Blood-Based DNA Methylation Analysis by Multiplexed OBBPA-ddPCR to Verify Indications for Prostate Biopsies in Suspected Prostate Cancer Patients.

Current prostate carcinoma (PCa) biomarkers, including total prostate-specific antigen (tPSA), have unsatisfactory diagnostic sensitivity and specificity resulting in overdiagnosis and overtreatment. Previously, we described an optimised bias-based preamplification-digital droplet PCR (OBBPA-ddPCR) technique, which detects tumour DNA in blood-derived cell-free DNA (cfDNA) of cancer patients. The current study investigated the performance of newly developed OBBPA-ddPCR-based biomarkers. Blood plasma samples from healthy individuals (n = 90, controls) and PCa (n = 39) and benign prostatic hyperplasia patients (BPH, n = 40) were analysed. PCa and BPH patients had tPSA values within a diagnostic grey area of 2-15 ng/mL, for whom further diagnostic validation is most crucial. Methylation levels of biomarkers RASSF1A, MIR129-2, NRIP3, and SOX8 were found significantly increased in PCa patients compared to controls. By combining classical PCa risk factors (percentage of free PSA compared to tPSA (QfPSA) and patient's age) with cfDNA-based biomarkers, we developed PCa risk scores with improved sensitivity and specificity compared to established tPSA and QfPSA single-marker analyses. The diagnostic specificity was increased to 70% with 100% sensitivity for clinically significant PCa patients. Thus, prostate biopsies could be avoided for 28 out of 40 BPH patients. In conclusion, the newly developed risk scores may help to confirm the clinical decision and prevent unnecessary prostate biopsy.

Early kinetics of C-reactive protein as prognosticator for survival in a real-world cohort of patients with metastatic renal cell cancer under first-line therapy with immune checkpoint inhibitors.

PURPOSE: This study investigated the prognostic potential of baseline C-reactive protein (CRP) levels and early CRP kinetics in a real-world cohort of patients with metastatic renal cell carcinoma (mRCC) under first-line (1L) therapy with immune checkpoint inhibitors (CPI). METHODS/PATIENTS: Analyses were performed retrospectively in a cohort of 61 mRCC patients under CPI-based 1L therapy. Patients were stratified based on baseline CRP (< 10 vs ≥ 10 mg/l) and CRP change within the initial three months of CPI therapy (normal: baseline < 10 mg/l, normalized: baseline ≥ 10 mg/l and nadir < 10 mg/l, non-normalized: baseline and nadir ≥ 10 mg/l). Finally, the association of baseline CRP and CRP change with progression-free (PFS) and overall survival (OS) was evaluated. RESULTS: Baseline CRP was not significantly associated with both PFS (p = 0.666) and OS (p = 0.143). Following stratification according to early CRP kinetics, 23, 25 and 13 patients exhibited normal, normalized and non-normalized CRP levels, respectively. Patients with normal and normalized CRP had a markedly prolonged PFS (p = 0.091) and OS (p = 0.008) compared to patients with non-normalized CRP. Consequently, significantly better PFS (p = 0.031) and OS (p = 0.002) were observed for the combined normal-normalized group. In multivariate analysis including ECOG and IMDC risk, normalized CRP kinetics alone or in combination with the normal group was identified as significant independent risk factor for OS, whereas a statistical trend was observed for PFS. CONCLUSIONS: The present study emphasizes the prognostic potential of early CRP kinetics in CPI-treated mRCC. As a standard laboratory parameter, CRP can be easily implemented into clinical routine to facilitate therapy monitoring.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

Acquired resistance to irradiation or docetaxel is not associated with cross-resistance to cisplatin in prostate cancer cell lines.

