mechanisms remain elusive. We report that non-canonical activation of estradiol (E 2 )-ERβ2 signaling axis primes growth, colony-forming ability and migration of the androgen receptor (AR)-null castration-resistant PC (CRPC) cells under androgen-deprived conditions (ADC). The non-classical E 2 -ERβ2 mediates phosphorylation and activation of Src-IGF-1R complex, which in turn triggers p65-dependent transcriptional upregulation of the androgen-regulated serine protease TMPRSS2:ETV5a/TMPRSS2:ETV5b gene fusions under ADC. siRNA silencing of TMPRSS2 and/or ETV5 suggests that TMPRSS2:ETV5 fusions facilitates the E 2 -ERβ induced growth and migration effects via NF-κB-dependent induction of cyclin D1 and MMP2 and MMP9 in PC-3 cells. Collectively, our results unravel the functional significance of oncogenic TMPRSS2:ETV5 fusions in mediating growth and migration of E 2 -ERβ2 signaling axis in CRPC cells. E 2 -ERβ2 signaling axis may have significant therapeutic and prognostic implications in patients with CRPC.

// Sen Liu 1, * , Qiuyang Zhang 1, * , Chong Chen 1 , Dongxia Ge 1 , Yine Qu 1 , Rongyi Chen 2 , Yi-Ming Fan 2 , Nan Li 2 , Wendell W. Tang 3 , Wensheng Zhang 4 , Kun Zhang 4 , Alun R. Wang 5 , Brian G. Rowan 1, 6, 7 , Steven M. Hill 1, 6 , Oliver Sartor 6, 8, 9 , Asim B. Abdel-Mageed 6, 8 , Leann Myers 10 , Qishan Lin 11 , Zongbing You 1, 6, 7, 12 1 Department of Structural and Cellular Biology, Tulane University, New Orleans, LA 70112, USA 2 Department of Dermatology, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China 3 Department of Pathology, Ochsner Clinic Foundation, New Orleans, LA 70130, USA 4 Department of Computer Science and Biostatistics Facility of RCMI Cancer Research Center, Xavier University of Louisiana, New Orleans, LA 70125, USA 5 Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA 70112, USA 6 Tulane Cancer Center, Louisiana Cancer Research Consortium, Tulane University, New Orleans, LA 70112, USA 7 Tulane Center for Stem Cell Research and Regenerative Medicine, Tulane University, New Orleans, LA 70112, USA 8 Department of Urology, Tulane University, New Orleans, LA 70112, USA 9 Department of Medicine, Tulane University, New Orleans, LA 70112, USA 10 Department of Biostatistics and Bioinformatics, Tulane University, New Orleans, LA 70112, USA 11 Proteomics/Mass Spectrometry Facility, University at Albany, Rensselaer, NY 12144, USA 12 Department of Orthopaedic Surgery and Tulane Center for Aging, Tulane University, New Orleans, LA 70112, USA * These authors contributed equally to this work Correspondence to: Zongbing You, e-mail: zyou@tulane.edu Keywords: hyperinsulinemia, obesity, prostate cancer, interleukin-17, GSK3 Received: December 02, 2015 Accepted: January 29, 2016 Published: February 10, 2016 ABSTRACT Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo . These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men.

Stem cell (SC) application is a promising area of research in regenerative medicine, with the potential to treat, prevent, and cure disease. In recent years, the number of studies focusing on SCs for the treatment of erectile dysfunction (ED) and other sexual dysfunctions has increased significantly.This review includes critical ED targets and preclinical studies, including the use of SCs and animal models in diabetes, aging, cavernous nerve injury, and Peyronie's disease. A literature search was performed on PubMed for English articles.Combination treatment offers better results than monotherapy to improve pathological changes in diabetic ED. Regenerative medicine is a promising approach for the maintenance of sexual health and erectile function later in life. Cavernous nerve regeneration and vascular recovery employing SC treatment may be focused on radical prostatectomy-induced ED. Notwithstanding, there are a number of hurdles to overcome before SC-based therapies for ED are considered in clinical settings. Paracrine action, not cellular differentiation, appears to be the principal mechanism of action underlying SC treatment of ED. Intracavernosal injection of a single SC type should be the choice protocol for future clinical trials.

