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Small-cell prostate carcinoma (SCPC) is an aggressive malignancy that is managed similarly to small-cell lung cancer. SCPC can evolve from prostate adenocarcinoma in response to androgen deprivation therapy, but, in rare cases, is present at initial cancer diagnosis. The molecular aetiology of de novo SCPC is incompletely understood, owing to the scarcity of tumour tissue and the short life-expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology, and the remainder had some admixed adenocarcinoma foci, but all were treated with first-line platinum-based chemotherapy. The median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin-fixed paraffin-embedded archival tumour tissue. We observed frequent biallelic deletion and/or mutation of the tumour suppressor genes TP53, RB1, and PTEN, similarly to what was found in treatment-related SCPC. Indeed, at the RNA level, pure de novo SCPC closely resembled treatment-related SCPC. However, five patients had biallelic loss of DNA repair genes, including BRCA1, BRCA2, ATM, and MSH2/6, potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harboured ETS gene rearrangements involving androgen-driven promoters, consistent with the evolution of de novo SCPC from an androgen-driven ancestor. Overall, our results reveal a highly aggressive molecular landscape that underlies this unusual pathological variant, and suggest opportunities for targeted therapy strategies in a disease with few treatment options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Refinement of parameters defining prostate cancer (PC) prognosis are urgently needed to identify patients with indolent versus aggressive disease. The Canadian Prostate Cancer Biomaker Network (CPCBN) consists of researchers from four Canadian provinces to create a validation cohort to address issues dealing with PC diagnosis and management., Methods: A total of 1512 radical prostatectomy (RP) specimens from five different biorepositories affiliated with teaching hospitals were selected to constitute the cohort. Tumoral and adjacent benign tissues were arrayed on tissue microarrays (TMAs). A patient clinical database was developed and includes data on diagnosis, treatment and clinical outcome., Results: Mean age at diagnosis of patients in the cohort was 61 years. Of these patients, 31% had a low grade (≤6) Gleason score (GS), 55% had GS 7 (40% of 3 + 4 and 15% of 4 + 3) and 14% had high GS (≥8) PC. The median follow-up of the cohort was 113 months. A total of 34% had a biochemical relapse, 4% developed bone metastasis and 3% of patients died from PC while 9% died of other causes. Pathological review of the TMAs confirmed the presence of tumor and benign tissue cores for > 94% of patients. Immunohistochemistry and FISH analyses, performed on a small set of specimens, showed high quality results and no biorepository-specific bias., Conclusions: The CPCBN RP cohort is representative of real world PC disease observed in the Canadian population. The frequency of biochemical relapse and bone metastasis as events allows for a precise assessment of the prognostic value of biomarkers. This resource is available, in a step-wise manner, for researchers who intend to validate prognostic biomarkers in PC. Combining multiple biomarkers with clinical and pathologic parameters that are predictive of outcome will aid in clinical decision-making for patients treated for PC.

Prostate cancer is the second leading cause of cancer-related deaths in men in North America and there is an urgent need for development of more effective therapeutic treatments against this disease. We have recently shown that diindolylmethane (DIM) and several of its halogenated derivatives (ring-DIMs) induce death and protective autophagy in human prostate cancer cells. However, the in vivo efficacy of ring-DIMs and the use of autophagy inhibitors as adjuvant therapy have not yet been studied in vivo. The objective of this study was to determine these effects on tumor growth in nude CD-1 mice bearing bioluminescent androgen-independent PC-3 human prostate cancer cells. We found that chloroquine (CQ) significantly sensitized PC-3 cells to death in the presence of sub-toxic concentrations of DIM or 4,4'-Br 2 DIM in vitro. Moreover, a combination of DIM (10 mg/kg) and CQ (60 mg/kg), 3× per week, significantly decreased PC-3 tumor growth in vivo after 3 and 4 weeks of treatment. Furthermore, 4,4'-Br 2 DIM at 10 mg/kg (3× per week) significantly inhibited tumour growth after 4 weeks of treatment. Tissues microarray analysis showed that DIM alone or combined with CQ induced apoptosis marker TUNEL; the combination also significantly inhibited the cell proliferation marker Ki67. In conclusion, we have confirmed that DIM and 4,4'-Br 2 DIM are effective agents against prostate cancer in vivo and shown that inhibition of autophagy with CQ enhances the anticancer efficacy of DIM. Our results suggest that including selective autophagy inhibitors as adjuvants may improve the efficacy of existing and novel drug therapies against prostate cancer.

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy., Competing Interests: The authors declare no conflict of interest.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management. Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Introduction: Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies like sunitinib, target the formation of new vessels allowing nutrient deprivation and cell death. However, recent studies have shown that patients develop resistance after approximately 1 year of treatment and show disease progression while on therapy. Therefore, we propose to identify the protein(s) responsible for increased migration with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease., Material and Methods: Human renal cancer cell lines (Caki-1, Caki-2, ACHN) were treated with increasing doses of sunitinib to develop a sunitinib-conditioned renal cell carcinoma cell line. mRNA microarray and qPCR were performed to compare the differences in gene expression between Caki-1 sunitinib-conditioned and non-conditioned cells. NTN1 was assessed in our in vivo sunitinib-conditioned mouse model using immunostaining. xCELLigence and scratch assays were used to evaluate migration and MTS was used to evaluate cell viability., Results: Human renal cell carcinoma sunitinib-conditioned cell lines showed upregulation of netrin-1 in microarray and q-PCR. Increased migration was demonstrated in Caki-1 sunitinib-conditioned cells when compared to the non-treated ones as well as, increased endothelial cell migration. Silencing of netrin-1 in sunitinib-conditioned Caki-1 cells did not demonstrate a significant reduction in cell migration., Conclusion: Netrin-1 is highly upregulated in renal cell carcinoma treated with sunitinib, but has no influence on cell viability or cell migration in metastatic RCC., Competing Interests: CONFLICTS OF INTEREST The authors state no conflicts on interest.