PURPOSE: Platinum chemotherapy can be considered to treat metastatic castration-resistant prostate cancer (mCRPC) with features of neuroendocrine differentiation. However, platinum compounds are generally only applied after the failure of multiple prior-line treatment options. This study investigated whether acquired resistance against ionizing radiation or docetaxel chemotherapy-two commonly applied treatment modalities in prostate cancer-influences the cisplatin (CDDP) tolerance in mCRPC cell line models. METHODS: Age-matched parental as well as radio- or docetaxel-resistant DU145 and PC-3 cell lines were treated with CDDP and their sensitivity was assessed by measurements of growth rates, viability, apoptosis, metabolic activity and colony formation ability. RESULTS: The data suggest that docetaxel resistance does not influence CDDP tolerance in all tested docetaxel-resistant cell lines. Radio-resistance was associated with sensitization to CDDP in PC-3, but not in DU145 cells. In general, DU145 cells tolerated higher CDDP concentrations than PC-3 cells regardless of acquired resistances. Furthermore, non-age-matched treatment-naïve PC-3 cells exhibited significantly different CDDP tolerances. CONCLUSION: Like patients, different mCRPC cell lines exhibit significant variability regarding CDDP tolerance. The presented in vitro data suggest that previous radiation treatment may be associated with a moderate sensitization to CDDP in an isogenic and age-matched setting. Therefore, previous radiotherapy or docetaxel chemotherapy might be no contraindication against initiation of platinum chemotherapy in selected mCRPC patients.

Comprehensive Evaluation of Multiple Approaches Targeting ABCB1 to Resensitize Docetaxel-Resistant Prostate Cancer Cell Lines.

Docetaxel (DTX) is a mainstay in the treatment of metastatic prostate cancer. Failure of DTX therapy is often associated with multidrug resistance caused by overexpression of efflux membrane transporters of the ABC family such as the glycoprotein ABCB1. This study investigated multiple approaches targeting ABCB1 to resensitize DTX-resistant (DTXR) prostate cancer cell lines. In DU145 DTXR and PC-3 DTXR cells as well as age-matched parental controls, the expression of selected ABC transporters was analyzed by quantitative PCR, Western blot, flow cytometry and immunofluorescence. ABCB1 effluxing activity was studied using the fluorescent ABCB1 substrate rhodamine 123. The influence of ABCB1 inhibitors (elacridar, tariquidar), ABCB1-specific siRNA and inhibition of post-translational glycosylation on DTX tolerance was assessed by cell viability and colony formation assays. In DTXR cells, only ABCB1 was highly upregulated, which was accompanied by a strong effluxing activity and additional post-translational glycosylation of ABCB1. Pharmacological inhibition and siRNA-mediated knockdown of ABCB1 completely resensitized DTXR cells to DTX. Inhibition of glycosylation with tunicamycin affected DTX resistance partially in DU145 DTXR cells, which was accompanied by a slight intracellular accumulation and decreased effluxing activity of ABCB1. In conclusion, DTX resistance can be reversed by various strategies with small molecule inhibitors representing the most promising and feasible approach.

Increased Sensitivity of Detection of RASSF1A and GSTP1 DNA Fragments in Serum of Prostate Cancer Patients: Optimisation of Diagnostics Using OBBPA-ddPCR.