Background Previous studies have documented improvement in erectile function after bilateral cavernous nerve injury (BCNI) in rats with the use of pioglitazone. Our group determined this improvement to be mediated by the insulin-like growth factor-1 (IGF-1) pathway. Aim To eliminate the systemic effects of pioglitazone and evaluate the local delivery of IGF-1 by polymeric microspheres after BCNI in the rat. Methods Male Sprague–Dawley rats aged 10–12 weeks were assigned at random to 3 groups: sham operation with phosphate buffered saline (PBS)-loaded microspheres (sham group), crush injury with PBS-loaded microspheres (crush group), and crush injury with IGF-1–loaded microspheres (IGF-1 group). Poly(lactic-co-glycolic) acid microspheres were injected underneath the major pelvic ganglion (MPG). IGF-1 was released at approximately 30 ng/mL/day per MPG per rat. Outcomes Functional results were demonstrated by maximal intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP). Protein-level analysis data of IGF-1 receptor (IGF-1R), extracellular signal–regulated kinase (ERK)-1/2, and neuronal nitric oxide synthase (nNOS) were obtained using Western blot analysis and immunohistochemistry for both the cavernosal tissue and the MPG and cavernous nerve (CN). Results At 2 weeks after nerve injury, animals treated with IGF-1 demonstrated improved erectile functional recovery (ICP/MAP) at all voltages compared with BCNI (2.5V, P = .001; 5V, P < .001; 7.5V, P < .001). Western blot results revealed that up-regulation of the IGF-1R and ERK-1/2 in both the nervous and erectile tissue was associated with improved erectile function recovery. There were no significant between-group differences in nNOS protein levels in cavernosal tissue, but there was an up-regulation of nNOS in the MPG and CN. Immunohistochemistry confirmed these trends. Clinical Translation Local up-regulation of the IGF-1R in the neurovascular bed at the time of nerve injury may help men preserve erectile function after pelvic surgery, such as radical prostatectomy, eliminating the need for systemic therapy. Strengths & Limitations This study demonstrates that local drug delivery to the MPG and CN can affect the CN tissue downstream, but did not investigate the potential effects of up-regulation of the growth factor receptors on prostate cancer tissue. Conclusion Stimulating the IGF-1R at the level of the CN has the potential to mitigate erectile dysfunction in men after radical prostatectomy, but further research is needed to evaluate the safety of this growth factor in the setting of prostate cancer.

Erectile Dysfunction (ED) is a common health condition occuring in roughly 50% of aging males (40-70 years old). Recent attention has related gene therapy to ED, and now there is an interest to further implement gene therapy concepts to ED treatment. This review is an attempt to analyze key challenges and emphasize primary areas, including mostly preclinical and a few clinical trials, cellular target(s), and different viral vectors/nanoparticles for gene delivery in ED. While overexpression of target genes can be silenced by RNA interference (RNAi), down-regulation of these mechanisms has been implicated in ED. Although many patients with ED demonstrate efficacy with phosphodiesterase type 5 inhibitors, this therapy is insufficient in approximately 30-40% of patients. Although several preclinical studies for ED treatment provided promising results, gene therapy has not shown success in clinical practice, due in part to technical limitations of gene therapy to address ED pathogenesis. Developments in small RNA, such as small interfering RNA (siRNA) may lead to significant benefit in the management of ED. Also, siRNA delivery into the corpus cavernosum seems a challenging issue and awaits further development. Several safety concerns of gene therapy, gene acquisition, preparation, and delivery are necessary to continue investigation before any widespread application is used in ED treatment.

Introduction Peyronie's disease (PD) has frequently been associated with erectile dysfunction (ED) and may further compromise coitus. Aim To investigate the efficacy of intratunical injection of genetically modified rat adipose tissue-derived stem cells (ADSCs) expressing human interferon α-2b (ADSCs-IFN) in decreasing fibrosis and restoring erectile function in a rat model of tunica albugineal fibrosis (TAF). Methods A total of 36 Sprague-Dawley rats (12 weeks old; 300–350 g) were randomly divided in six equal groups: (i) sham group (50 μL saline-injected into the tunica albuginea [TA]); (ii) TAF group (transforming growth factor [TGF]-β1 [0.5 μg/50 μL] injected into the TA); (iii) TGF-β1 plus 5 × 105 control ADSCs injected same day; (iv) TGF-β1 plus 5 × 105 ADSCs-IFN injected same day; (v) TGF-β1 plus 5 × 105 control ADSCs injected after 30 days; and (vi) TGF-β1 plus 5 × 105 ADSCs-IFN injected after 30 days. Rat allogeneic ADSCs were harvested from inguinal fat tissue. Main Outcome Measures Forty-five days following the TGF-β1 injection, erectile function was assessed, and penile tissues were harvested for further evaluations. Results In the same-day injection groups, intratunical injection of ADSCs and ADSC-IFN improved erectile response observed upon stimulation of cavernous nerve compared with TAF group. Intratunical ADSC-IFN injection at day 30 improved erectile responses 3.1, 1.8, and 1.3 fold at voltages of 2.5, 5.0, and 7.0, respectively, when compared with TAF group. Furthermore, at voltages of 2.5 and 5.0, treatment on day 30 with ADSCs-IFN improved erectile responses 1.6- and 1.3-fold over treatment with ADSCs alone. Local injection of ADSCs or ADSCs-IFN reduced Peyronie's-like manifestations, and these effects might be associated with a decrease in the expression of tissue inhibitors of metalloproteinases. Conclusion This study documents that transplantation of genetically modified ADSCs, with or without human IFN α-2b, attenuated Peyronie's-like changes and enhanced erectile function in a rat model of TAF.