We have explored the effects of 20(S)-protopanaxadiol (aPPD), a naturally derived ginsenoside, against androgen receptor (AR) positive castration resistant prostate cancer (CRPC) xenograft tumors and have examined its interactions with AR. In silico docking studies for aPPD binding to AR, alongside transactivation bioassays and in vivo efficacy studies were carried out in the castration-resistant C4-2 xenograft model. Immunohistochemical (IHC) and Western blot analyses followed by evaluation of AR, apoptotic, cell cycle and proliferative markers in excised tumors was performed. The growth of established CRPC tumors was inhibited by 53% with aPPD and a corresponding decrease in serum PSA was seen compared to controls. The IHC data revealed that Ki-67 was significantly lower for aPPD treated tumors and was associated with elevated p21 and cleaved caspase-3 expression, compared to vehicle treatment. Furthermore, aPPD decreased AR protein expression in xenograft tumors, while significantly upregulating p27 and Bax protein levels. In vitro data supporting this suggests that aPPD binds to and significantly inhibits the N-terminal or the DNA binding domains of AR. The AR androgen binding site docking score for androgen (dihydrotestosterone) was -11.1, while that of aPPD was -7.1. The novel findings described herein indicate aPPD potently inhibits PCa in vivo partly via inhibition of a site on the AR N-terminal domain. This manifested as cell cycle arrest and concurrent induction of apoptosis via an increase in Bax, cleaved-caspase-3, p27 and p21 expression., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing financial interest.

Background: Cisplatin-based chemotherapy significantly improved the survival of patients with germ cell testicular cancer. However, long term side effects of chemotherapy have non-negligible impact on the quality of life of these young patients, who have a long life expectancy after being successfully treated., Materials and Methods: 25-OH vitamin D, testosterone, FSH and LH of patients with testicular cancer were retrospectively evaluated and for each patient clinical information were collected. The tissue of 52 patients with germ cell tumors was analyzed for VDR expression by immunohistochemistry. The serum 25-OH vitamin D and VDR expression were correlated to the patients 'clinical characteristics., Results: 25-OH vitamin D was analyzed in 82 patients. Insufficient (< 30 ng/ml) levels were detected in 65%-85%, mild deficient (< 20 ng/ml) in 25%-36% and severe deficient (< 10 ng/ml) in 6%-18% of the patients over a median follow-up of 48 months. No difference in serum 25-OH vitamin D was detected over the follow-up time points. No correlation with histology, stage and type of treatment was found. The 25-OH vitamin D levels were not correlated to testosterone, FSH and LH levels. Interestingly, the expression of VDR was much higher in non seminoma than in seminoma tissue., Conclusions: Patients with testicular cancer have reduced vitamin D levels after the treatment of the primary cancer. Since long term hypovitaminosis D leads to high risk of fractures, infertility and cardiovascular diseases, we envision that vitamin D should be regularly checked in patients with testicular cancer and replaced if needed., Competing Interests: CONFLICTS OF INTEREST The authors have declared no conflicts of interest.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

The introduction of serum Prostate Specific Antigen (PSA) testing nearly 30 years ago has been associated with a significant shift towards localized disease and decreased deaths due to prostate cancer. Recognition that PSA testing has caused over diagnosis and over treatment of prostate cancer has generated considerable controversy over its value, and has spurred efforts to identify prognostic biomarkers to distinguish patients who need treatment from those that can be observed. Recent studies show that cancer is heterogeneous and forms a hierarchy of tumor cell populations. We developed a method of identifying prostate cancer differentiation states related to androgen signaling using Boolean logic. Using gene expression data, we identified two markers, CD38 and ARG2, that group prostate cancer into three differentiation states. Cancers with CD38-, ARG2- expression patterns, corresponding to an undifferentiated state, had significantly lower 10-year recurrence-free survival compared to the most differentiated group (CD38+ARG2+). We carried out immunohistochemical (IHC) staining for these two markers in a single institution (Stanford; n = 234) and multi-institution (Canary; n = 1326) cohorts. IHC staining for CD38 and ARG2 in the Stanford cohort demonstrated that combined expression of CD38 and ARG2 was prognostic. In the Canary cohort, low CD38 protein expression by IHC was significantly associated with recurrence-free survival (RFS), seminal vesicle invasion (SVI), extra-capsular extension (ECE) in univariable analysis. In multivariable analysis, ARG2 and CD38 IHC staining results were not independently associated with RFS, overall survival, or disease-specific survival after adjusting for other factors including SVI, ECE, Gleason score, pre-operative PSA, and surgical margins., Competing Interests: CONFLICTS OF INTEREST None.

