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The supplementary figures file contains additional data that are not provided in the main text (because of space constraints). Fig. S1, Data on the miR-520f expression models, incl. phase-contrast images; Fig. S2, Schematic representation of SOEing PCR; Fig. S3, Reporter and cell line models to study EMT-reversal; Fig. S4, Membranous E-cadherin staining in PANC-1 cells; Fig. S5, Overview of the 3'-UTR reporter vectors; Fig. S6, Additional data on the in vivo (mouse) experiments.

The supplementary materials and methods file contains a more detailed description of methods (cell culture, real-time and stem loop RT-PCR, Western blot analysis, and 3'-UTR reporter assays) that are described in the main text in brief. Methods that have not been described in the main text, because the data are not shown or only provided in the supplementary figures (Sanger DNA sequence analysis, phase-contrast imaging, and immunocytochemisty) are also described here.

The supplementary table file contains a table with the names and origin of the cell lines used in this study (Suppl. Table S1). It also contains four tables with nucleotide sequences of primers and oligonucleotides used for qPCR and DNA sequence analysis, and for vector cloning (Suppl. Tables S2, S3, S5 and S6), and a table describing the origin of the miRNA mimics and siRNAs used in this study (Suppl. Table S4).

The use of anticancer drugs targeting specific molecular tumor characteristics is rapidly increasing in clinical practice, but selecting patients to benefit from these remains a challenge. It has been suggested that organoid cultures would be ideally suited to test drug responses in vitro. Here we describe and characterize in depth a case of ETV6-NTRK3 gene fusion-positive secretory carcinoma of the salivary glands and corresponding organoid cultures that responded and subsequently acquired resistance to TRK targeting therapy with larotrectinib. This case-culture-characterization illustrates the advances made in precision oncology, but also exposes important caveats in using organoids to predict treatment response.

BACKGROUND: Various germline genetic variants are associated with the prognosis of non-muscle invasive bladder cancer (NMIBC). Germline variants in genes frequently somatically mutated in bladder cancer have not been studied thoroughly in relation to risk of recurrence or progression in NMIBC. OBJECTIVE: To identify germline DNA variants in bladder carcinogenesis-related genes associated with recurrence or progression in NMIBC. METHODS: We analysed associations between single-nucleotide polymorphisms (SNPs) and NMIBC recurrence and progression using data from the Nijmegen Bladder Cancer Study (NBCS, 1,443 patients). We included 5,053 SNPs within 46 genes known to have mutation, overexpression or amplification in bladder cancer. We included all recurrences in the statistical analysis and performed both single variant analysis and gene-based analysis. SNPs and genes that showed significant or suggestive association (false discovery rate P value

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled

Introduction: Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations, such as NTRK gene fusions. This research aimed to identify these actionable aberrations by combining NGS-based analysis of RNA (gene fusions) and DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. Methods: RNA and DNA were extracted from archival tissue of 121 patients with various SGC subtypes. Gene fusion analysis was performed using a customized RNA-based targeted NGS panel. DNA was sequenced using a targeted NGS panel encompassing 523 cancer-related genes. Cross-validation of NGS-based NTRK fusion detection and pan-TRK immunohistochemistry (IHC) was performed. Results: Fusion transcripts were detected in 50% of the cases and included both known (MYB-NFIB, MYBL1-NFIB, CRTC1-MAML2) and previously unknown fusions (including transcripts involving RET, BRAF or RAD51B). Only one NTRK fusion transcript was detected, in a secretory carcinoma case. Pan-TRK IHC (clone EPR17341) was false positive in 74% of cases. The proportion of patients with targets for genetically matched therapies differed among subtypes (salivary duct carcinoma: 82%, adenoid cystic carcinoma 28%, mucoepidermoid carcinoma 50%, acinic cell carcinoma 33%). Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Conclusions: Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR‐positive UCC‐derived cell line model, UM‐UC‐3‐AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell‐like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen‐stimulated UM‐UC‐3‐AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer., Hormonal factors have been implicated in the aetiology of urothelial cell carcinoma (UCC). Here, we explored the role of androgen receptor (AR) signalling in UCC progression. We detected AR expression in a subset of patients with UCC. Androgen treatment increased clonogenicity and migration of a UCC‐derived cell line, while AR blockade reverted these effects. Finally, androgens regulated the expression of nearly 300 genes, some of which correlated with AR expression in UCC tumour tissues. ​

Purpose Prostate cancer (PCa) is a leading cause of cancer-related death. Upon androgen-deprivation therapy, the disease may progress further to castration-resistant PCa (CRPC) with a poor prognosis. MicroRNAs (miRNAs) are small non-coding RNAs, which play crucial roles in gene regulation. The aim of our study is to find CRPC-associated miRNAs and to evaluate their functional role. Methods In this study, 23 benign prostatic hyperplasia (BPH), 76 primary PCa, and 35 CRPC specimens were included. Total RNA extracted from tissue sections was used for miRNA profiling on the Affymetrix GSC 3000 platform. Subsequently, stem-loop RT-qPCR analysis was performed to validate the expression levels of selected miRNAs. PCa cell lines were transfected with miRNA mimics or inhibitors to evaluate the effects on cell proliferation, cell migration and cell invasion. Results In our profiling study, several miRNAs were found to be deregulated in CRPC compared to primary PCa tissue, of which miR-205 (− 4.5-fold; p = 0.0009), miR-92b (− 3.1 fold; p p p = 0.0006) and miR-4417 (5.0-fold; p = 0.0005) were most upregulated. While KLK3, miR-21 and miR-141 expression levels in androgen-treated VCaP and LNCaP cells were increased, the expression levels of miR-3687 and miR-4417 were reduced. None of the miRNAs were androgen-regulated in the AR-negative PC3 cell line. Overexpression of miR-3687 reduced cell migration and cell invasion, whilst miR-3195 enhanced cell migration. Conclusion We have identified several novel deregulated miRNAs in CRPC tissue, including two microRNAs that are potentially involved in tumor invasion. Our data support the hypothesized involvement of miRNAs in PCa tumorigenesis and progression to CRPC. The applicability of these miRNAs as novel biomarkers for CRPC remains to be further investigated.