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

Introduction It is common for men to develop erectile dysfunction after radical prostatectomy. The anatomy of the rat allows the cavernous nerve (CN) to be identified, dissected, and injured in a controlled fashion. Therefore, bilateral CN injury (BCNI) in the rat model is routinely used to study post-prostatectomy erectile dysfunction. Aim To compare and contrast the available literature on pharmacologic intervention after BCNI in the rat. Methods A literature search was performed on PubMed for cavernous nerve and injury and erectile dysfunction and rat . Only articles with BCNI and pharmacologic intervention that could be grouped into categories of immune modulation, growth factor therapy, receptor kinase inhibition, phosphodiesterase type 5 inhibition, and anti-inflammatory and antifibrotic interventions were included. Main Outcome Measures To assess outcomes of pharmaceutical intervention on erectile function recovery after BCNI in the rat model. The ratio of maximum intracavernous pressure to mean arterial pressure was the main outcome measure chosen for this analysis. Results All interventions improved erectile function recovery after BCNI based on the ratio of maximum intracavernous pressure to mean arterial pressure results. Additional end-point analysis examined the corpus cavernosa and/or the major pelvic ganglion and CN. There was extreme heterogeneity within the literature, making accurate comparisons between crush injury and therapeutic interventions difficult. Conclusions BCNI in the rat is the accepted animal model used to study nerve-sparing post-prostatectomy erectile dysfunction. However, an important limitation is extreme variability. Efforts should be made to decrease this variability and increase the translational utility toward clinical trials in humans. Haney NM, Nguyen HMT, Honda M, et al. Bilateral Cavernous Nerve Crush Injury in the Rat Model: A Comparative Review of Pharmacologic Interventions. Sex Med Rev 2017;X:XXX–XXX.

Active surveillance (AS) is a treatment strategy for prostate cancer (PCa) whereby patients diagnosed with PCa undergo ongoing characterization of their disease with the intent of avoiding radical treatment. Previously, AS has been demonstrated to be a reasonable option for men with low-risk PCa, but existing cohorts largely consist of Caucasian Americans. Because African Americans have a greater incidence, more aggressive, and potentially more lethal PCa than Caucasian Americans, it is unclear if AS is appropriate for African Americans. We performed a review of the available literature on AS with a focus on African Americans.

In multicellular organisms, effective communication between cells is a crucial part of cellular and tissue homeostasis. This communication mainly involves direct cell–cell contact as well as the secretion of molecules that bind to receptors at the recipient cells. However, a more recently characterized mode of intercellular communication — the release of membrane vesicles known as exosomes — has been the subject of increasing interest and intensive research over the past decade. Following the discovery of the exosome-mediated immune activation, the pathophysiological roles of exosomes have been recognized in different diseases, including cancer. In this review, we describe the biogenesis and main physical characteristics that define exosomes as a specific population of secreted vesicles, with a special focus on their role in oncogenic transformation and cancer progression.

Prostate cancer (PC) is the most common and the second leading cause of cancer-related death among American men. Early diagnosis is a prerequisite to improving therapeutic benefits. However, the current clinical biomarkers for PC do not reliably decipher indolent PC from other urogenital disorders. Thus, effective clinical intervention necessitates development of new biomarkers for early detection of PC. The present study aimed to identify the miRNA signature in organ-confined (Gleason Score 6) prostate tumors. MicroRNA (miRNA/miR) array analysis identified 118 upregulated and 73 downregulated miRNAs in microdissected tumors in comparison to matched neighboring normal prostate epithelium. The miRs-Plus-A1083, -92b-5p, -18a-3p, -19a-3p, -639, -3622b-3p, -3189-3p, -155-3p, -410, -1179, 548b-5p, and -4469 are predominantly expressed (7–11-fold), whereas miRs-595, 4490, -3120-5p, -1299, -21-5p, -3677-3, -let-7b-5p, -5189, 3-121-5p, -4518, -200a-5p, -3682-5p, -3689d, -3149 represent the most downregulated (12–113-fold) miRNAs in microdissected prostate tumors. The array expression profile of selected miRNA signature and their potential mRNA targets was validated by qRT-PCR analysis in PC cell lines. Integrated in silico and computational prediction analyses demonstrated that the dysregulated miRNA signature map to key regulatory factors involved in tumorigenesis, including cell cycle, apoptosis, and p53 pathways. The newly identified miRNA signature has potential clinical utility as biomarkers, prognostic indicators, and therapeutic targets for early detection of PC. Further studies are needed to assess the functional significance and clinical usefulness of the identified miRNAs. Impact Statement To our knowledge his is the first study of identifying miRNA signatures in microdissected indolent (Gleason score 6) prostate cancer in comparison to matched normal prostate epithelium. By employing in silico and computational prediction analysis, the study provides a landscape of potential miRNA targets and key cellular pathways involved in prostate tumorigenesis. Identification if miRNAs and their relevant targets and pathways pave the way for underpinning their mechanistic role of miRNAs in human prostate tumorigenesis, and possibly other human cancers. Importantly, the outcome of the study has important clinical implications for the management of prostate cancer, including the use of miRNA(s) as biomarkers for early detection of prostate cancer.

Introduction: We sought to evaluate the therapeutic effect of adipose tissue-derived stem cells (ADSCs) in a rat model of urethral fibrosis.Methods: Eighteen (18) male Sprague-Dawley rats (300‒350 g) were divided into three groups: (1) sham (saline injection); (2) urethral fibrosis group (10 μg transforming growth factor beta 1 (TGF-β1) injection); and (3) ADSCs group (10 μg TGF-β1 injection plus 2 x 105 ADSCs). Rat ADSCs were harvested from rat inguinal fat pads. All study animals were euthanized at two weeks afterurethral injection. Following euthanasia, rat urethral tissue was were quantitated by Western blot analysis. Results: TGF-β1 injection induced significant urethral fibrosis and increased collagen type I and III expression (p