Darolutamide (ODM-201) is a novel androgen receptor (AR) antagonist with a chemical structure distinctly different from currently approved AR antagonists that targets both wild-type and mutated ligand binding domain variants to inhibit AR nuclear translocation. Here, we evaluate the activity of darolutamide in enzalutamide-resistant castration resistant prostate cancer (CRPC) as well as in AR mutants detected in patients after treatment with enzalutamide, abiraterone, or bicalutamide. Darolutamide significantly inhibited cell growth and AR transcriptional activity in enzalutamide-resistant MR49F cells in vitro, and led to decreased tumor volume and serum prostate-specific antigen levels in vivo, prolonging survival in mice bearing enzalutamide-resistant MR49F xenografts. Moreover, darolutamide inhibited the transcriptional activity of AR mutants identified in the plasma of CRPC patients progressing on traditional therapies. In particular, darolutamide significantly inhibited the transcriptional activity of the F877L, H875Y/T878A, F877L/T878A, and the previously unreported T878G AR mutants, that transform enzalutamide into a partial agonist. In silico cheminformatics computer modeling provided atomic level insights confirming darolutamide antagonist effect in F877L and T878G AR mutants. In conclusion, our results provide a rationale for further clinical evaluation of darolutamide in enzalutamide-resistant CRPC, in particular in combination with circulating tumor DNA assays that detect AR mutants sensitive to darolutamide, in a precision oncology setting., Patient Summary: In this study we evaluated the novel drug darolutamide in preclinical models of prostate cancer. We found that darolutamide delays growth of enzalutamide-resistant prostate cancer, in particular in cells with mutated forms of the androgen receptor after previous treatment. Our data supports further evaluation of darolutamide in clinical trials., (Copyright © 2017. Published by Elsevier B.V.)

This study investigates whether Genomic Organization at Large Scales (which we propose to call GOALS) as quantified via nuclear phenotype characteristics and cell sociology features (describing cell organization within tissue) collected from prostate tissue microarrays (TMAs) can separate biochemical failure from biochemical nonevidence of disease (BNED) after radical prostatectomy (RP). Of the 78 prostate cancer tissue cores collected from patients treated with RP, 16 who developed biochemical relapse (failure group) and 16 who were BNED patients (nonfailure group) were included in the analyses (36 cores from 32 patients). A section from this TMA was stained stoichiometrically for DNA using the Feulgen-Thionin methodology, and scanned with a Pannoramic MIDI scanner. Approximately 110 nuclear phenotypic features, predominately quantifying large scale DNA organization (GOALS), were extracted from each segmented nuclei. In addition, the centers of these segmented nuclei defined a Voronoi tessellation and subsequent architectural analysis. Prostate TMA core classification as biochemical failure or BNED after RP using GOALS features was conducted (a) based on cell type and cell position within the epithelium (all cells, all epithelial cells, epithelial >2 cell layers away from basement membrane) from all cores, and (b) based on epithelial cells more than two cell layers from the basement membrane using a Classifier trained on Gleason 6, 8, 9 (16 cores) only and applied to a Test set consisting of the Gleason 7 cores (20 cores). Successful core classification as biochemical failure or BNED after RP by a linear classifier was 75% using all cells, 83% using all epithelial cells, and 86% using epithelial >2 layers. Overall success of predicted classification by the linear Classifier of (b) was 87.5% using the Training Set and 80% using the Test Set. Overall success of predicted progression using Gleason score alone was 75% for Gleason >7 as failures and 69% for Gleason >6 as failures. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

Purpose: The combination of docetaxel chemotherapy and androgen deprivation therapy (ADT) has become a standard treatment for patients with metastatic prostate cancer. The recently accrued phase III CALGB 90203 trial was designed to investigate the clinical effectiveness of this treatment approach earlier in the disease. Specimens from this trial offer a unique opportunity to interrogate the acute molecular response to docetaxel and ADT and identify potential biomarkers. Experimental Design: We evaluated baseline clinical data, needle biopsies, and radical prostatectomy (RP) specimens from 52 (of 788) patients enrolled on CALGB 90203 at one high volume center. Pathology review, tumor and germline-targeted DNA sequencing ( n = 72 genes), and expression profiling using NanoString platform ( n = 163 genes) were performed to explore changes in critical prostate cancer pathways linked to aggression and resistance. Results: Three of 52 patients had only microfocal residual cancer at prostatectomy. The most common alterations included TMPRSS2-ERG fusion ( n = 32), TP53 mutation or deletion ( n = 11), PTEN deletion ( n = 6), FOXA1 ( n = 6), and SPOP ( n = 4) mutation, with no significant enrichment in posttreated specimens. We did not observe AR amplification or mutations. The degree of AR signaling suppression varied among treated tumors and there was upregulation of both AR and AR-V7 expression as well as a subset of neuroendocrine and plasticity genes. Conclusions: These data support the feasibility of targeted and temporal genomic and transcriptome profiling of neoadjuvant-treated prostate cancer with limited formalin-fixed paraffin embedded tissue requirement. Characterization of the heterogeneity of treatment response and molecular outliers that arise posttreatment provides new insight into potential early markers of resistance. Clin Cancer Res; 23(22); 6802-11. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: To determine the relationship between parameters measured from luminal water imaging (LWI), a new magnetic resonance imaging (MRI) T 2 mapping technique, and the corresponding tissue composition in prostate., Materials and Methods: In all, 17 patients with prostate cancer were examined with a 3D multiecho spin echo sequence at 3T prior to undergoing radical prostatectomy. Maps of seven MR parameters, called N, T 2-short , T 2-long , A short , A long , geometric mean T 2 time (gmT 2 ), and luminal water fraction (LWF), were generated using nonnegative least squares (NNLS) analysis of the T 2 decay curves. MR parametric maps were correlated to digitized whole-mount histology sections. Percentage area of tissue components, including luminal space, nuclei, and cytoplasm plus stroma, was measured on the histology sections by using color-based image segmentation. Spearman's rank correlation test was used to evaluate the correlation between MR parameters and the corresponding tissue components, with particular attention paid to the correlation between LWF and percentage area of luminal space., Results: N, T 2-short , A long , gmT 2 , and LWF showed significant correlation (P < 0.05) with percentage area of luminal space and stroma plus cytoplasm. T 2-short and gmT 2 also showed significant correlation (P < 0.05) with percentage area of nuclei. Overall, the strongest correlation was observed between LWF and luminal space (Spearman's coefficient of rank correlation = 0.75, P < 0.001)., Conclusion: Results of this study show that LWF measured with MRI is strongly correlated with the fractional amount of luminal space in prostatic tissue. This result suggests that LWI can potentially be applied for evaluation of prostatic diseases in which the extent of luminal space differs between normal and abnormal tissues., Level of Evidence: 1 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;46:861-869., (© 2017 International Society for Magnetic Resonance in Medicine.)