Rationale: Treatment options for recurrent and/or metastatic (R/M) adenoid cystic carcinoma (ACC) and salivary duct carcinoma (SDC), major subtypes of salivary gland cancer, are limited. Both tumors often show overexpression of prostate-specific membrane antigen (PSMA). In prostate cancer, PSMA-ligands labeled with 68Ga or 177Lu are used for imaging and therapy, respectively. Primary aim of this study in R/M ACC and SDC patients was to systematically investigate 68Ga-PSMA-uptake by PET/CT imaging to determine if PSMA radionuclide therapy could be a treatment option. Methods: In a prospective phase II study, PET/CT imaging was performed 1 h post injection of 68Ga-PSMA-HBED-CC in 15 ACC patients and 10 SDC patients. Maximum standardized uptake values (SUV) were determined in tumor lesions. Immunohistochemical PSMA expression was scored in primary tumors and metastatic tissue. Standard imaging (MRI or CT) was performed for comparison. Results: In ACC patients, SUVmax ranged from 1.1 to 30.2 with a tumor/liver-ratio >1 in 13 out of 14 evaluable patients (93%). In SDC patients, SUVmax ranged from 0.3 to 25.9 with a tumor/liver-ratio >1 in 4 out of 10 patients (40%). We found a large intra-patient inter-metastatic variation in uptake of 68Ga-PSMA, and immunohistochemistry did not predict ligand uptake in ACC and SDC. Finally, PSMA-PET detected additional bone metastases compared to CT in 2 ACC patients with unexplained pain. Conclusion: In 93% of ACC patients and 40% of SDC patients we detected relevant PSMA-ligand uptake, which warrants to study PSMA radionuclide therapy in these patients. Additionally, our data provide arguments for patient selection and treatment timing. Finally, PSMA-PET imaging has added diagnostic value compared to CT in selected patients.

Contains fulltext : 248260.pdf (Publisher’s version ) (Open Access) BACKGROUND: Cell-penetrating peptides (CPPs) are a promising approach for delivering antisense oligonucleotides (AONs) as they form nanosized complexes through noncovalent interactions that show efficient cellular uptake. Previously, we have designed an AON system to correct splicing of the androgen receptor (AR) pre-mRNA, thereby preventing the generation of the splice variant AR-V7 mRNA. AON-mediated knockdown of AR-V7 resulted in inhibition of androgen-independent cell proliferation. In this study, we evaluated the CPP-mediated delivery of this AON into castration-resistant prostate cancer cell line models 22Rv1, DuCaP (dura mater cancer of the prostate), and VCaP (vertebral cancer of the prostate). METHODS: Nanoparticles (polyplexes) of AONs and CPPs were formed through rapid mixing. The impact of the peptide carrier, the formulation parameters, and cell incubation conditions on cellular uptake of fluorescently labeled AONs were assessed through flow cytometry. The cytotoxic activity of these formulations was measured using the CellTiter-Glo cell viability assay. The effectivity of CPP-mediated delivery of the splice-correcting AON-intronic splicing enhancer (ISE) targeting the ISE in the castration-resistant prostate cancer (CRPC)-derived 22Rv1, DuCaP, and VCaP cells was determined by measuring levels of AR-V7 mRNA normalized to those of the human heterochromatin protein 1 binding protein 3 (HP1BP3). Western blot analysis was used to confirm AR-V7 downregulation at a protein level. The cellular distribution of fluorescently labeled AON delivered by a CPP or a transfection reagent was determined through confocal laser scanning microscopy. RESULTS: The amphipathic and stearylated CPP PepFect 14 (PF14) showed higher uptake efficiency than arginine-rich CPPs. Through adjustment of formulation parameters, concentration and incubation time, an optimal balance between carrier-associated toxicity and delivery efficiency was found with a formulation consisting of an amino/phosphate ratio of 3, 0.35 μM AON concentration and 30 min incubation time of the cells with polyplexes. Cellular delivery of AON-ISE directed against AR pre-mRNA achieved significant downregulation of AR-V7 by 50%, 37%, and 59% for 22Rv1, DuCaP, and VCaP cells, respectively, and reduced androgen-independent cell proliferation of DuCaP and VCaP cells. CONCLUSIONS: This proof-of-principle study constitutes the basis for further development of CPP-mediated delivery of AONs for targeted therapy in prostate cancer.

Background: Non-muscle-invasive bladder cancer (NMIBC) is characterized by frequent recurrences and a risk of progression in stage and grade. Increased knowledge of underlying biological mechanisms is needed.Objective: To identify single nucleotide polymorphisms (SNPs) associated with recurrence-free (RFS) and progression-free (PFS) survival in NMIBC.Design, setting, and participants: We analyzed outcome data from 3400 newly diagnosed NMIBC patients from the Netherlands, the UK, Canada, and Spain. We generated genome-wide germline SNP data using Illumina OmniExpress and Infinium Global Screening Array in combination with genotype imputation.Outcome measurements and statistical analysis: Cohort-specific genome-wide association studies (GWASs) for RFS and PFS were performed using a Cox proportional hazard model. Results were combined in a fixed-effect inverse-variance weighted meta-analysis. Candidate genes for the identified SNP associations were prioritized using functional annotation, gene-based analysis, expression quantitative trait locus analysis, and transcription factor binding site databases. Tumor expression levels of prioritized genes were tested for association with RFS and PFS in an independent NMIBC cohort.Results and limitations: This meta-analysis revealed a genome-wide significant locus for RFS on chromosome 14 (lead SNP rs12885353, hazard ratio [HR] C vs T allele 1.55, 95% confidence interval [CI] 1.33-1.82, p = 4.0 x 10(-8)), containing genes G2E3 and SCFD1. Higher expression of SCFD1 was associated with increased RFS (HR 0.70, 95% CI 0.59-0.84, p(FDR) = 0.003). Twelve other loci were suggestively associated with RFS (p < 10(-5)), pointing toward 18 additional candidate genes. For PFS, ten loci showed suggestive evidence of association, indicating 36 candidate genes. Expression levels of ten of these genes were statistically significantly associated with PFS, of which four (IFT140, UBE2I, FAHD1, and NME3) showed directional consistency with our meta-analysis results and published literature.Conclusions: In this first prognostic GWAS in NMIBC, we identified several novel candidate loci and five genes that showed convincing associations with recurrence or progression.Patient summary: In this study, we searched for inherited DNA changes that affect the outcome of non-muscle-invasive bladder cancer (NMIBC). We identified several genes that are associated with disease recurrence and progression. The roles and mechanisms of these genes in NMIBC prognosis should be investigated in future studies. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology.

Contains fulltext : 287575.pdf (Publisher’s version ) (Open Access) Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Contains fulltext : 287577.pdf (Publisher’s version ) (Open Access) OBJECTIVE: Three-dimensional organoid cell cultures have been established for a variety of human cancers. For most rare cancers, including salivary gland cancer (SGC), these models are lacking, despite the great unmet need to study cancer biology in these diseases. Therefore, we aimed to develop patient-derived organoid (PDO) models for different subtypes of SGC. METHODS: Tumor samples of SGC patients were processed and embedded in Matrigel. Successful PDOs (expandable > 1*10(6) cells) were phenotypically characterized using immunohistochemistry (IHC) and genotypically by gene fusion analysis and by targeted and whole-exome sequencing. Successfully established PDOs were subjected to small-scale drug screening. RESULTS: Out of 37 attempts, 7 viable short-term PDOs were established (19 % success rate; 3 salivary duct carcinoma, 3 adenoid cystic carcinoma and 1 mucoepidermoid carcinoma). Each PDO showed close phenotypical mimicry to parental tissue. Genotypic characterization revealed that in each PDO > 97.6 % of all COSMIC annotated variants and all MYB, MYBL1 and NFIB gene rearrangements were retained. Drug screening was proven feasible in all PDOs. CONCLUSION: We present the first comprehensively characterized short-term SGC PDO models for three subtypes of SGC with close phenotypic and genotypic resemblance to parental tissue, which can be used for drug screening applications.