Prostate cancer (PCa) cells utilize androgen for their growth. Hence, androgen deprivation therapy (ADT) using anti-androgens, e.g. bicalutamide (BIC) and enzalutamide (ENZ), is a mainstay of treatment. However, the outgrowth of castration resistant PCa (CRPC) cells remains a significant problem. These CRPC cells express androgen receptor (AR) and utilize the intratumoral androgen towards their continued growth and invasion. Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, can decrease AR protein levels. In the present study, we tested the combined efficacy of anti-androgens and SFN in suppressing PCa cell growth, motility and clonogenic ability. Both androgen-dependent (LNCaP) and androgen-independent (C4-2B) cells were used to monitor the effects of BIC and ENZ, alone and in combination with SFN. Co-exposure to SFN significantly (p

Introduction: The de novo synthesis and “intracrine” production of androgens by prostate tumors provides a significant survival advantage leading to the outgrowth of castration resistant tumors. Currently, targeting residual androgens in metastatic prostate cancer (PC) microenvironment is the toughest challenge in the clinical management of advanced PC patients treated with androgen ablation therapy (AAT), as they are implicated for development of castration-resistant PC (CRPC). Our previous studies (Stem Cells, 2014) demonstrate that PC-derived adipose derived stem cells (pASCs) promote tumor growth and undergo PC mimicry through neoplastic reprogramming. The present study examines the role of pASCs in synthesizing and producing and rogens and its potential in supporting growth of hormone-dependent PC cells under castrate conditions in vitro and in vivo. Further more, the study attempts to identify key players among the and rogen-metabolizing enzymes (AMEs) in the backdoor pathway for targeting and degradation of androgen synthesis in the prostate tumor microenvironment, by both in vitro and in vivo assays. Methods: Based on an IRB approved protocol, the pASCs were isolated from fatt issues procured from PC patients undergoing radical prostatectomies. The purity of the pASC isolates was validated by FACS analysis and differentiation assays. The ability of pASCs exposed to PC-derived conditioned media (CM) to produce androgens was monitored by expression of AMEs (PCR) and androgen release (EIAkit). The ability of pASC CM to support growth of androgen-dependent LNCaP cells was assessed by MTT assay and growth of LNCaP tumors co-transplanted with pASCs in castrated mice in vivo. Normal donor-derived adipose derived stem cells (nASCs) were enriched for tumor tropicity by a transwell system. Next, we subcloned in a GFP bicistronic IRES lentivirus construct (pLVX-IRES-ZsGreen1) the rat androgen hydrolyzing enzyme 3α-hydroxysteroid dehydrogenase (Type I, 3α-HSD), as known as AKRIC14, in-frame with IL-2SS sequence at the N-terminus. R1C14. The ILL2SS sequencec enables the AKR1C14 by the transduced cells. Upon transduction, the enriched nASC population was monitored for the transgene expression and release by qRT-PCR, immunoblotting and EIA kit. The efficacy and functional activity of the recombinant enzyme and the nASC-released ANR1C14 enzyme were monitored by MTT, LDH and luciferase reporterassays in the androgen-dependent LNCaP cells in vitro. The tumor-homing potential of the GFP-AKRC14 expressing nASCs infused by IV route was validated by their engraftment in LNCaP tumor-bearing mice. To examine the efficacy of secreted enzyme to hydrolyze androgens andinduce tumor regression in vivo, the GFP-AKR1C14 transduced nASCs were injected in mice bearing LNCaP tumors and tumor volumes were measured weekly. Results: We demonstrate here in that unlike normal adipose-tissue derived stem cells (nASCs), the PC cell microenvironment subverts tumor-tropic PC patients' derived ASCs (pASCs) to synthesize and produce androgens in the tumor microenvironment. The pASC-mediated hormone production was sufficient to support the growth of androgen-dependent LNCaP PC cells in vitro under hormone-deprivation conditions as well as in castrated nude mice. Comparative analysis of gene expression of AMEs in microdissected cells versus the adjacent normal tissue revealed AKR1C4, an alpha keto reductase, to be the most promising candidate for targeting the backdoor pathway in dihydrotestesterone (DHT) synthesis. Type 1 3α-HSD enzyme has been shown to readily oxidize testosterone to Δ4-androstene-3, 17-dione in a substrate dependent reaction. The recombinant AKR1C4 induced cytotoxicity and apoptosis in androgen-dependent PC cells in vitro. Next, using a GFP bicistronic IRES lentivirus construct, we demonstrate that the genetically engineered tumor-tropic nASCs enable expression and secretion of AKR1C14. The results showed that the engineered nASCs are capable of expressing the enzyme, which in turn catabolizes androgens in vitro and induce apoptosis in androgen-dependent LNCaP cells. The AKR1C14 transduced nASCs successfully engrafted in and reduced growth of LNCaP tumors in nude mice. Conclusions: Together, our data demonstrates that selective delivery of alpha keto reductases by nASCs could eliminate residual androgens and further paves the way for their potential use in combination with AAT for treatment of CRPC.

Although a spectrum of options is available for erectile dysfunction (ED) treatment, ED in diabetics, post-prostatectomy patients, and those with Peyronie's disease (PD) may be more severe in degree and less likely to respond to conventional medical therapies. Unfortunately, there have been limited breakthroughs in therapeutic options for severe ED during the past decade. However, one of the more fascinating strategies in preclinical development to treat ED is stem cell transplantation. Depending on the cell type, recent research has demonstrated that with transplantation, these stem cells can exert a paracrine effect on surrounding penile tissues and differentiate into smooth muscle, endothelium, and neurons. Adipose tissue-derived stem cells (ADSCs) have become a valuable resource because of their abundance and ease of isolation. It is evident that ADSCs may provide a realistic, therapeutic modality for the treatment of ED. In this review, we will cover the literature that has evaluated ADSCs in the treatment of ED.