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγ High /RXRα S427F/Y impairs CD8 + T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

Background: Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting., Objective: To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy., Design, Setting, and Participants: An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients., Intervention: An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC., Outcome Measurements and Statistical Analysis: Antineoplastic effects of targeting ofCS., Results and Limitations: In situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC 50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design., Conclusions: Targeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC., Patient Summary: Cisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin., (Copyright © 2017 European Association of Urology. All rights reserved.)

Objective: To better understand the effects of double J stenting on ureteral physiology and function., Materials and Methods: In total, 24 pigs were stented cystoscopically unilaterally for 48 hours, 1, 2, 4, and 7 weeks. Controls consisted of un-stented animals (n = 4) or the contralateral un-stented ureter in pigs. Ureters were harvested and tested in tissue baths to evaluate their contractility. Ureteral inflammation and expression of Sonic Hedgehog (Shh) and the transcriptional activator Gli1 (the downstream target of active Hedgehog signaling) were assessed histologically and by immunohistochemistry, respectively., Results: Indwelling ureteral stents were found to abolish normal ureteral function in all animals. Specifically, ureteral smooth muscle (SM) activity was significantly diminished within 48 hours after stenting and persisted at the 1-week time point. Furthermore, ureteral SM dysfunction was associated with increasing ureteral dilation due to the indwelling stent. Simultaneously, we observed a loss of Gli1 expression in SM cells, with a concomitant increase in ureteral inflammation. Expression of Shh was restricted to the urothelium and was not different between controls, stented, and contralateral ureters., Conclusion: Stent-induced aperistalsis was associated with diminished SM contractility, increased tissue inflammation, and reduced Gli1 expression in ureteral SM cells, independent of Shh expression. The present study is the first to show that indwelling stents negatively affect ureteral SM activity and identify a role for specific molecular mechanisms involved., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Purpose: Androgen deprivation therapy (ADT) commonly leads to incomplete cell death and the fate of persistent cells involves, in part, a senescent phenotype. Senescence is terminal growth arrest in response to cell stress that is characterized by increased lysosomal-β-galactosidase (GLB1) the origin of senescence associated-β-gal activity (SA-β-gal). In the current study senescence is examined in vivo after ADT use in a neoadjuvant trial., Methods and Materials: Tissue microarrays were generated from prostate cancer specimens (n = 126) from a multicenter neoadjuvant ADT trial. Arrays were subjected to multiplexed immunofluorescent staining for GLB1, Ki67, cleaved caspase 3 (CC3) and E-cadherin. Automated quantitative imaging was performed using Vectra™ and expression correlated with clinicopathologic features., Results: Tissue was analyzed from 59 patients treated with neoadjuvant ADT and 67 receiving no therapy preoperatively. Median follow-up was 85.3 mo and median ADT treatment was 5 mo. In PC treated with neoadjuvant ADT, GLB1 expression increased in intermediate Gleason score (GS 6-7; p = 0.001), but not high grade (GS 8-10) cancer. Significantly higher levels of GLB1 were seen in tissues undergoing neoadjuvant ADT longer than 5 months compared to untreated tissues (p = 0.002). In contrast, apoptosis significantly increased earlier (1-4 mo) after ADT treatment (p<0.5)., Conclusions: Increased GLB1 after neoadjuvant ADT occurs primarily among more clinically favorable intermediate grade cancers and enrichment of the phenotype occurs in a temporally prolonged fashion. Senescence may explain the persistence of PCa cells after ADT. Given concerns for the detrimental longer term presence of senescent cells, targeting these cells for removal may improve outcomes.

Background: LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO)., Methods: A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies., Results: The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts., Conclusions: These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor-resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo Mechanistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with classic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors., Significance: Understanding the contribution of the AR to the emergence of highly lethal, drug-resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discov; 7(1); 54-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1., (©2016 American Association for Cancer Research.)

Epigenetic factors play critical roles in prostate cancer (PCa) development. However, how they contribute to neuroendocrine differentiation (NED) and castration-resistant PCa (CRPC) is not fully understood. Using bioinformatics and biochemical approaches to analyze cell-based models of NED and CRPC, we found a cluster of epigenetic factors whose expression is downregulated during NED and upregulated in CRPC (i.e. follow a Down-Up pattern). Two histone demethylases within this cluster, PHF8 and KDM3A, are post-transcriptionally regulated by c-MYC through miR-22, which targets both PHF8 and KDM3A. We also found that the c-MYC/miR-22/PHF8 axis is downstream of androgen receptor (AR) signaling in CRPC cells. The co-expression of PHF8 with AR in clinical CRPC samples, normal mouse prostate, and adenocarcinomas of the prostate during PCa progression in a transgenic (TRAMP) mouse model supports the connection between PHF8 and AR. Knockdown of PHF8 impedes cell cycle progression in CRPC cells and has more profound effects on their growth than on the parental LNCaP cell line. Furthermore, PHF8 knockdown sensitizes LNCaP-Abl cells to the AR antagonist enzalutamide. Our data reveal novel mechanisms that underlie the regulation of PHF8 and KDM3A during NED and in CRPC, and support the candidacy of PHF8 as a therapeutic target in CRPC.