Circulating RNAs extracted from liquid biopsies represent a promising source of cancer‐ and therapy‐related biomarkers. We screened whole blood from patients with metastatic castration‐resistant prostate cancer (mCRPC) following their first‐line treatment with abiraterone acetate and prednisone (AA‐P) to identify circulating RNAs that may correlate with progression‐free survival (PFS). In a prospective multicenter observational study, 53 patients with mCRPC were included after they started first‐line AA‐P treatment. Blood was drawn at baseline, 1, 3, and 6 months after treatment initiation. The levels of predefined circulating RNAs earlier identified as being upregulated in patients with mCRPC (e.g., microRNAs, long noncoding RNAs, and mRNAs), were analyzed. Uni‐ and multivariable Cox regression and Kaplan–Meier analyses were used to analyze the prognostic value of the various circulating RNAs for PFS along treatment. Detectable levels of kallikrein‐related peptidase 3 (KLK3) mRNA at baseline were demonstrated to be an independent prognostic marker for PFS (201 vs 501 days, P = 0.00054). Three months after AA‐P treatment initiation, KLK3 could not be detected in the blood of responding patients, but was still detectable in 56% of the patients with early progression. Our study confirmed that KLK3 mRNA detection in whole blood is an independent prognostic marker in mCRPC patients receiving AA‐P treatment. Furthermore, the levels of circulating KLK3 mRNA in patients receiving AA‐P treatment might reflect treatment response or early signs of progression., Circulating RNAs extracted from liquid biopsies represent a promising source of cancer‐ and therapy‐related biomarkers. KLK3 mRNA detection in whole blood is an independent prognostic marker for progression‐free survival in mCRPC patients. KLK3 mRNA levels probably correspond to the number of circulating tumor cells. Therefore, KLK3 detection may be a potential biomarker to monitor treatment response but prospective clinical validation is needed.

Contains fulltext : 237678.pdf (Publisher’s version ) (Open Access) Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Item does not contain fulltext PURPOSE: Laparoscopic adrenalectomy is standard treatment for patients with unilateral aldosterone-producing adenomas, but surgeons are increasingly tempted to perform partial adrenalectomy, disregarding potential multinodularity of the adrenal. We assess the diagnostic value of endoscopic ultrasound for differentiating solitary adenomas from multinodularity by examining in-depth adrenal pathology with ex vivo 11.7 T magnetic resonance imaging and immunohistochemistry. MATERIALS AND METHODS: In 15 primary aldosteronism patients, we performed intraoperative endoscopic ultrasound, ex vivo magnetic resonance imaging and histopathological examination. Every adrenal was intraoperatively and postoperatively assessed for solitary adenomas or multinodular hyperplasia. After unblinding for ex vivo magnetic resonance imaging results a second detailed histopathological examination, including immunohistochemistry analysis with CYP11B2 (aldosterone synthase) and chemokine receptor 4 (CXCR4), a new marker for aldosterone-producing adenomas, was performed. Finally, presence of somatic mutations linked to aldosterone-producing adenomas was assessed. RESULTS: The sensitivity and specificity of endoscopic ultrasound to identify multinodularity were 46% and 50%, respectively. We found multinodular hyperplasia in 87% of adrenals with ex vivo magnetic resonance imaging combined with detailed histopathology, and 6 adrenals contained multiple CYP11B2-producing nodules. Every CYP11B2 positive nodule and 61% of CYP11B2 negative nodules showed CXCR4 staining. Finally, in 4 adrenals (27%) we found somatic mutations. In multinodular glands, only 1 nodule harbored this mutation. CONCLUSIONS: Intraoperative endoscopic ultrasound in primary aldosteronism patients has low accuracy to identify multinodularity. Ex vivo magnetic resonance imaging can serve as a tool to direct detailed histopathological examination, which frequently shows CYP11B2 production in multiple nodules. Therefore, partial adrenalectomy is inappropriate in primary aldosteronism as multiple aldosterone-producing nodules easily stay behind.

Simple Summary Androgen receptor signaling seems pivotal in aggressive salivary duct carcinoma. However, androgen deprivation therapy (ADT) frequently fails, emphasizing the need for biomarkers to predict treatment response. Here, the activities of several possible tumor-driving pathways were quantified and related to clinical outcome in a large cohort of SDC patients. Our results indicated that AR pathway activity and SRD5A1 expression levels can be used to predict response to ADT, which could prevent overtreatment in clinical practice. Abstract Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

Objectives: Salivary duct carcinoma (SDC) is a rare and aggressive subtype of salivary gland cancer. Androgen receptor (AR) (96%) and HER2 (29-46%) expression, and a high propensity for regional lymph node metastases are hallmarks of the disease. We hypothesized that c-MET, E-cadherin, PSMA tumor and PSMA neovascular expression may be prognostic factors in SDC.Materials and methods: Expression levels of these proteins were established on tissue microarrays containing 165 primary SDC tumor specimens. Association with survival was studied with Kaplan-Meier curves, and univariable and multivariable Cox regression models. Furthermore, association with lymph node status, AR and HER2 expression, and gender was studied.Results: We found that patients with high PSMA tumor expression showed a significantly longer overall survival (OS) (median 83 vs. 43 months, P = 0.022), a trend towards a longer DFS (median 51 vs. 22 months, P = 0.094), and significantly reduced hazard ratio for death in the univariable Cox regression model (HR 0.46, P = 0.034). In the multivariable model only a high number of tumor-positive lymph nodes and high age (> 80) at diagnosis were prognostic for poor OS. High PSMA tumor expression was also significantly associated with low N-stage (P = 0.001) and expression was higher in women versus men (P = 0.029). High PSMA tumor expression and Ecadherin loss were significantly associated with strong and weak AR-expression, respectively (P = 0.033 and P = 0.007). None of the factors were significantly associated with HER2 expression.Conclusion: c-MET, E-cadherin, and tumor and neovascular PSMA expression are no independent prognostic factors in SDC.