Breast cancer tissue is a heterogeneous cellular milieu comprising cancer and host cells. The interaction between breast malignant and non-malignant cells takes place in breast tumor microenvironment (TM), and has a crucial role in breast cancer progression. In addition to cellular component of TM, it mainly consists of cytokines released by tumor cells. The tumor-tropic capacity of mesenchymal stem cells (MSCs) and their interaction with breast TM is an active area of investigation. In the present communication, the interplay between the breast resident adipose tissue-derived MSCs (B-ASCs) and breast TM was studied. It was found that a distinct subset of B-ASCs display a strong affinity for conditioned media (CM) from two breast cancer cell lines, MDA-MB 231 (MDA-CM) and MCF-7 (MCF-CM). The expressions of several cytokines including angiogenin, GM-CSF, IL-6, GRO-α and IL-8 in MDA-CM and MCF-CM have been identified. Upon functional analysis a crucial role for GRO-α and IL-8 in B-ASCs migration was detected. The B-ASC migration was found to be via negative regulation of RECK and enhanced expression of MMPs. Furthermore, transcriptome analysis showed that migratory subpopulation express both pro- and anti-tumorigenic genes and microRNAs (miRNA). Importantly, we observed that the migratory cells exhibit similar gene and miRNA attributes as those seen in B-ASCs of breast cancer patients. These findings are novel and suggest that in breast cancer, B-ASCs migrate to the proximity of tumor foci. Characterization of the molecular mechanisms involved in the interplay between B-ASCs and breast TM will help in understanding the probable role of B-ASCs in breast cancer development, and could pave way for anticancer therapies.

The contributions of interleukin (IL)-17 to cancer remain unclear and somewhat controversial. We took a genetic approach to explore its role in prostate cancers by interbreeding IL-17 receptor C (IL-17RC)–deficient mice with mice that are conditionally mutant for PTEN, one established preclinical model for prostate cancer. Mice that were IL-17RC–deficient (IL-17RC−) displayed prostates that were smaller than mice that maintained IL-17RC expression (IL-17RC+). In addition, IL-17RC− mice developed a reduced number of invasive prostate adenocarcinomas with lower rates of cellular proliferation and higher apoptosis than IL-17RC+ mice. Moreover, the fibromuscular stroma surrounding prostatic glands was relatively thicker in IL-17RC− mice and was associated with decreased matrix metalloproteinase (Mmp)7 expression and increased Timp1, 2, and 4 expression, whereas administration of recombinant mouse IL-17 induced prostatic expression of Mmp7. Taken together, our results suggested that IL-17 promotes the formation and growth of prostate adenocarcinoma, and that an IL-17–MMP7 signaling axis is required for the transition of prostatic intraepithelial neoplasia to frank adenocarcinoma. Cancer Res; 72(10); 2589–99. ©2012 AACR.

Prostate cancer (PCa) is a leading cause of morbidity and mortality in elderly men in the United States. Despite androgen deprivation therapy (ADT), the recurrence of castration resistant prostate cancer (CRPC) remains a significant challenge. Augmented signaling via the full-length androgen receptor (AR) and the constitutively active AR splice variants, especially ARv7, is associated with ADT-resistance. Therefore, a proper understanding of augmented AR signaling and adjunct strategies to suppress both AR and ARv7 levels are critically needed. ADT-induced oxidative stress (Ox-stress) can facilitate CRPC outgrowth. We previously documented that the Ox-stress induced transcription factor, Nrf2 can suppress AR expression and AR-transactivation function in PCa cells. Recently, we also showed that the Nrf2-inducing natural compound, sulforaphane (SFN) can decrease both AR and ARv7 levels in PCa cells, and co-exposure to SFN enhanced the efficacy of anti-androgens. In this study, we examined whether a synthetic triterpenoid drug, Bardoxolone-methyl (CDDO-me), a potent enhancer of Nrf2, can similarly decrease AR and ARv7 expression. Exposure to nanomolar (nM) concentrations of CDDO-me rapidly downregulated AR levels in the androgen-dependent LNCaP cells and androgen-independent C4-2B cells. Exposure to CDDO-me also suppressed both AR and ARv7 levels in the CRPC line, CWR22Rv1. A biphasic effect of CDDO-me on reactive oxygen species (ROS) levels was observed. Pre-exposure to the antioxidant, n-acetyl cysteine (NAC) blocked the AR-suppressive effect of CDDO-me. Molecular mechanistic studies using actinomycin-D (transcription inhibitor), cycloheximide (translation inhibitor), and MG132 (proteasomal inhibitor) suggested that CDDO-me may decrease AR gene expression and increase degradation of AR and ARv7 proteins. Furthermore, exposure to CDDO-me increased proteasomal activity in PCa cells and suppressed the levels of several AR splicing factors, i.e. the heteronuclear ribonucleoproteins A1 and H1 (hnRNP-A1 and hnRNP-H1). Co-exposure to CDDO-me increased the anti-cancer efficacy of enzalutamide (ENZ) as evident by decreased cell-viability (MTT), migration (scratch-wound) and colony forming unit (CFU) assays. Therefore, adjunct treatment with low-dose CDDO-me may be an effective strategy to suppress AR and ARv7 levels and augment the efficacy of anti-androgens. Most importantly, since CDDO-me is in late-stage clinical trials for chronic kidney disease (CKD), its repositioning as a potent AR suppressive agent may be of significant translational value in PCa patients undergoing ADT. Citation Format: Namrata Khurana, Partha Chandra, Hogyoung Kim, Asim B Abdel-Mageed, Suresh Sikka, Debasis Mondal. Bardoxolone-methyl suppresses both androgen receptor and its splice-variant ARv7 in prostate cancer cells to enhance the anti-cancer efficacy of enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4809.