Identification of aberrant DNA methylation is a promising tool in prostate cancer (PCa) diagnosis and treatment. In this study, we evaluated a two-step method named optimised bias-based preamplification followed by digital PCR (OBBPA-dPCR). The method was used to identify promoter hypermethylation of 2 tumour suppressor genes RASSF1A and GSTP1 in the circulating cell-free DNA (cfDNA) from serum samples of PCa patients (n = 75), benign prostatic hyperplasia (BPH, n = 58), and healthy individuals (controls, n = 155). The PCa cohort was further subdivided into subgroups comprising (I) patients with Gleason Scores (GS) ≤ 7 (n = 55), (II) GS ≥ 8 (n = 10), and (III) patients with metastatic PCa diagnosis (n = 10). We found that RASSF1A methylation levels were significantly increased in all 3 PCa subgroups compared to the controls and BPH cohorts (p < 0.01 for all comparisons). Fractional abundances of methylated GSTP1 DNA fragments were significantly increased in subgroup III of metastatic PCa patients (p < 0.001). RASSF1A methylation analysis was found to be beneficial as a complementary biomarker where further diagnostic validation is most crucial. In combination with free PSA, RASSF1A methylation status helps to identify PCa patients with GS ≥ 8 and grey-zone total PSA values between 2-10 ng/mL. In our study, PCR biases between 80-90% were sufficient to detect minute amounts of tumour DNA with high signal-to-noise ratios as well as high analytical sensitivity and specificity. Both RASSF1A and GSTP1 exhibited strongly increased DNA methylation levels in all metastatic PCa patients. Our data indicates a superior sensitivity of epigenetic biomarker analyses in early detection of PCa metastases that should also help to improve PCa therapy.

Serum miRNAs Support the Indication for MRI-Ultrasound Fusion-Guided Biopsy of the Prostate in Patients with Low-PI-RADS Lesions.

Multiparametric MRI (mpMRI) and targeted biopsy of the prostate enhance the tumor detection rate. However, the prediction of clinically significant prostate cancer (PCa) is still limited. Our study tested the additional value of serum levels of selected miRNAs in combination with clinical and mpMRI information for PCa prediction and classification. A total of 289 patients underwent targeted mpMRI-ultrasound fusion-guided prostate biopsy complemented by systematic biopsy. Serum miRNA levels of miRNAs (miR-141, miR-375, miR-21-5p, miR-320b, miR-210-3p, let-7c, and miR-486) were determined by quantitative PCR. Detection of any PCa and of significant PCa were the outcome variables. The patient age, pre-biopsy PSA level, previous biopsy procedure, PI-RADS score, and serum miRNA levels were covariates for regularized binary logistic regression models. The addition of miRNA expression of miR-486 and let-7c to the baseline model, containing only clinical parameters, increased the predictive accuracy. Particularly in patients with PI-RADS ≤3, we determined a sensitivity for detecting significant PCa (Gleason score ≥ 7a corresponding to Grade group ≥2) of 95.2%, and an NPV for absence of significant PCa of 97.1%. This accuracy could be useful to support patient counseling in selected cases.

Evaluation of MicroRNAs as Non-Invasive Diagnostic Markers in Urinary Cells from Patients with Suspected Prostate Cancer.

Currently used tumor markers for early diagnosis of prostate cancer (PCa) are often lacking sufficient specificity and sensitivity. Therefore, the diagnostic potential of selected microRNAs in comparison to serum PSA levels and PSA density (PSAD) was explored. A panel of 12 PCa-associated microRNAs was quantified by qPCR in urinary sediments from 50 patients with suspected PCa undergoing prostate biopsy, whereupon PCa was detected in 26 patients. Receiver operating characteristic (ROC) curve analyses revealed a potential for non-invasive urine-based PCa detection for miR-16 (AUC = 0.744, p = 0.012; accuracy = 76%) and miR-195 (AUC = 0.729, p = 0.017; accuracy = 70%). While serum PSA showed an insufficient diagnostic value (AUC = 0.564, p = 0.656; accuracy = 50%) in the present cohort, PSAD displayed an adequate diagnostic performance (AUC = 0.708, p = 0.031; accuracy = 70%). Noteworthy, the combination of PSAD with the best candidates miR-16 and miR-195 either individually or simultaneously improved the diagnostic power (AUC = 0.801-0.849, p < 0.05; accuracy = 76-90%). In the sub-group of patients with PSA ≤ 10 ng/mL (n = 34), an inadequate diagnostic power of PSAD alone (AUC = 0.595, p = 0.524; accuracy = 68%) was markedly surpassed by miR-16 and miR-195 individually as well as by their combination with PSAD (AUC = 0.772-0.882, p < 0.05; accuracy = 74-85%). These findings further highlight the potential of urinary microRNAs as molecular markers with high clinical performance. Overall, these results need to be validated in a larger patient cohort.