Androgen deprivation therapy (ADT) is first‐line palliative treatment in androgen receptor‐positive (AR+) salivary duct carcinoma (SDC), and response rates are 17.6–50.0%. We investigated potential primary ADT resistance mechanisms for their predictive value of clinical benefit from ADT in a cohort of recurrent/metastatic SDC patients receiving palliative ADT (n = 30). We examined mRNA expression of androgen receptor (AR), AR splice variant‐7, intratumoral androgen synthesis enzyme‐encoding genes AKR1C3, CYP17A1, SRD5A1 and SRD5A2, AR protein expression, ERBB2 (HER2) gene amplification and DNA mutations in driver genes. Furthermore, functional AR pathway activity was determined using a previously reported Bayesian model which infers pathway activity from AR target gene expression levels. SRD5A1 expression levels and AR pathway activity scores were significantly higher in patients with clinical benefit from ADT compared to those without benefit. Survival analysis showed a trend toward a longer median progression‐free survival for patients with high SRD5A1 expression levels and high AR pathway activity scores. The AR pathway activity analysis, and not SRD5A1 expression, also showed a trend toward better disease‐free survival in an independent cohort of locally advanced SDC patients receiving adjuvant ADT (n = 14) after surgical tumor resection, and in most cases a neck dissection (13/14 patients) and postoperative radiotherapy (13/14 patients). In conclusion, we are the first to describe that AR pathway activity may predict clinical benefit from ADT in SDC patients, but validation in a prospective study is needed., What's new? Androgen deprivation therapy (ADT) is a leading treatment strategy in the palliative care of patients with androgen receptor (AR)‐positive salivary duct carcinoma (SDC). However, while as many as half of patients may respond to ADT, resistance frequently emerges, undermining its use. In this investigation of primary ADT resistance mechanisms, expression of the androgen synthesis enzyme‐encoding gene SRD5A1 and functional activity of the AR pathway were found to predict clinical benefit from ADT in SDC patients. High AR pathway activity scores were further linked to improved disease‐free survival in SDC patients with locally advanced disease who received adjuvant ADT.

Contains fulltext : 225075.pdf (Publisher’s version ) (Open Access) BACKGROUND: Salivary duct carcinoma (SDC) is an aggressive subtype of salivary gland cancer. Approximately half of SDC patients will develop recurrences or metastases. Therapeutic palliative therapy is therefore often needed. The majority of SDC tumors expresses the androgen receptor (AR) and one-third expresses human epidermal growth factor receptor 2 (HER2), both are potential therapeutic targets. The aim of this paper is to systematically review and summarize the evidence on systemic palliative therapy for SDC and to provide treatment recommendations. MATERIALS AND METHODS: Electronic libraries were systematically searched with a broad search strategy to identify studies where SDC patients received systemic therapy. Due to the rarity of SDC no restrictions were placed on study designs. RESULTS: The search resulted in 2014 articles of which 153 were full-text analyzed. Forty-five studies were included in the analysis, which included in total 256 SDC patients receiving systemic therapy. Two phase 2 trials primarily including SDC patients were identified. The majority of the studies were case series or case reports, resulting in an overall low quality of available evidence. Based on studies including ≥ 5 SDC patients, objective responses to HER2 targeting agents were observed in 60-70%, to AR pathway agents in 18-53% and to chemotherapy in 10-50%. CONCLUSION: For AR or HER2 positive SDC, agents targeting these pathways are the cornerstone for palliative treatment. Regarding chemotherapy, the combination of carboplatin combined with a taxane is best studied. Regarding other targeted agents and immunotherapy evidence is anecdotal, limiting formulation of treatment recommendations for these antineoplastic agents.

Contains fulltext : 218088.pdf (Publisher’s version ) (Open Access) Introduction: Prostate cancer (PCa) is one of the most common malignancies in men and a major cause of cancer deaths among men worldwide. Prostate specific antigen (PSA) monitoring and histopathological examination of tumor biopsies remain gold standards in PCa diagnostics. These clinical parameters are not well suited for patient stratification, predicting and monitoring treatment response. On the other hand, liquid biopsies offer a unique opportunity to easily isolate tumor-derived material for longitudinal clinical assessment.Areas covered: In this review we focus on the clinical application of novel liquid biomarkers that have the potential to monitor and stratify patients in order to achieve better therapeutic effects and improve clinical outcomes. Enumeration and characterization of circulating tumor cells (CTCs), tumor-educated platelets, exosomes, and cell-free nucleic acids have been studied for their clinical utility in PCa diagnostics, prognostics, monitoring treatment response and guiding treatment choice.Expert opinion: Liquid biomarkers have high potential to be used for prognosis, monitoring treatment response and guiding treatment selection. Although there is a remarkable progress in PCa biomarker discovery, their clinical validation is very limited. Research should be focused on biomarker validation and the incorporation of these biomarkers in clinical practice.

Background Several treatment options were recently added for metastatic castration-resistant prostate cancer (mCRPC). However, response to therapy is variable, and biomarkers that can guide treatment selection and response evaluation are lacking. Circulating RNAs are a promising source of biomarkers. We explored messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) as potential biomarkers in liquid biopsies of patients with mCRPC treated with enzalutamide. Methods Forty patients were included in this prospective multicenter observational study. Whole blood was drawn at baseline and 1, 3, and 6 months after start of therapy. Four mRNAs, 6 miRNAs, and 5 lncRNAs were analyzed by quantitative PCR. RNA levels in 30 healthy individuals were used as controls. RNA expression data were analyzed by Kaplan–Meier and Cox regression analyses, and the primary end point was progression-free survival. Clinical factors were included in the multivariable Cox regression analysis. Results Levels of 2 miRNAs, miR-375 and miR-3687, and 1 lncRNA, N-acetylated alpha-linked acidic dipeptidase like 2 antisense RNA 2 (NAALADL2-AS2), were more than 2-fold higher in patients with mCRPC compared with healthy volunteers. Patients with higher levels of miR-375 or miR-3687 showed a shorter time to progression. Patients with higher levels of NAALADL2-AS2 showed a longer time to progression. In the multivariable Cox regression analysis, higher miR-375, miR-3687 and serum prostate-specific antigen concentrations were shown to be independent predictors for shorter time to progression. Conclusions We identified miR-3687 as a novel prognostic marker for response in patients with CRPC treated with enzalutamide, and we confirmed the prognostic value of miR-375.

Contains fulltext : 174182.pdf (Publisher’s version ) (Closed access) Reversing epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, but a limited number of broadly comprehensive investigations of miRNAs involved in this process have been conducted. In this study, we screened a library of 1120 miRNA for their ability to transcriptionally activate the E-cadherin gene CDH1 in a promoter reporter assay as a measure of EMT reversal. By this approach, we defined miR-520f as a novel EMT-reversing miRNA. miR-520f expression was sufficient to restore endogenous levels of E-cadherin in cancer cell lines exhibiting strong or intermediate mesenchymal phenotypes. In parallel, miR-520f inhibited invasive behavior in multiple cancer cell systems and reduced metastasis in an experimental mouse model of lung metastasis. Mechanistically, miR-520f inhibited tumor cell invasion by directly targeting ADAM9, the TGFbeta receptor TGFBR2 and the EMT inducers ZEB1, ZEB2, and the snail transcriptional repressor SNAI2, each crucial factors in mediating EMT. Collectively, our results show that miR-520f exerts anti-invasive and antimetastatic effects in vitro and in vivo, warranting further study in clinical settings. Cancer Res; 77(8); 2008-17. (c)2017 AACR.