Background: The uncertainties inherent in clinical measures of prostate cancer (CaP) aggressiveness endorse the investigation of clinically validated tissue biomarkers. MUC1 expression has been previously reported to independently predict aggressive localized prostate cancer. We used a large cohort to validate whether MUC1 protein levels measured by immunohistochemistry (IHC) predict aggressive cancer, recurrence and survival outcomes after radical prostatectomy independent of clinical and pathological parameters., Material and Methods: MUC1 IHC was performed on a multi-institutional tissue microarray (TMA) resource including 1,326 men with a median follow-up of 5 years. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazard models and the Log-rank test., Results: The presence of MUC1 expression was significantly associated with extracapsular extension and higher Gleason score, but not with seminal vesicle invasion, age, positive surgical margins or pre-operative serum PSA levels. In univariable analyses, positive MUC1 staining was significantly associated with a worse recurrence free survival (RFS) (HR: 1.24, CI 1.03-1.49, P = 0.02), although not with disease specific survival (DSS, P>0.5). On multivariable analyses, the presence of positive surgical margins, extracapsular extension, seminal vesicle invasion, as well as higher pre-operative PSA and increasing Gleason score were independently associated with RFS, while MUC1 expression was not. Positive MUC1 expression was not independently associated with disease specific survival (DSS), but was weakly associated with overall survival (OS)., Conclusion: In our large, rigorously designed validation cohort, MUC1 protein expression was associated with adverse pathological features, although it was not an independent predictor of outcome after radical prostatectomy., Competing Interests: The authors have declared that no competing interests exist.

Castration-resistant prostate cancer (CRPC) is characterized by a shift in androgen receptor (AR) signaling from androgen-dependent to androgen (ligand)-independent. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens has been reported in androgen-dependent prostate cancer, its roles in regulating AR signaling transformation and CRPC progression remain unknown. In this study, we show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis, and CRPC progression. UGT2B17 expression and activity were higher in androgen-independent compared to androgen-dependent cell lines. UGT2B17 stimulated cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. Gene microarray analysis indicated that UGT2B17 suppressed androgen-dependent AR transcriptional activity and enhanced of ligand-independent transcriptional activity at genes associated with cell mitosis. These UGT2B17 actions were mainly mediated by activation of the c-Src kinase. In CRPC tumors, UGT2B17 expression was associated positively with c-Src activation. These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling to activate cell mitosis in cancer cells. Cancer Res; 76(22); 6701-11. ©2016 AACR., (©2016 American Association for Cancer Research.)

Histologic grading remains the gold standard for prognosis in prostate cancer, and assessment of Gleason score plays a critical role in active surveillance management. We sought to optimize the prognostic stratification of grading and developed a method of recording and studying individual architectural patterns by light microscopic evaluation that is independent of standard Gleason grade. Some of the evaluated patterns are not assessed by current Gleason grading (eg, reactive stromal response). Individual histologic patterns were correlated with recurrence-free survival in a retrospective postradical prostatectomy cohort of 1275 patients represented by the highest-grade foci of carcinoma in tissue microarrays. In univariable analysis, fibromucinous rupture with varied epithelial complexity had a significantly lower relative risk of recurrence-free survival in cases graded as 3+4=7. Cases having focal "poorly formed glands," which could be designated as pattern 3+4=7, had lower risk than cribriform patterns with either small cribriform glands or expansile cribriform growth. In separate multivariable Cox proportional hazard analyses of both Gleason score 3+3=6 and 3+4=7 carcinomas, reactive stromal patterns were associated with worse recurrence-free survival. Decision tree models demonstrate potential regrouping of architectural patterns into categories with similar risk. In summary, we argue that Gleason score assignment by current consensus guidelines are not entirely optimized for clinical use, including active surveillance. Our data suggest that focal poorly formed gland and cribriform patterns, currently classified as Gleason pattern 4, should be in separate prognostic groups, as the latter is associated with worse outcome. Patterns with extravasated mucin are likely overgraded in a subset of cases with more complex epithelial bridges, whereas stromogenic cancers have a worse outcome than conveyed by Gleason grade alone. These findings serve as a foundation to facilitate optimization of histologic grading and strongly support incorporating reactive stroma into routine assessment.

Background: Given the uncertainties inherent in clinical measures of prostate cancer aggressiveness, clinically validated tissue biomarkers are needed. We tested whether Alpha-2-Glycoprotein 1, Zinc-Binding (AZGP1) protein levels, measured by immunohistochemistry, and RNA expression, by RNA in situ hybridization (RISH), predict recurrence after radical prostatectomy independent of clinical and pathological parameters., Methods: AZGP1 IHC and RISH were performed on a large multi-institutional tissue microarray resource including 1,275 men with 5 year median follow-up. The relationship between IHC and RISH expression levels was assessed using the Kappa analysis. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazards models and the Log-rank test., Results: Absent or weak expression of AZGP1 protein was associated with worse recurrence free survival (RFS), disease specific survival, and overall survival after radical prostatectomy in univariable analysis. AZGP1 protein expression, along with pre-operative serum PSA levels, surgical margin status, seminal vesicle invasion, extracapsular extension, and Gleason score predicted RFS on multivariable analysis. Similarly, absent or low AZGP1 RNA expression by RISH predicted worse RFS after prostatectomy in univariable and multivariable analysis., Conclusions: In our large, rigorously designed validation cohort, loss of AZGP1 expression predicts RFS after radical prostatectomy independent of clinical and pathological variables. Prostate 76:1409-1419, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