Reactive oxygen species (ROS) signaling has recently sparked a surge of interest as being the molecular underpinning for cancer cell survival, but the precise mechanisms involved have not been completely elucidated. This review covers the possible roles of two ROS-induced transcription factors, Nrf1 and Nrf2, and the antioxidant proteins peroxiredoxin-1 (Prx-1) and Thioredoxin-1 (Txn-1) in modulating AR expression and signaling in aggressive prostate cancer (PCa) cells. In androgen independent (AI) C4-2B cells, in comparison to the parental androgen dependent (AD) LNCaP cells, we present evidence of high Nrf1 and Prx-1 expression and low Nrf2 expression in these aggressive PCa cells. Furthermore, in DHT treated C4-2B cells, increased expression of the p65 (active) isoform of Nrf1 correlated with enhanced AR transactivation. Our findings implicate a crucial balance of Nrf1 and Nrf2 signaling in regulating AR activity in AI-PCa cells. Here we will discuss how understanding the mechanisms by which oxidative stress may affect AR signaling may aid in developing novel therapies for AI-PCa.

Purpose: The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men. Experimental Design: We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade–matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored. Results: Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo. Conclusions: Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men. Clin Cancer Res; 22(7); 1744–56. ©2015 AACR.

Summary Chronic genitourinary inflammation results in Leukocytospermia (LCS), an elevated number of white blood cells (WBCs) in semen, which, in association with oxidative stress, may suppress sperm function, and manifest as male factor infertility. The current clinical diagnosis of LCS employs manual enumeration of WBCs and requires complex staining and laboratory skills or measurement of inflammatory cytokines and chemokines levels. Many patients with idiopathic infertility are asymptomatic. In search of better inflammatory markers for LCS, we evaluated expression of toll-like receptors 2 and 4 (TLR-2/4), cyclooxygenase-2 (COX-2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) in semen samples of age-matched infertile patients with and without LCS. We employed the usage of specific Western blot evaluation, cytokine array; immunofluorescence microscopy (IFM) followed by computer-based analysis, and other molecular approaches. As compared with non-LCS patients (n = 38), semen samples from LCS patients (n = 47) displayed significantly lower total sperm count (p

We sought to develop a reproducible TGF-β1 injection technique to induce urethral fibrosis in the rat urethra.A total of 32 male Sprague Dawley® rats weighing 300 to 350 gm were anesthetized with ketamine/xylazine intraperitoneally. Using a 5 mm penoscrotal incision the rat urethra was exposed. In the experimental group varying doses of TGF-β1 (5, 10 and 25 μg) were injected in each side of the urethral wall. Normal saline infiltration was used in the sham treated group. Rats were sacrificed 2 and 4 weeks following TGF-β1 injection. Urethral specimens were stained with hematoxylin and eosin, and Masson trichrome, and Western blot evaluations were performed. Normal and strictured urethral tissues from patients were collected and evaluated in the same fashion.There was no evidence of urethral wall thickening or fibrosis in the sham treated group. Varied histological evidence of fibrosis was noted in all experimental groups. There was a significant increase in collagen type I expression 2 weeks after injection of 5, 10 and 25 μg TGF-β1. Collagen type III expression was significantly increased 2 weeks after injecting 10 and 25 μg of TGF-β1, which persisted to 28 days after injection.TGF-β1 injection can successfully generate a reproducible rat model of urethral spongiofibrosis. This technique is simple, inexpensive and reproducible. Our series is a proof of concept study. Additional studies in larger animals are needed to further confirm our findings.

The exciting field of embryonic stem cell research emerged in 1981 with the study of mouse stem cells [1,2]. The Thompson group from the University ofWisconsin reported on human embryonic stem cells in 1998 [3]. Because ethical concerns and the regulatory environment have inhibited thewidespread use of human embryonic stem cells, interest has focused on successful reprogramming of adult stem cells as an alternative strategy. Mesenchymal stem cells are a type of adult stem cell that can be found in many tissues, including bone marrow, adipose tissue, umbilical cord blood, placenta, and dental pulp. It has been shown that autotransplantation of adipose-derived stem cells (ADSCs) is of benefit in treating erectile dysfunction, Peyronie’s disease, and urethral stricture disease (USD) in animal models [4–6]. In this issue of European Urology, Castiglione et al [7] present a compelling and timely study that adds to the literature regarding the use of ADSCs for therapy in USD. It adds cystometric analysis, which was lacking in prior animal models. It also represents a modification of our rat TGF-b [2_TD$DIFF]1 model of USD [8]. In this study, after injection of TGF-b [3_TD$DIFF]1, transmural injury to the urethral wall of rats was induced using a 23-gauge needle to puncture the urethra four times until the urethral catheter was visible. The authors do not justify their use of trauma to the urethra in combination with the use of TGF-b[4_TD$DIFF]1. Furthermore, they state that USD models based on physical injury have not been reproducible, but still add physical injury to themodel. A dose of 1 mg of TGF-b [2_TD$DIFF]1 ismuch lower than that used in our established TGF-b[2_TD$DIFF]1 rat model of USD.