Aim Salivary duct carcinoma (SDC), an aggressive subtype of salivary gland cancer, is androgen receptor (AR)–positive in 67–96% of cases. In patients with locally recurrent and metastatic (R/M) AR-positive SDC, androgen deprivation therapy (ADT) has an overall response rate of 18–64.7%. In this study, we describe the efficacy of adjuvant ADT in patients with poor-risk (stage 4a) AR-positive SDC. Methods This is a retrospective cohort study in which patients with stage 4a AR-positive SDC were offered adjuvant ADT, i.e. bicalutamide, luteinizing hormone-releasing hormone (LHRH) analogue or a combination of these after tumour resection. In the control group, data were collected on patients with stage 4a SDC who underwent a tumour resection but did not receive adjuvant ADT. Results Twenty-two AR-positive SDC patients were treated with adjuvant ADT for a median duration of 12 months. The control group consisted of 111 SDC patients. After a median follow-up of 20 months in the ADT-treated patients and 26 months in the control group, the 3-year disease-free survival (DFS) was estimated as 48.2% (95% confidence interval [CI] 14.0–82.4%) and 27.7% (95% CI 18.5–36.9%) (P = 0.037). Multivariable Cox regression analysis showed a hazard ratio of 0.138 (95% CI 0.025–0.751, P = 0.022) for DFS and 0.064 (95% CI 0.005–0.764, P = 0.030) for overall survival (OS) in favour of the ADT-treated patients. Conclusion Poor-risk, AR-positive SDC patients who received adjuvant ADT have a significantly longer DFS compared with patients in the control group, who did not receive adjuvant ADT. For OS, this was just below and above the significance level, in case there was or was no correction for confounders.

Contains fulltext : 204684.pdf (Publisher’s version ) (Closed access) Accumulating evidence has shown that intracrinology in prostate cancer (PCa) has a pivotal role in survival of cancer cell. PCa cells are able to produce androgens from different androgen precursors, such as dehydroepiandrosterone, thereby maintaining androgen receptor signaling. Several drugs have been developed that target intracrinology, some of which are now being used as standard treatment for the so-called castrate-resistant prostate cancer (CRPC) patients. Recently, the US FDA approval has changed the indication of drugs targeting intracrinology, e.g., abiraterone and enzalutamide where it evolved from post-chemotherapy CRPC to hormone-naive metastatic PCa cases. This approval raises question whether those drugs can also be used as the first-line treatment in localized stage PCa cases. In addition, development of additional drugs targeting major components of intracrinology is ongoing. Application of these new drugs and administration of combinations of existing drugs will ultimately lead to an increase in the efficacy of such treatments as well as to reduce the toxicity of the therapy and to prevent the risk of resistance.

One of the mechanisms by which advanced prostate cancer develops resistance to androgen deprivation therapy is the elevated expression of C-terminally truncated androgen receptor (AR) variants. These variants, such as AR-V7, originate from aberrant splicing of the AR pre-mRNA and the inclusion of a cryptic exon containing a premature stop codon in the mRNA. The resulting loss of the ligand-binding domain allows AR-V7 to act as a constitutively active transcription factor. Here, we designed two antisense oligonucleotides (AONs) directed against cryptic splicing signals within the AR pre-mRNA. These two AONs, AON-ISE and AON-ESE, demonstrated high efficiency in silencing AR-V7 splicing without affecting full-length AR expression. The subsequent downregulation of AR-V7-target gene UBE2C was accompanied by inhibition of androgen-independent cell proliferation and induction of apoptosis in castration-resistant prostate cancer (CRPC)-derived cell line models 22Rv1, DuCaP, and VCaP. Our results show that splicing-directed AONs can efficiently prevent expression of AR-V7, providing an attractive new therapeutic option for the treatment of CRPC.

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.

Purpose Genome-wide association studies (GWAS) have led to the identification of a bladder cancer susceptibility variant (rs710521) in a non-coding intergenic region between the TP63 and LEPREL1 genes on chromosome 3q28, suggesting a role in the transcriptional regulation of these genes. In this study, we aimed to functionally characterize the 3q28 bladder cancer risk locus. Methods Fine-mapping was performed by focusing on the region surrounding rs710521, and variants were prioritized for further experiments using ENCODE regulatory data. The enhancer activity of the identified region was evaluated using dual-luciferase assays. CRISPR/Cas9-mediated deletion of the enhancer region was performed and the effect of this deletion on cell proliferation and gene expression levels was evaluated using CellTiter-Glo and RT-qPCR, respectively. Results Fine-mapping of the GWAS signal region led to the identification of twenty SNPs that showed a stronger association with bladder cancer risk than rs710521. Using publicly available data on regulatory elements and sequences, an enhancer region containing the bladder cancer risk variants was identified. Through reporter assays, we found that the presence of the enhancer region significantly increased ΔNTP63 promoter activity in bladder cancer-derived cell lines. CRISPR/Cas9-mediated deletion of the enhancer region reduced the viability of bladder cancer cells by decreasing the expression of ΔNTP63 and p63 target genes. Conclusions Taken together, our data show that bladder cancer risk-associated variants on chromosome 3q28 are located in an active enhancer region. Further characterization of the allele-specific activity of the identified enhancer and its target genes may lead to the identification of novel signaling pathways involved in bladder carcinogenesis. Electronic supplementary material The online version of this article (10.1007/s13402-018-0393-5) contains supplementary material, which is available to authorized users.

Approximately 20% of patients with bladder cancer are diagnosed with muscle‐invasive disease (MIBC). The treatment involves radical cystectomy, but almost 50% of patients with MIBC eventually relapse and develop metastasis. The use of platinum‐based chemotherapy in the neoadjuvant setting or for metastatic patients has been shown to improve the overall survival in a subset of patients. Unfortunately, no biomarkers are available to select patients with MIBC who will benefit from chemotherapy or to monitor the efficacy of the treatment. Recently, long noncoding RNAs (lncRNAs) were shown to regulate a variety of processes involved in the development and progression of cancer, including bladder cancer. Moreover, several lncRNAs have been shown to play a role in chemotherapy resistance. Here, we analyzed lncRNA expression associated with response to platinum‐based chemotherapy in metastatic MIBC using data from the MiTranscriptome lncRNA expression database. Expression of the lncRNA, LINC00857, was found to be upregulated in tumors from patients that did not respond to platinum‐based chemotherapy. Moreover, high expression of LINC00857 is correlated with shorter recurrence‐free and overall survival of patients with MIBC. Knockdown of LINC00857 significantly decreased cell viability of bladder cancer cell lines through the induction of apoptosis. Furthermore, LINC00857 knockdown sensitized UM‐UC‐3 and T24 bladder cancer cells to cisplatin, via the negative regulation of the LMAN1 gene. Our data indicate that LINC00857 plays an important role in the regulation of response to platinum‐based chemotherapy. LINC00857 potentially could serve as a novel prognostic and predictive biomarker and might be a therapeutic target to overcome cisplatin resistance in patients with MIBC.