A new paradigm for understanding immune-surveillance and immune escape in cancer is described here. Metastatic carcinomas express reduced levels of IL-33 and diminished levels of antigen processing machinery (APM), compared to syngeneic primary tumours. Complementation of IL-33 expression in metastatic tumours upregulates APM expression and functionality of major histocompatibility complex (MHC)-molecules, resulting in reduced tumour growth rates and a lower frequency of circulating tumour cells. Parallel studies in humans demonstrate that low tumour expression of IL-33 is an immune biomarker associated with recurrent prostate and kidney renal clear cell carcinomas. Thus, IL-33 has a significant role in cancer immune-surveillance against primary tumours, which is lost during the metastatic transition that actuates immune escape in cancer.

PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

Introduction: Urological dogma dictates that washings collected from the urinary tract for cytological assessment must be performed without interference from contrast agents that may alter cellular integrity and diagnostic interpretation. In practice, the initial contrast used to outline the upper tracts is commonly discarded with subsequent saline washings sent for cytology. We hypothesize that contrast washings do not affect the morphology of urothelial carcinoma cells or the integrity of cytology interpretation., Methods: Samples obtained from (1) human bladder cell lines; (2) urine from a human xenograft bladder cancer model using UC-3 cells; and (3) patients with urothelial carcinoma were subjected to various experimental solutions (water, saline, urine, and dilutions of contrast media) for different exposure times. After exposure to various different solutions, samples underwent cytological analysis to assess morphologic and degenerative changes., Results: No cytological differences were seen when cells were exposed to ionic, hyperosmolar, or non-ionic low-osmolar contrast agents for any exposures up to five minutes. Cells exposed to mixtures of contrast agents and urine also demonstrated no evidence of degenerative change. Cells exposed to water for greater than one minute demonstrated significant hydropic degeneration impacting cytological interpretation. At 40 minutes or later, all reagents caused severe degeneration when evaluating urine samples from the mouse bladder cancer model and from patients undergoing urothelial carcinoma., Conclusions: Commonly used contrast agents have no effect on urinary cytology up to five minutes. Contrast washings of the urinary tract should not be discarded and can be sent for cytological diagnosis if fixed within this time period.

In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ . Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ . rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody ( anti -AR). As the K D value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.

Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: Recent molecular analyses of bladder cancer open the door to significant advances in targeted therapies. NOTCH has been identified as a tumor suppressor in bladder cancer, but prior reports have focused on NOTCH1 Here we hypothesized that NOTCH2 is an oncogene suitable for therapeutic targeting in bladder cancer., Experimental Design: We studied genomic aberrations of NOTCH, compared survival and tumor progression according to NOTCH2 expression levels, and studied NOTCH2 function in vitro and vivo, Results: We report a high rate of NOTCH2 copy number gain in bladder cancer. High NOTCH2 expression was identified especially in the basal subtype and in mesenchymal tumors. NOTCH2 activation correlated with adverse disease parameters and worse prognosis by immunohistochemistry. Forced overexpression of the intracellular domain of NOTCH2 (N2ICD) induced cell growth and invasion by cell-cycle progression, maintenance of stemness and epithelial-to-mesenchymal transition (EMT). These effects were abrogated by silencing of CSL, indicating that the effects were mediated through the canonical NOTCH signaling pathway. In an orthotopic xenograft model, forced overexpression of N2ICD increased growth, invasion, and metastasis. To explore the potential for therapeutic targeting of NOTCH2, we first silenced the receptor with shRNA and subsequently treated with a specific inhibitory antibody. Both interventions decreased cell growth, invasion, and metastasis in vitro and in the orthotopic xenograft model., Conclusions: We have demonstrated that NOTCH2 acts as an oncogene that promotes bladder cancer growth and metastasis through EMT, cell-cycle progression, and maintenance of stemness. Inhibition of NOTCH2 is a rational novel treatment strategy for invasive bladder cancer. Clin Cancer Res; 22(12); 2981-92. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: The management of castration-resistant prostate cancer (CRPC) is a major challenge in the clinic. Androgen receptor signaling-directed strategies are not curative in CRPC therapy, and new strategies targeting alternative, key cancer properties are needed. Using reprogrammed glucose metabolism (aerobic glycolysis), cancer cells typically secrete excessive amounts of lactic acid into their microenvironment, promoting cancer development, survival, and progression. Cellular lactic acid secretion is thought to be predominantly mediated by MCT4, a plasma membrane transporter protein. As such, the MCT4 gene provides a unique, potential therapeutic target for cancer., Experimental Design: A tissue microarray of various Gleason grade human prostate cancers was stained for MCT4 protein. Specific, MCT4-targeting antisense oligonucleotides (MCT4 ASO) were designed and candidate MCT4 ASOs checked for effects on (i) MCT4 expression, lactic acid secretion/content, glucose consumption, glycolytic gene expression, and proliferation of human CRPC cells and (ii) growth of PC-3 tumors in nude mice., Results: Elevated MCT4 expression was associated with human CRPC and an earlier time to relapse. The treatment of PC-3, DU145, and C4-2 CRPC cultures with candidate MCT4 ASOs led to marked inhibition of MCT4 expression, lactic acid secretion, to increased intracellular lactic acid levels, and markedly reduced aerobic glycolysis and cell proliferation. Treatment of PC-3 tumor-bearing nude mice with the MCT4 ASOs markedly inhibited tumor growth without inducing major host toxicity., Conclusions: MCT4-targeting ASOs that inhibit lactic acid secretion may be useful for therapy of CRPC and other cancers, as they can interfere with reprogrammed energy metabolism of cancers, an emerging hallmark of cancer. Clin Cancer Res; 22(11); 2721-33. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: PTEN is the most commonly deleted tumor suppressor gene in primary prostate cancer (PCa) and its loss is associated with poor clinical outcomes and ERG gene rearrangement., Objective: We tested whether PTEN loss is associated with shorter recurrence-free survival (RFS) in surgically treated PCa patients with known ERG status., Design Setting and Participants: A genetically validated, automated PTEN immunohistochemistry (IHC) protocol was used for 1275 primary prostate tumors from the Canary Foundation retrospective PCa tissue microarray cohort to assess homogeneous (in all tumor tissue sampled) or heterogeneous (in a subset of tumor tissue sampled) PTEN loss. ERG status as determined by a genetically validated IHC assay was available for a subset of 938 tumors., Outcome Measurements and Statistical Analysis: Associations between PTEN and ERG status were assessed using Fisher's exact test. Kaplan-Meier and multivariate weighted Cox proportional models for RFS were constructed., Results and Limitations: When compared to intact PTEN, homogeneous (hazard ratio [HR] 1.66, p = 0.001) but not heterogeneous (HR 1.24, p = 0.14) PTEN loss was significantly associated with shorter RFS in multivariate models. Among ERG-positive tumors, homogeneous (HR 3.07, p < 0.0001) but not heterogeneous (HR 1.46, p = 0.10) PTEN loss was significantly associated with shorter RFS. Among ERG-negative tumors, PTEN did not reach significance for inclusion in the final multivariate models. The interaction term for PTEN and ERG status with respect to RFS did not reach statistical significance ( p = 0.11) for the current sample size., Conclusions: These data suggest that PTEN is a useful prognostic biomarker and that there is no statistically significant interaction between PTEN and ERG status for RFS., Patient Summary: We found that loss of the PTEN tumor suppressor gene in prostate tumors as assessed by tissue staining is correlated with shorter time to prostate cancer recurrence after radical prostatectomy.