Assessment of STAT5 as a potential therapy target in enzalutamide-resistant prostate cancer.

Despite enzalutamide's efficacy in delaying the progression of metastatic castration-resistant prostate cancer (CRPC), resistance to this anti-androgen inevitably occurs. Several studies have revealed that the signal transducer and activator of transcription (STAT) 5 plays a role in tumour progression and development of drug resistance such as enzalutamide. Data mining revealed heterogeneous expression of STAT5 in enzalutamide-treated mCRPC patients and enzalutamide-resistant prostate cancer (PCa). Isobologram analysis revealed that the STAT5 inhibitor pimozide combined with enzalutamide has? additive and synergistic inhibitory effects on cell viability in the used models. Functional analysis with siRNA-mediated STAT5 knockdown yielded divergent results. The LNCaP-derived cell line MR49F could be resensitised to enzalutamide by siRNA-mediated STAT5b-knock-down. In contrast, neither STAT5a nor STAT5b knockdown resensitised enzalutamide-resistant LAPC4-EnzaR cells to enzalutamide. In conclusion, our results indicate that STAT5 may be a possible target in a subgroup of enzalutamide-resistant PCa. However, based on the data presented here, a general role of STAT5 in enzalutamide-resistance and its potential as a therapeutic target could not be shown.

ProstaTrend-A Multivariable Prognostic RNA Expression Score for Aggressive Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is the most prevalent solid cancer among men in Western Countries. The clinical behavior of localized PCa is highly variable. Some cancers are aggressive leading to death, while others can even be monitored safely. Hence, there is a high clinical need for precise biomarkers for identification of aggressive disease in addition to established clinical parameters. OBJECTIVE: To develop an RNA expression-based score for the prediction of PCa prognosis that facilitates clinical decision making. DESIGN, SETTING, AND PARTICIPANTS: We assessed 233 tissue specimens of PCa patients with long-term follow-up data from fresh-frozen radical prostatectomies (RPs), from formalin-fixed and paraffin-embedded RP specimens and biopsies by transcriptome-wide next-generation sequencing and customized expression microarrays. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We applied Cox proportional hazard models to the cohorts from different platforms and specimen types. Evidence from these models was combined by fixed-effect meta-analysis to identify genes predictive of the time to death of disease (DoD). Genes were combined by a weighted median approach into a prognostic score called ProstaTrend and transferred for the prediction of biochemical recurrence (BCR) after RP in an independent cohort of The Cancer Genome Atlas (TCGA). RESULTS AND LIMITATIONS: ProstaTrend comprising ∼1400 genes was significantly associated with DoD in the training cohort of PCa patients treated by RP (leave-one-out cross-validation, Cox regression: p=2e-09) and with BCR in the TCGA validation cohort (Cox regression: p=3e-06). The prognostic impact persisted after multivariable Cox regression analysis adjusting for Gleason grading group (GG) ≥3 and resection status (p=0.001; DoD, training cohort) and for GG≥3, pathological stage ≥T3, and resection state (p=0.037; BCR, validation cohort). CONCLUSIONS: ProstaTrend is a transcriptome-based score that predicts DoD and BCR in cohorts of PCa patients treated with RP. PATIENT SUMMARY: ProstaTrend provides molecular patient risk stratification after radical prostatectomy.

Urinary MicroRNAs as Potential Markers for Non-Invasive Diagnosis of Bladder Cancer.