Background Recently 3D organoid cell cultures have been established for a variety of human cancers. Salivary gland cancer (SGC) is a rare cancer with 22 different subtypes and few treatment options. Our aim is to generate a large repertoire of patient-derived SGC organoids with different phenotypes, which will facilitate pre-clinical and pharmacological studies. Methods Fresh tissues of resected primary tumours and/or metastases of SGC patients were collected. Tissues were minced and digested with collagenase type 2 and TrypLE to generate single cells. After washing, the cells were seeded in growth factor reduced Matrigel. Organoid medium, containing Rho kinase inhibitor Y27632, was refreshed twice a week. Viable organoids were characterized by immunohistochemistry and by DNA/RNA sequencing. Moreover, the organoids were used to evaluate various treatments that are or may be relevant for the treatment of SGC. Results Between 2016 and 2018 we obtained tissue of 17 SGC patients, of which 16 contained sufficient material for processing: 10 salivary duct carcinoma (SDC), 3 adenoid cystic carcinoma (ACC), 2 muco-epidermoid carcinoma (MEC) and 1 parotid adenocarcinoma not otherwise specified (NOS). For 5 tumors (2 SDC, 2 ACC, 1 adenocarcinoma NOS) viable organoids were established, which could be passaged at least three times. Organoids cultures derived from the other tumours stopped growing after the first passage. Viable organoids showed resemblance with the original primary tumour, based on morphology, protein expression, and growth pattern. Various drugs were tested at a single dose, and showed reduced organoid cell growth compared to untreated controls. Moreover, a dose-response relationship was established for an ALK-positive MEC organoid that was treated with crizotinib. Finally, epithelial-mesenchymal transition was shown in SDC organoids of a primary tumour and a lymph node metastasis of the same patient. Conclusions We are the first that have successfully developed and characterized long-term organoid cultures for ACC and SDC. These organoid cell lines will facilitate pre-clinical and pharmacological studies. Other SGC organoid cultures do not grow infinitely, but do provide an in vitro model to study different treatments during initial growth. Legal entity responsible for the study Radboud University Medical Center. Funding Dutch Salivary Gland Cancer Patient Platform. Disclosure All authors have declared no conflicts of interest.

BACKGROUND: Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored. METHODS: We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes. RESULTS: HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients. CONCLUSIONS: HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy. Prostate 75:1868-1876, 2015. (c) 2015 Wiley Periodicals, Inc.

Item does not contain fulltext PURPOSE: Dihydrotestosterone is the main active androgen in the prostate and it has a role in prostate cancer progression. After androgen deprivation therapy androgen receptor signaling is still active in tumor cells. Persistent intratumor steroidogenesis and androgen receptor changes are responsible for this continued activity, which influences the efficacy of prostate cancer treatment. We hypothesized that combining a 5alpha-reductase inhibitor and an antiandrogen would block intratumor androgen synthesis and androgen receptor protein activity. Thus, it would act synergistically to reduce tumor cell proliferation. MATERIALS AND METHODS: The expression level of 5alpha-reductase and androgen receptor in endocrine therapy naive prostate cancer and castration resistant prostate cancer tissues, and cell line models was determined by microarray and quantitative polymerase chain reaction analysis. Intracellular androgen was measured with radioimmunoassay. Tumor cell proliferation was determined using coloric MTT assay. The synergistic effects of combination treatments on tumor cell proliferation were calculated using the Chou-Talalay equation. RESULTS: In all prostate cancer cases 5alpha-reductase-1 and 3 were up-regulated. Androgen receptor was up-regulated in metastatic prostate cancer and castration resistant prostate cancer cases. The 5alpha-reductase inhibitor dutasteride effectively decreased dihydrotestosterone production in prostate cancer and castration resistant prostate cancer cell lines. Furthermore, dutasteride combined with the novel antiandrogen enzalutamide synergistically suppressed endocrine therapy naive prostate cancer and castration resistant prostate cancer cell proliferation. CONCLUSIONS: In this study the combination of a 5alpha-reductase inhibitor and (novel) antiandrogens synergistically inhibited tumor cell proliferation. These findings support clinical studies of combinations of a 5alpha-reductase inhibitor and (novel) antiandrogens as first line treatment of prostate cancer and castration resistant prostate cancer.

// Mohammed Alshalalfa 1 , Gerald W. Verhaegh 2, 3 , Ewan A. Gibb 1 , Maria Santiago-Jimenez 1 , Nicholas Erho 1 , Jennifer Jordan 1 , Kasra Yousefi 1 , Lucia L.C. Lam 1 , Tyler Kolisnik 1 , Jijumon Chelissery 1 , Roland Seiler 1, 4 , Ashley E. Ross 5 , R. Jeffrey Karnes 6 , Edward M. Schaeffer 7 , Tamara T. Lotan 8 , Robert B. Den 9 , Stephen J. Freedland 10 , Elai Davicioni 1 , Eric A. Klein 11 and Jack A. Schalken 2, 3 1 GenomeDx Biosciences Inc., Vancouver, BC, Canada 2 Department of Urology, Radboud University Medical Center, Nijmegen, The Netherlands 3 Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands 4 Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada 5 James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD, USA 6 Department of Urology, Mayo Clinic, Rochester, MN, USA 7 Department of Urology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA 8 Department of Pathology and Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA 9 Sidney Kimmel Cancer Centre, Thomas Jefferson University, Philadelphia, PA, USA 10 Department of Surgery, Division of Urology, Center of Integrated Research on Cancer and Lifestyle, Samuel Oschin Comprehensive Cancer Center, Cedars Sinai Medical Center, Los Angeles, CA, USA 11 Glickman Urological & Kidney Institute, Cleveland Clinic, Cleveland, OH, USA Correspondence to: Mohammed Alshalalfa, email: mohamed@genomedx.com Keywords: prostate cancer, PCA3, initial biopsy, prognosis, under-diagnosis Received: November 16, 2016 Accepted: January 10, 2017 Published: February 07, 2017 ABSTRACT Background: Prostate cancer antigen 3 ( PCA3 ) is a prostate cancer diagnostic biomarker that has been clinically validated. The limitations of the diagnostic role of PCA3 in initial biopsy and the prognostic role are not well established. Here, we elucidate the limitations of tissue PCA3 to predict high grade tumors in initial biopsy. Results: PCA3 has a bimodal distribution in both biopsy and radical prostatectomy (RP) tissues, where low PCA3 expression was significantly associated with high grade disease (p