Ureteropelvic junction obstruction (UPJO) is defined as an impediment to urinary flow from the renal pelvis into the ureter. The exact cause remains an enigma despite investigations along embryologic, anatomic, and histologic lines. Our goal was to investigate in vivo the expression profile of cytokines in hyperplastic urothelial cells as a means of determining the source of UPJO.Cellular proteomes of matched normal and hyperplastic urothelial cells were analyzed by laser capture microdissection (LCM) and tissue microdissection and human cytokine proteomic chips. All specimens (N = 9) were surgically obtained from patients undergoing laparoscopic dismembered pyeloplasty and were immediately embedded in O.C.T. solution and flash-frozen in liquid nitrogen. Tissue sections (6 microm) were mounted on uncoated glass slides using a cryostat, fixed in 70% ethanol, stained with hematoxylin and eosin, and sequentially dehydrated in ethanol and xylene. Typically, paired samples of normal and hyperplastic urothelial cells were procured on separate caps from serial sections of each specimen by the Arcturus PixCell II LCM system using 3000 laser pulses and a spot diameter of 30 microm. Total proteins were harvested and quantitated. Differential expression profile analysis of 43 cytokines in normal and hyperplastic cells were performed using human protein chips. Briefly, the membranes were initially probed with protein (150 ng) from normal or hyperplastic cells and sequentially reacted with a cocktail of biotinylated cytokine antibodies and horseradish peroxidase-conjugated streptavidin. The membranes were developed using enhanced chemiluminescence and analyzed by densitometry.Comparative densitometric analysis revealed twofold to fourfold upregulation of growth-related oncogene alpha (GRO-alpha), interleukin (IL)-1alpha, interferon (INF)-gamma, IL-8, tumor necrosis factor (TNF)-alpha, RANTES, and macrophage inflammatory protein-1beta (MIP-1beta) and twofold to fourfold downregulation of transforming growth factor (TGF)-beta and IL-10 in hyperplastic urothelial cells compared with paired control cells.We here report the efficient application of LCM and proteomic array chips for expression profile analysis of cytokines in vivo. Because the etiology and pathogenesis of UPJO are still fragmentary, the marked heterogeneity of the observed cytokine alterations reported here may be of significance. Further studies are required to elucidate the functional significance of the differentially expressed cytokines in the pathogenesis of the disease.

We determined whether adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis of streptozotocin induced diabetic rats could improve the impaired erectile response.Two experimental groups of animals were transfected with adenoviruses, including streptozotocin (Sigma Chemical Company, St. Louis, Missouri) diabetic rats with AdCMVbetagal and streptozotocin diabetic rats with AdCMVeNOS. At 1 to 2 days after transfection these study animals underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age matched control rats. In control and transfected streptozotocin diabetic rats eNOS and neuronal NOS (nNOS) were examined by Western blot analysis. Constitutive and inducible NOS activities were evaluated in the presence and absence of calcium by L-arginine to L-citrulline conversion and nitrate plus nitrite levels were measured. In control and streptozotocin diabetic penes beta-galactosidase activity and localization were determined.After transfection with AdCMVbetagal beta-galactosidase was localized to the endothelium and smooth muscle cells of the streptozotocin diabetic rat penis. Streptozotocin diabetic rats had a significant decrease in erectile function, as determined by peak and total intracavernous pressure (area under the curve) after cavernous nerve stimulation compared with control rats. Streptozotocin diabetic rats transfected with AdCMVeNOS had peak intracavernous pressure and area under the curve similar to those in control animals. This change in erectile function was a result of eNOS over expression with an increase in eNOS protein expression and constitutive NOS activity as well as an increase in nitric oxide biosynthesis, as reflected by an increase in cavernous nitrate plus nitrite formation. There was no change in nNOS protein expression or calcium independent conversion of NOS (inducible NOS activity).Adenoviral gene transfer of eNOS significantly increased peak and total intracavernous pressure to cavernous nerve stimulation in streptozotocin diabetic rats to a value similar to the response observed in control rats. Our results suggest that eNOS contributes significantly to the physiology of penile erection. These data demonstrate that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the streptozotocin diabetic rat.