Currently, voided urine cytology (VUC) serves as the gold standard for the detection of bladder cancer (BCa) in urine. Despite its high specificity, VUC has shortcomings in terms of sensitivity. Therefore, alternative biomarkers are being searched, which might overcome these disadvantages as a useful adjunct to VUC. The aim of this study was to evaluate the diagnostic potential of the urinary levels of selected microRNAs (miRs), which might represent such alternative biomarkers due to their BCa-specific expression. Expression levels of nine BCa-associated microRNAs (miR-21, -96, -125b, -126, -145, -183, -205, -210, -221) were assessed by quantitative PCR in urine sediments from 104 patients with primary BCa and 46 control subjects. Receiver operating characteristic (ROC) curve analyses revealed a diagnostic potential for miR-96, -125b, -126, -145, -183, and -221 with area under the curve (AUC) values between 0.605 and 0.772. The combination of the four best candidates resulted in sensitivity, specificity, positive and negative predictive values (NPV), and accuracy of 73.1%, 95.7%, 97.4%, 61.1%, and 80.0%, respectively. Combined with VUC, sensitivity and NPV could be increased by nearly 8%, each surpassing the performance of VUC alone. The present findings suggested a diagnostic potential of miR-125b, -145, -183, and -221 in combination with VUC for non-invasive detection of BCa in urine.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.

New markers in prostate cancer: Genomics.

Prostate cancer (PCa) is a very heterogeneous disease with unknown outcome at the time of first diagnosis. Multiple clinicopathological parameters andmodern imaging approaches are currently used to detect PCa and to assess the necessity of early or delayed treatment according to the predicted aggressiveness of the tumor. Despite regular adjustments of predictive systems based on histopathological factors such as the Gleason grading system or based on prostate MRI such as the Prostate Imaging Reporting and Data System (PIRADS) these tools for risk stratification of PCa patients still harbor significant limitations with regard to the accuracy of PCa outcome prediction. Therefore, great hopes have been placed on the use of biomolecular markers which might be more closely associated with the underlying biological characteristics of this tumor entity and able to predict the course of the disease better than clinical parameters. Such biomarkers are expected to serve as valuable tools not only to improve PCa diagnostics but also to enhance pre- and posttreatment risk stratification which could finally facilitate therapeutic decisions.In this review, current literature on genomic biomarkers used for PCa detection and early prediction of the tumor aggressiveness is examined. First, germline mutations and single nucleotide polymorphisms which might influence PCa onset are discussed in relation to the usefulness of targeted PCa screening approaches. Moreover, different urine- and tissue-based diagnostic tests assessing PCa-associated alterations on genetic, epigenetic and transcriptional level are reviewed. Most of these genomic biomarker assays were validated in large patient cohorts and their potential clinical usability could be proven. They provide useful diagnostic information to facilitate decisions with regard to screen men at risk to develop PCa or to repeat diagnostics in men with negative biopsy results, but persistent suspicion of cancer.Several assays can assist the early identification of patients with high-risk PCa, who potentially would need treatment, and may facilitate the selection of patients suitable for active surveillance. More evidence of the clinicalusability of such genomic PCa detection assays has to be provided by further prospective studies to support the transferability of these new diagnostic approaches to daily clinical practice.