Contains fulltext : 170668.pdf (Publisher’s version ) (Open Access) Development of castration-resistant prostate cancer (CRPC) in a low androgen environment, arising from androgen deprivation therapy (ADT), is a major problem in patients with advanced prostate cancer (PCa). Several mechanisms have been hypothesized to explain the progression of PCa to CRPC during ADT, one of them is so called persistent intratumoral steroidogenesis. The existence of intratumoral steroidogenesis was hinted based on the residual levels of intraprostatic testosterone (T) and dihydrotestosterone (DHT) after ADT. Accumulating evidence has shown that the intraprostatic androgen levels after ADT are sufficient to induce cancer progression. Several studies now have demonstrated that PCa cells are able to produce T and DHT from different androgen precursors, such as cholesterol and the adrenal androgen, dehydroepiandrosterone (DHEA). Furthermore, up-regulation of genes encoding key steroidogenic enzymes in PCa cells seems to be an indicator for active intratumoral steroidogenesis in CRPC cells. Currently, several drugs are being developed targeting those steroidogenic enzymes, some of which are now in clinical trials or are being used as standard care for CRPC patients. In the future, novel agents that target steroidogenesis may add to the arsenal of drugs for CRPC therapy.

Reversing epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, but a limited number of broadly comprehensive investigations of miRNAs involved in this process have been conducted. In this study, we screened a library of 1120 miRNA for their ability to transcriptionally activate the E-cadherin gene

Contains fulltext : 171608.pdf (Publisher’s version ) (Closed access) AIM: The molecular interactions between Transient Receptor Potential Vanilloid subtype 4 channels (TRPV4) and cell junction formation were investigated in the human and mouse urogenital tract. MATERIALS AND METHODS: A qualitative study was performed to investigate TRPV4 channels, adherence junctions (AJ's) and tight junctions (TJ's) in kidney, ureter and bladder tissues from humans and wild type and transgenic TRPV4 knockout (-/-) mice with immunohistochemistry, western blotting, immunoprecipitation and reverse-trasnscription-PCR. Cell junction formation in the wild type and TRPV4 knockout (-/-) mouse was evaluated with immunohistochemistry and Transmission Electron Microscope (TEM) techniques. RESULTS: TRPV4 channels are predominantly located in membranes of epithelial cells of the bladder, ureter and the collecting ducts of the kidney. There is a molecular interaction between the TRPV4 channel and the AJ. TEM evaluation showed that AJ formation is disrupted in the TRPV4 -/- mouse resulting in deficient intercellular connections and integrity of the epithelium. CONCLUSIONS: TRPV4 is believed to be a mechanoreceptor in the bladder. This study demonstrates that TRPV4 is also involved in intercellular connectivity and structural integrity of the epithelium. This article is protected by copyright. All rights reserved.

Item does not contain fulltext BACKGROUND: Castration-resistant prostate cancer (CRPC) represents a therapeutic challenge for current medications. METHODS: In order to explore the molecular mechanisms involved in CRPC progression and to identify new therapeutic targets, we analyzed a unique sample set of 11 CRPCs and 7 advanced tumors by array-CGH and gene expression microarrays. The genome-wide DNA and RNA data were integrated to identify genes whose overexpression was driven by their amplification. To assess the functional role of these genes, their expression was analyzed in a transcriptional data set of 329 clinical prostate cancers and the corresponding gene products were silenced using RNA interference in prostate cancer cells. RESULTS: Six recurrent genetic targets were identified in the CRPCs; ATP1B1, AR, FAM110B, LAS1L, MYC, and YIPF6. In addition to AR and MYC, FAM110B emerged as a potential key gene involved in CRPC progression in a subset of the tumors. FAM110B was able to regulate AR signaling in prostate cancer cells and FAM110B itself was regulated by androgens. FAM110B siRNA inhibited the growth of prostate cancer cells in vitro, and this effect was substantially enhanced in androgen deficient conditions. Ectopic FAM110B expression in non-cancerous epithelial prostate cells induced aneuploidy and impaired antigen presentation. CONCLUSIONS: The DNA/RNA gene outlier detection combined with siRNA cell proliferation assay identified FAM110B as a potential growth promoting key gene for CRPC. FAM110B appears to have a key role in the androgen signaling and progression of CRPC impacting multiple cancer hallmarks and therefore highlighting a potential therapeutic target.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field. Three genome-wide association studies in Europe and the USA have reported eight urinary bladder cancer (UBC) susceptibility loci. Using extended case and control series and 1000 Genomes imputations of 5 340 737 single-nucleotide polymorphisms (SNPs), we searched for additional loci in the European GWAS. The discovery sample set consisted of 1631 cases and 3822 controls from the Netherlands and 603 cases and 37 781 controls from Iceland. For follow-up, we used 3790 cases and 7507 controls from 13 sample sets of European and Iranian ancestry. Based on the discovery analysis, we followed up signals in the urea transporter (UT) gene SLC14A. The strongest signal at this locus was represented by a SNP in intron 3, rs17674580, that reached genome-wide significance in the overall analysis of the discovery and follow-up groups: odds ratio = 1.17, P = 7.6 × 10(-11). SLC14A1 codes for UTs that define the Kidd blood group and are crucial for the maintenance of a constant urea concentration gradient in the renal medulla and, through this, the kidney's ability to concentrate urine. It is speculated that rs17674580, or other sequence variants in LD with it, indirectly modifies UBC risk by affecting urine production. If confirmed, this would support the 'urogenous contact hypothesis' that urine production and voiding frequency modify the risk of UBC. info:eu-repo/grantAgreement/EC/FP7/018827 info:eu-repo/grantAgreement/EC/FP7/218071 Radboud University Nijmegen Medical Centre (RUNMC) Prinses Beatrix Fonds VSB Fonds National Institute of Mental Health (NIH/NIMH) MH078075 Cancer Research UK Yorkshire Cancer Research European Union 513943 Compagnia di San Paolo-Human Genetics Foundation (HuGeF) Italian Association for Cancer Research, Italy Piedmont Region Progetti di Ricerca Sanitaria Finalizzata Flemish government Belgian province of Limburg Swedish Cancer Society Swedish Research Council Shiraz Institute for Cancer Research Shiraz, Iran ICR-87-502 Red Tematica de Investigacion Cooperativa en Cancer RD06/0020/1054