Increased superoxide anion (O[Formula: see text]·) may contribute to vascular dysfunction in aging. In aged cavernosal tissue, lucigenin-enhanced chemiluminescence demonstrated a threefold increase in superoxide formation, and the oxidative fluorescent probe hydroethidine indicated higher superoxide levels throughout the aged penis. This increase in superoxide was associated with impaired cavernosal nerve-mediated and agonist-induced erectile responses, increased nitrotyrosine staining, and lower cGMP levels, but no compensatory change in cavernosal extracellular (EC)-superoxide dismutase (EC-SOD) mRNA or protein. In vivo adenoviral (Ad) gene transfer of EC-SOD to the penis resulted in higher expression of EC-SOD mRNA, protein, SOD activity, cGMP levels, and lower nitrotyrosine staining. Transfection with AdCMVEC-SOD resulted in a significant increase in erectile response to cavernosal nerve stimulation, ACh, and zaprinast to a magnitude similar to young rats. These data provide evidence in support of the hypothesis that erectile dysfunction associated with aging is related in part to an increase in cavernosal O[Formula: see text]· formation. Gene-transfer of EC-SOD reduces superoxide formation and restores age-associated erectile function and may represent a novel therapeutic target for the treatment of erectile dysfunction.

The etiologies of erectile dysfunction (ED) after nerve-sparing radical prostatectomy have not been clearly elucidated. The aim of this study was to evaluate the effects of cavernous nerve injury on cavernous fibrosis, and to consider measures to prevent irreversible damage to the cavernous tissues. Twenty male Sprague-Dawley rats constituted the study population. The animals were divided into 2 groups; group 1 consisted of sham-operated rats (n = 10), and group 2 consisted of rats that underwent incision of both cavernous nerves (n = 10). Three months later, all rats underwent intracavernous papaverine injection (300 and 600 mg), and intracorporal pressures were recorded. Transforming growth factor-beta(1) (TGF-beta(1)) messenger RNA (mRNA) expression from rat penile tissue was measured using reverse transcriptase-polymerase chain reaction. Hypoxia-inducible factor-1alpha (HIF-1alpha), TGF-beta(1), and collagen I and III protein expressions were determined by Western blot analysis and immunohistochemical staining. Erectile function as studied with intracavernosal papaverine injection and histological analysis of penile cross-sections at 3 months was similar in both groups. TGF-beta(1) mRNA expression, HIF-1alpha, TGF-beta(1), and collagen I and III protein expressions were significantly greater in the neurotomy group. Immunohistochemical staining for TGF-beta(1), HIF-1alpha, and collagen III were qualitatively more positive in the neurotomy group, whereas collagen I staining was similar. This study demonstrates an increase in TGF-beta(1), HIF-1alpha, and collagen III synthesis in rat cavernosal smooth musculature after cavernous neurotomies. In theory, cavernous fibrosis may be reduced by employing various vasoactive agents or interventions that increase oxygenation to the corporal tissues during the postoperative period.

In an effort to better understand the pathophysiology of ureteropelvic junction (UPJ) obstruction and to determine possible predisposing factors for endopyelotomy failures, we compared the activation of the nuclear factor NF-kappa B and proinflammatory cytokines in patients who failed endopyelotomy and post-primary pyeloplasty patients. We hypothesized that an imbalance toward proinflammatory cytokines may promote fibrosis prior to and after endopyelotomy in patients with severe hydronephrosis.The charts of patients who underwent open pyeloplasty at our institution were reviewed. Group I was the control group, consisting of 10 patients who had undergone radical nephrectomy for renal-cell carcinoma without involvement of the renal pelvis. Group II was the endopyelotomy failure group and included 11 patients over the age of 15 years treated for symptomatic UPJ obstruction. Group III included six patients who underwent primary pyeloplasty. Paraffin-embedded blocks of UPJ segments from each of these patients were obtained, and immunohistochemical detection of NF-kappa B activation, interleukin (IL)-6, and hypoxia-inducing factor (HIF) was performed. As an in-vitro model, activation of NF-kappa B and cytokine gene expression were also monitored in human bladder T24 urothelial cells 24 hours after exposure to hypoxia (1% O(2)) in the presence and absence of NF-kappa B inhibitor. The activation of NF-kappa B was determined by immunocytochemical analysis, whereas cytokine gene expression was measured using reverse transcriptase-polymerase chain reaction.Immunoreactivity to NF-kappa B was observed in the nuclei of the urothelium and muscle layer in all patients in group II. Such immunostaining suggests increased nuclear translocation and activation of this transcription factor. Those patients with increased expression of NF-kappa B demonstrated increases in IL-6 expression as well. Hypoxia-inducing factor was identified in all the tissue samples tested in group II. Stimulation of the human urothelial cells by hypoxia, known to activate NF-kappa B, resulted in an increase in the levels of IL-1 and IL-6 transcripts compared with hypoxia-exposed cells in the presence of NF-kappa B inhibitors.The NF-kappa B factor was upregulated and proinflammatory cytokines were activated in patients with UPJ obstruction who failed endopyelotomy. Proinflammatory cytokines upregulated by this nuclear factor can result in fibrosis and affect healing after endopyelotomy. Hypoxia appears to activate this nuclear factor. Further studies correlating the degree of hydronephrosis with the activation of HIF are necessary to clarify the role of severe hydronephrosis and its management in UPJ obstruction.

Despite its side-effects, docetaxel (DTX) remains a first-line treatment against castration resistant prostate cancer (CRPC). Therefore, strategies to increase its anti-tumor efficacy and decrease its side effects are critically needed. Targeting of the constitutive endoplasmic reticulum (ER) stress in cancer cells is being investigated as a chemosensitization approach. We hypothesized that the simultaneous induction of ER-stress and suppression of PI3K/AKT survival pathway will be a more effective approach. In a CRPC cell line, C4-2B, we observed significant (p