El cáncer de próstata (CaP) es una enfermedad muy heterogénea con una evolución desconocida en el momento del primer diagnóstico. Actualmentese utilizan múltiples parámetros clínico-patológicos y abordajes radiológicos modernos para detectar el CaP y evaluar la necesidad de tratamiento temprano o diferido de acuerdo con la agresividad predecible del tumor.A pesar de los ajustes regulares de los sistemas predictivos basados en factores histopatológicos, como la escala de Gleason, o en la RMN prostática, como elPI-RADS (Prostate Imaging Reporting and Data System), estas herramientas de estratificación de riesgos del CaP todavía tienen importantes limitaciones con respecto a la precisión de predicción de la evolución del CaP. Por lo tanto, se han depositado grandes esperanzas en el uso de marcadores biomoleculares, los cuales podrían estar más íntimamente relacionados con las característicasbiológicas subyacentes de esta entidad tumoral y serían capaces de predecir el curso de la enfermedad mejor que los parámetros clínicos. Se espera que dichos biomarcadores sirvan como valiosas herramientas no sólo para mejorar el diagnóstico de CaP sino también mejorar la estratificación de riesgos pre- y post-tratamiento lo que podría facilitar las decisiones terapéuticas.En esta revisión, examinamos la literatura actual sobre biomarcadores genómicos utilizados para la detección del CaP y la predicción temprana de la agresividad tumoral. Primero, se discuten las mutaciones de líneasgerminales y polimorfismos de un único nucleótido que podrían influenciar el inicio del CaP en relación con la utilidad de abordajes de cribado de CaP dirigidos.Además, se revisan diferentes pruebas diagnósticas en orina y tejidos que evalúan las alteraciones al nivel genético, epigenético y transcripcional asociadas con CaP. La mayoría de estas pruebas de biomarcadores genéticos fueron validadas en grandes cohortes de pacientes y su potencial usabilidad clínica puede probarse. Suministran información diagnóstica útil para facilitar las decisiones en relación con el cribado de varones en riesgo de desarrollar CaP o repetir pruebas diagnósticas en varones con biopsia negativa y sospecha persistente de cáncer. Varios tests pueden asistir en la identificación temprana de pacientes con CaP de alto riesgo, que potencialmente necesitarían tratamiento, y pueden facilitar la selección de pacientes adecuados para vigilancia activa. Para apoyar la transferibilidad de estos nuevos abordajes diagnósticos a la práctica es necesaria más evidencia sobre la utilidad clínica de dichas pruebas de detección genómica de CaP mediante nuevos estudios prospectivos.

Linking NRP2 With EMT and Chemoradioresistance in Bladder Cancer.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFß1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFß1, associate with TGFß receptors, and enhance TGFß1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2.

Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer.

BACKGROUND: Cell-free DNA (cfDNA) is proposed to be a valuable source of biomarkers in liquid biopsies for various diseases as it is supposed to partially originate from tumor cells. However, data about the diagnostic implications of cfDNA in urine for the detection of bladder cancer (BCa) is sparse. METHODS: We evaluated the usability of urinary cfDNA for diagnostic purposes compared to urine sediment DNA (sDNA) in 53 BCa patients and 36 control subjects by analyzing two abundant point-mutations (C228T/C250T) in the TERT promoter using Next-Generation Sequencing. RESULTS: Mutations were detected in 77% of the urinary sDNA compared to 63% of the cfDNA samples. Moreover, the TERT mutation allele frequencies (MAF) were highly correlated in cfDNA and sDNA. In comparison, the accuracy of the TERT assay was higher in sDNA (84%) compared to cfDNA or voided urine cytology (both 77%). Interestingly, MAFs from leukocyte-rich urines were higher in cfDNA than in sDNA, indicating a diagnostic advantage of cfDNA in such urines. CONCLUSIONS: Urine-based mutation detection has the ability to augment and surpass voided urine cytology as the current gold-standard for the non-invasive detection and surveillance of BCa. The analysis of cell-free DNA provides no general diagnostic advantage compared to urine sediment DNA.

Role of WNT5A receptors FZD5 and RYK in prostate cancer cells.

Prostate cancer is the most common malignancy in men and has a high propensity to metastasize to bone. WNT5A has recently been implicated in the progression of prostate cancer, however, the receptors that mediate its effects remain unknown. Here, we identified Wnt receptors that are highly expressed in prostate cancer and investigated which of these receptors mediate the anti-tumor effects of WNT5A in prostate cancer in vitro. Extensive in vitro analyses revealed that the WNT5A receptors FZD5 and RYK mediate the anti-tumor effects of WNT5A on prostate cancer cells. Knock-down of FZD5 completely abrogated the anti-proliferative effect of WNT5A in PC3 cells. In contrast, knock-down of RYK and FZD8 did not rescue the inhibition of proliferation after WNT5A overexpression. In contrast, RYK knock-down inhibited the pro-apoptotic effect of WNT5A in PC3 cells by 60%, whereas the knock-down of either FZD5 or FZD8 further stimulated apoptosis after WNT5A overexpression (by 33% and 234%, respectively). Surface plasmon resonance analysis indicated that WNT5A has a 30% stronger binding response to FZD5 than to RYK. Further investigations using a tissue microarray revealed that expression of RYK is increased in advanced prostate cancer tumor stages, but is not associated with survival of prostate cancer patients. In contrast, patients with low local FZD5 expression, in particular in combination with low WNT5A expression, showed a longer disease-specific survival. In conclusion, WNT5A/FZD5 and WNT5A/RYK signaling are both involved in mediating the pro-apoptotic and anti-proliferative effects of WNT5A in prostate cancer.