BACKGROUND: The occurrence of the retrovirus xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been reported in prostate tissue of patients with prostate cancer (PrCa). Considering the potential great medical and social relevance of this discovery, we investigated whether this finding could be confirmed in an independent group of Dutch sporadic PrCa cases. METHODS: We investigated the occurrence of XMRV in fresh-frozen PrCa specimens of 74 PrCa patients from The Netherlands. Total RNA and DNA were isolated, subjected to cDNA synthesis, and analyzed by real-time PCR targeting conserved XMRV sequences. RESULTS: Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences in 100,000 cells by real-time PCR, demonstrating high sensitivity of the assay. XMRV sequences were detected in 3 out of 74 (i.e., 4%) PrCa specimens. The number of XMRV containing cells was extremely low (1 in 600-7,000 cells). This was corroborated by the fact that XMRV could not be detected in consecutive tissue sections of the initial XMRV-positive cases. CONCLUSIONS: XMRV was rarely detected, and at extremely low levels, in sporadic PrCa samples from Dutch patients. When XMRV would play a causal role in prostate carcinogenesis, integration of the provirus could be expected to be present in, at least, a proportion of tumor cells. Therefore, our data do not support the claim that there is an association between XMRV infection and PrCa in Dutch PrCa patients.

Contains fulltext : 88881.pdf (Publisher’s version ) (Closed access) BACKGROUND: PCA3 is one of the most prostate cancer (PrCa)-specific markers described so far. Recently, a new genomic structure of PCA3 as well as new flanking and overlapping gene transcripts has been identified. Furthermore, a co-regulation of PCA3 and its overlapping gene PRUNE2(BMCC1) has been suggested. Our aim was to assess the diagnostic performance of a new PCA3 isoform (PCA3-TS4) and to study the interactions between PCA3 and BMCC1 in PrCa. METHODS: We used SYBR Green quantitative (q)PCR with specific primers to compare PCA3 and BMCC1 expression of normal versus tumor tissue of human prostate. PCA3-TS4 plasmid was created to calculate the absolute amounts of PCA3 transcripts. The androgen regulation of PCA3 and BMCC1 expression was studied in LNCaP and 22Rv1 cells stimulated with 5alpha-dihydrotestosterone. RESULTS: We have not found any relevant diagnostic advantage of the PCA3-TS4 isoform over the "classical" PCA3 isoform in our group of PrCa patients. Additionally, PCA3-TS4 appears to be only a minor PCA3 transcript. We were also unable to confirm the hypothesis that BMCC1 isoforms are androgen-induced in vitro. CONCLUSIONS: Despite the presence of the recently identified marginal PCA3 transcripts in human PrCa, the previously described major PCA3 isoform still constitutes the best target for diagnostic purposes. PCA3 and BMCC1 are overlapping genes in reverse orientation that do not appear to be co-regulated.

Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored.We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes.HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients.HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy.

Contains fulltext : 50445.pdf (Publisher’s version ) (Closed access) AIM: E-cadherin plays a role in carcinogenesis. For two genetic polymorphisms in the gene (CDH1) promoter, a reduced transcription has been reported: a C/A single nucleotide polymorphism (SNP) and a G/GA SNP at -160 bp and -347 bp, respectively, upstream of the transcriptional start site. We studied the association between both polymorphisms and the risk of bladder cancer. METHODS: One hundred and ninety-seven patients with bladder cancer and 344 population controls were genotyped and haplotyped for both SNPs. RESULTS: A borderline significantly increased risk for bladder cancer was found for A allele carriers (OR 1.36; 95% CI: 0.96-1.94). We did not find any association between the -347 G/GA SNP and bladder cancer. Haplotype analyses did not yield much stronger associations with bladder cancer than the -160 C/A genotype analyses. CONCLUSION: This study supports earlier suggestions that the -160 C/A SNP in the CDH1 promoter is a risk factor for bladder cancer.

INTRODUCTION AND OBJECTIVES: The loss of cell-cell contacts between epithelial cells is an important step in cancer progression. Alpha-catenin plays a crucial role in E-cadherin mediated cell-cell adhesion. We identified a novel protein FAM110A that interacts with alpha-catenin. FAM110A is a member of FAM110 gene family that was originally identified in a search for centrosome and spindle pole-associated proteins, but whose function in cancer is still unknown. We hypothesized that FAM110A plays a role in prostate cancer (PrCa) progression. The objective of this study is to analyze the expression and biological functions of FAM110A in PrCa. METHODS: Expression of the FAM110A gene in tumor cell lines and in human PrCa tissues was analyzed by quantitative real-time PCR. We created an EGFP-tagged FAM110A expression vector to observe the localization of FAM110A in cancer cells. Co-immunoprecipitation experiments were performed to confirm the genuine interaction between FAM110A and alpha-catenin. We established a doxycycline-inducible FAM110A expressing A431 cell line. This cell line was used to study the effect of FAM110A on cell viability, migration and invasion, using CellTiter-Glo , wound healing and Matrigel invasion assays, respectively. RESULTS: In human prostate tissues, we found that FAM110A mRNA expression in primary PrCa was unchanged, but was significantly increased in PrCa metastasis (1.5-fold increase compared to normal prostate tissue, p1⁄40.037). The immunofluorescence staining showed that FAM110A was co-localized with E-cadherin and betacatenin at cell-cell adherence junctions. In doxycycline-inducible FAM110A cells, FAM110A did neither affect cell viability nor cell migration, but cell invasiveness was significantly increased after FAM110A induction (1.9-fold compared to non-induced cells, p1⁄40.002). FAM110A expressing cells had a more spindle-like cell morphology, and had impaired F-actin bundle formation. CONCLUSIONS: FAM110A gene expression analysis in prostate tissues suggests that FAM110A plays a role in PrCa metastasis. Furthermore, results of functional assays indicate that binding of FAM110A to alpha-catenin may change the interaction between alpha-catenin and the actin cytoskeleton, which results in a more mesenchymal-like cell morphology and the acquisition of an invasive phenotype.

Item does not contain fulltext Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50

Contains fulltext : 117920.pdf (Publisher’s version ) (Open Access) Genome-wide association studies (GWAS) have been successful in the identification of the several urinary bladder cancer (UBC) susceptibility loci, pointing towards novel genes involved in tumor development. Despite that, functional characterization of the identified variants remains challenging, as they mostly map to poorly understood, non-coding regions. Recently, two of the UBC risk variants (PSCA and UGT1A) were confirmed to have functional consequences. They were shown to modify bladder cancer risk by influencing gene expression in an allele-specific manner. Although the role of the other UBC risk variants is unknown, it can be hypothesized-based on studies from different cancer types-that they influence cancer susceptibility by alterations in regulatory networks. The insight into UBC heritability gained through GWAS and further functional studies can impact on cancer prevention and screening, as well as on the development of new biomarkers and future personalized therapies.