Bipedalism in humans requires regionalization of myometrial function with a contracted lower uterine segment and a relaxed fundus during pregnancy to prevent fetal pressure on the cervix and reversal of this phenotype during labor. The HoxA13 gene is highly expressed in the lower uterine segment before term labor and regulates the regionalization of myometrium contractility. However, how HoxA13 regulates signal pathways to exert its functions remains unclear. Using a gene microarray technique, we profiled HoxA13 transcriptome in myometrial cells containing immune response genes (eg, IL-1β, IL-6, and IL-8) and contraction-associated proteins (CAPs) such as cyclooxygenase-2 (Cox-2) and connexin-43. IL-1β is responsible for mediating HoxA13 actions in up-regulating IL-6, IL-8, Cox-2, and connexin-43 expression. Blocking IL-1β with its inhibitor abolishes these HoxA13 actions. HoxA13-induced IL-1β stimulates the recruitment of activated THP-1 monocytes to myometrial cells, which in turn amplify the secretion of IL-1β, IL-6, and IL-8 through a mutual feed-forward loop between these cell types. As a result, Cox-2 expression is dramatically enhanced. These findings lead us to conclude that HoxA13 increases myometrial cell contractility by enhancing the secretion of IL-1β, resulting in an up-regulation of CAP and other proinflammatory cytokine expression. HoxA13-induced IL-1β in myometrial cells also prompts leukocyte recruitment and further amplifies CAP expression.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Purpose: Clinical evidence suggests increased cancer stem cells (CSCs) in a tumor mass may contribute to the failure of conventional therapies because CSCs seem to be more resistant than differentiated tumor cells. Thus, unveiling the mechanism regulating CSCs and candidate target molecules will provide new strategy to cure the patients., Experimental Design: The stem-like cell properties were determined by a prostasphere assay and dye exclusion assay. To find critical stem cell marker and reveal regulation mechanism, basic biochemical and molecular biologic methods, such as quantitative real-time PCR, Western blot, reporter gene assay, and chromatin immunoprecipitation assay, were used. In addition, to determine the effect of combination therapy targeting both CSCs and its progeny, in vitro MTT assay and in vivo xenograft model was used., Results: We demonstrate immortalized normal human prostate epithelial cells, appeared nontumorigenic in vivo, become tumorigenic, and acquire stem cell phenotype after knocking down a tumor suppressor gene. Also, those stem-like cells increase chemoresistance to conventional anticancer reagent. Mechanistically, we unveil that Wnt signaling is a key pathway regulating well-known stem cell marker CD44 by directly interacting to the promoter. Thus, by targeting CSCs using Wnt inhibitors synergistically enhances the efficacy of conventional drugs. Furthermore, the in vivo mouse model bearing xenografts showed a robust inhibition of tumor growth after combination therapy., Conclusions: Overall, this study provides strong evidence of CSC in castration-resistant prostate cancer. This new combination therapy strategy targeting CSC could significantly enhance therapeutic efficacy of current chemotherapy regimen only targeting non-CSC cells., (©2015 American Association for Cancer Research.)