Quantification of MicroRNAs in Urine-Derived Specimens.

MicroRNAs are small noncoding RNAs which regulate the expression of genes involved in a multitude of cellular processes. Dysregulation of microRNAs and-in consequence-of the affected pathways is frequently observed in numerous pathologies including cancers. Therefore, tumor-related alterations in microRNA expression and function can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers.In particular, microRNAs with differential expression in bladder cancer (BCa) might represent promising tools for noninvasive tumor detection in urine. This would be helpful not only for diagnostic and monitoring purposes but also for therapeutic decisions. Detection and quantification of BCa-associated microRNAs in urine can be performed using the cellular sediment, which also contains BCa cells, or in exosomes originating from those cells. Methods for isolation of exosomes from urine, extraction of total RNA from cells and exosomes as well as techniques for RNA quantification, reverse transcription, and qPCR-based quantification of microRNA expression levels are described herein.

An orthotopic xenograft model for high-risk non-muscle invasive bladder cancer in mice: influence of mouse strain, tumor cell count, dwell time and bladder pretreatment.

BACKGROUND: Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. METHODS: Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106-5.0 × 106) and tumor cell dwell time in the murine bladder (30 min - 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). RESULTS: Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration - both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. CONCLUSIONS: With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Induction of alpha-methylacyl-CoA racemase by miR-138 via up-regulation of ß-catenin in prostate cancer cells.

PURPOSE: Alpha-methylacyl-CoA racemase (AMACR) is highly overexpressed in prostate cancer (PCa) and its transcriptional regulators include various transcription factors and CTNNB1/ß-catenin. Our previous findings suggested a post-transcriptional regulation by the tumor-suppressive microRNA miR-138 in PCa. Thus, the aim of this study was to demonstrate the direct interaction of miR-138 with the 3'-UTR of AMACR. Furthermore, the influence of miR-138 on the expression of AMACR and selected AMACR regulators was investigated in PCa cells. METHODS: Using DU-145, PC-3, and LNCaP PCa cells, the effect of exogenous miR-138 on AMACR and selected AMACR regulators was determined by quantitative PCR and Western blot. Luciferase reporter assays were used to verify target and promoter interaction. RESULTS: Using a luciferase reporter assay a direct interaction of miR-138 with the AMACR-3'-UTR was confirmed. Surprisingly, AMACR expression was up-regulated by up to 125% by exogenous miR-138 in PCa cells. The lack of any miR-138 binding sites within the AMACR promoter suggested an indirect mechanism of up-regulation. Therefore, the effect of miR-138 on selected AMACR regulators including CTNNB1/ß-catenin, RELA, SMAD4, SP1, and TCF4 was evaluated. MiR-138 solely evoked an up-regulation of CTNNB1 mRNA expression and ß-catenin protein levels by up to 75%. Further in silico analysis revealed a binding site for miR-138 within the CTNNB1 promoter. MiR-138 could enhance the activity of the CTNNB1 promoter, which in turn could contribute to the observed AMACR up-regulation. CONCLUSIONS: The present findings suggest that miR-138 can indirectly up-regulate AMACR via transcriptional induction of CTNNB1, at least in vitro in PCa cells.