The underlying mechanisms responsible for the development of castration-resistant prostate cancer (CRPC) in patients who have undergone androgen deprivation therapy are not fully understood. This is the first study to address whether β2-adrenergic receptor (ADRB2)- mediated signaling may affect CRPC progression in vivo. By immunohistochemical analyses, we observed that low levels of ADRB2 is associated with a more rapid development of CRPC in a Norwegian patient cohort. To elucidate mechanisms by which ADRB2 may affect CRPC development, we stably transfected LNCaP cells with shRNAs to mimic low and high expression of ADRB2. Two UDP-glucuronosyltransferases, UGT2B15 and UGT2B17, involved in phase II metabolism of androgens, were strongly downregulated in two LNCaP shADRB2 cell lines. The low-ADRB2 LNCaP cell lines displayed lowered glucuronidation activities towards androgens than high-ADRB2 cells. Furthermore, increased levels of testosterone and enhanced androgen responsiveness were observed in LNCaP cells expressing low level of ADRB2. Interestingly, these cells grew faster than high-ADRB2 LNCaP cells, and sustained their low glucuronidation activity in castrated NOD/SCID mice. ADRB2 immunohistochemical staining intensity correlated with UGT2B15 staining intensity in independent TMA studies and with UGT2B17 in one TMA study. Similar to ADRB2, we show that low levels of UGT2B15 are associated with a more rapid CRPC progression. We propose a novel mechanism by which ADRB2 may affect the development of CRPC through downregulation of UGT2B15 and UGT2B17.

The PP2A signaling axis regulates multiple oncogenic drivers of castration resistant prostate cancer (CRPC). We show that targeting the endogenous PP2A regulator, SET (I2PP2A), is a viable strategy to inhibit prostate cancers that are resistant to androgen deprivation therapy. Our data is corroborated by analysis of prostate cancer patient cohorts showing significant elevation of SET transcripts. Tissue microarray analysis reveals that elevated SET expression correlates with clinical cancer grading, duration of neoadjuvant hormone therapy (NHT) and time to biochemical recurrence. Using prostate regeneration assays, we show that in vivo SET overexpression is sufficient to induce hyperplasia and prostatic intraepithelial neoplasia. Knockdown of SET induced significant reductions in tumorgenesis both in murine and human xenograft models. To further validate SET as a therapeutic target, we conducted in vitro and in vivo treatments using OP449 - a recently characterized PP2A-activating drug (PAD). OP449 elicits robust anti-cancer effects inhibiting growth in a panel of enzalutamide resistant prostate cancer cell lines. Using the Pten conditional deletion mouse model of prostate cancer, OP449 potently inhibited PI3K-Akt signaling and impeded CRPC progression. Collectively, our data supports a critical role for the SET-PP2A signaling axis in CRPC progression and hormone resistant disease.

Purpose: Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model., Experimental Design: Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models., Results: LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO., Conclusions: LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics., (©2015 American Association for Cancer Research.)

Vascular endothelial growth factor (VEGF)-targeted antiangiogenic therapy significantly inhibits the growth of clear cell renal cell carcinoma (RCC). Eventually, therapy resistance develops in even the most responsive cases, but the mechanisms of resistance remain unclear. Herein, we developed two tumor models derived from an RCC cell line by conditioning the parental cells to two different stresses caused by VEGF-targeted therapy (sunitinib exposure and hypoxia) to investigate the mechanism of resistance to such therapy in RCC. Sunitinib-conditioned Caki-1 cells in vitro did not show resistance to sunitinib compared with parental cells, but when tested in vivo, these cells appeared to be highly resistant to sunitinib treatment. Hypoxia-conditioned Caki-1 cells are more resistant to hypoxia and have increased vascularity due to the upregulation of VEGF production; however, they did not develop sunitinib resistance either in vitro or in vivo. Human endothelial cells were more proliferative and showed increased tube formation in conditioned media from sunitinib-conditioned Caki-1 cells compared with parental cells. Gene expression profiling using RNA microarrays revealed that several genes related to tissue development and remodeling, including the development and migration of endothelial cells, were upregulated in sunitinib-conditioned Caki-1 cells compared with parental and hypoxia-conditioned cells. These findings suggest that evasive resistance to VEGF-targeted therapy is acquired by activation of VEGF-independent angiogenesis pathways induced through interactions with VEGF-targeted drugs, but not by hypoxia. These results emphasize that increased inhibition of tumor angiogenesis is required to delay the development of resistance to antiangiogenic therapy and maintain the therapeutic response in RCC., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC.

Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Systemic therapy for advanced bladder cancer has not changed substantially in more than 2 decades and mortality rates remain high. The recognition of HER2 over expression in bladder cancer has made HER2 a promising therapeutic target. T-DM1, a new drug consisting of the HER2 antibody trastuzumab conjugated with a cytotoxic agent, has been shown in breast cancer to be superior to trastuzumab. We tested T-DM1 in preclinical models of bladder cancer., Materials and Methods: We evaluated the effect of T-DM1 compared to trastuzumab in different in vitro and in vivo models of HER2 over expressing bladder cancer., Results: RT4V6 was the highest HER2 expressing bladder cancer cell line and it showed higher growth inhibition with T-DM1 compared to trastuzumab. T-DM1 but not trastuzumab induced apoptosis of RT4V6 cells after G2/M arrest on cell cycle analysis. HER2 expression was higher in cell lines with acquired cisplatin resistance compared to the corresponding parental cell lines. Resistant cells showed higher sensitivity to T-DM1 by the induction of apoptosis. In addition, cells cultured in anchorage independent conditions increased HER2 expression compared to cells cultured in adherent conditions and T-DM1 significantly inhibited colony formation in soft agar compared to trastuzumab. In an orthotopic bladder cancer xenograft model tumor growth of cisplatin resistant RT112 was significantly inhibited by T-DM1 via the induction of apoptosis compared to treatment with control IgG or trastuzumab., Conclusions: T-DM1 has promising antitumor effects in preclinical models of HER2 over expressing bladder cancer., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.

Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)