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To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000., Genomics, Proteomics and Bioinformatics, 18 (2)

To answer the increasing need for detecting and validating protein biomarkers in clinical specimens, proteomic techniques are required that support the fast, reproducible and quantitative analysis of large clinical sample cohorts. Targeted mass spectrometry techniques, specifically SRM, PRM and the massively parallel SWATH/DIA technique have emerged as a powerful method for biomarker research. For optimal performance, they require prior knowledge about the fragment ion spectra of targeted peptides. In this report, we describe a mass spectrometric (MS) pipeline and spectral resource to support data-independent acquisition (DIA) and parallel reaction monitoring (PRM) based biomarker studies. To build the spectral resource we integrated common open-source MS computational tools to assemble an open source computational workflow based on Docker. It was then applied to generate a comprehensive DIA pan-human library (DPHL) from 1,096 data dependent acquisition (DDA) MS raw files, and it comprises 242,476 unique peptide sequences from 14,782 protein groups and 10,943 SwissProt-annotated proteins expressed in 16 types of cancer samples. In particular, tissue specimens from patients with prostate cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, squamous cell lung carcinoma, diseased thyroid, glioblastoma multiforme, sarcoma and diffuse large B-cell lymphoma (DLBCL), as well as plasma samples from a range of hematologic malignancies were collected from multiple clinics in China, the Netherlands and Singapore and included in the resource. This extensive spectral resource was then applied to a prostate cancer cohort of 17 patients, consisting of 8 patients with prostate cancer (PCa) and 9 with benign prostate hyperplasia (BPH), respectively. Data analysis of DIA data from these samples identified differential expressions of FASN, TPP1 and SPON2 in prostate tumors. Thereafter, PRM validation was applied to a larger PCa cohort of 57 patients and the differential expressions of FASN, TPP1 and SPON2 in prostate tumors were validated. As a second application, the DPHL spectral resource was applied to a patient cohort consisting of samples from 19 DLBCL patients and 18 healthy individuals. Differential expressions of CRP, CD44 and SAA1 between DLBCL cases and healthy controls were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supported that DIA-PRM MS pipeline enables robust protein biomarker discoveries.

Altres ajuts: The study was supported by grants AECC-JB-2013-02 of Asociación Española Contra el Cáncer, 7th Framework Programe, IRSES (PROTBIOFLUID −269285) - Belgium, pre-doctoral fellowship of Vall d'Hebron Research Institute (VHIR) and cofinanced by the European Regional Development Fund(ERDF). Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.

Background: The role of statins in prostate cancer (PCa) remains unclear. Conflicting evidence has been found concerning risk reduction with the use of statins on biochemical recurrence (BCR). In this study, we evaluated whether statin use decreases the incidence of advanced PCa in males with elevated prostate-specific antigen (PSA; ≥4.0 ng/mL) levels and determined whether statin use reduces the risk of BCR after radical prostatectomy (RP). Methods: Patients visiting the outpatient urology clinic of the VU Medical Center between 2006 and 2018 with elevated PSA were retrospectively analyzed. Biochemical recurrence after RP was defined as a PSA level of ≥0.2 ng/mL (measured twice). Results: A total of 1566 patients were included, of which 1122 (72%) were diagnosed with PCa. At the time of diagnosis, 252 patients (23%) used statins compared to 83 patients (19%) in the non-malignancy group (p = 0.10). No differences were found in the use of statins between the different risk groups. No correlation was found between the risk of BCR after RP and the use of statins in the total (p = 0.20), the intermediate-risk group (p = 0.63) or the high-risk group (p = 0.14). Conclusion: The use of statins does not affect PCa development/progression in patients with elevated PSA levels, nor the development of BCR after RP.

Prostate cancer (PCa) is the most common type of cancer and the second leading cause of cancer-related death in men. Despite extensive research, the molecular mechanisms underlying PCa initiation and progression remain unclear, and there is increasing need of better biomarkers that can distinguish indolent from aggressive and life-threatening disease. With the advent of advanced genomic technologies in the last decade, it became apparent that the human genome encodes tens of thousands non-protein-coding RNAs (ncRNAs) with yet to be discovered function. It is clear now that the majority of ncRNAs exhibit highly specific expression patterns restricted to certain tissues and organs or developmental stages and that the expression of many ncRNAs is altered in disease and cancer, including cancer of the prostate. Such ncRNAs can serve as important biomarkers for PCa diagnosis, prognosis, or prediction of therapy response. In this review, we give an overview of the different types of ncRNAs and their function, describe ncRNAs relevant for the diagnosis and prognosis of PCa, and present emerging new aspects of ncRNA research that may contribute to the future utilization of ncRNAs as clinically useful therapeutic targets.

Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultracentrifugation. The urinary EV isolation protocol using the Vn96-peptide is easier, time convenient (≈1.5 h) and no special equipment is needed, in contrast to ultracentrifugation protocol (>3.5 h), making this protocol clinically feasible. We compared the isolated vesicles of both ultracentrifugation and Vn96-peptide by proteome profiling using mass spectrometry-based proteomics (n = 4 per method). We reached a depth of >3000 proteins, with 2400 proteins that were commonly detected in urinary EVs from different donors. We show a large overlap (>85%) between proteins identified in EVs isolated by ultracentrifugation and Vn96-peptide. Addition of the detergent NP40 to Vn96-peptide EV isolations reduced levels of background proteins and highly increased the levels of the EV-markers TSG101 and PDCD6IP, indicative of an increased EV yield. Thus, the Vn96-peptide-based EV isolation procedure is clinically feasibly and allows largescale protein profiling of urinary EV biomarkers.

Expression of the neuroendocrine peptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs), and activation of the CT-CTR axis in non-invasive PC cells induces an invasive phenotype. We aimed to link CT/CTR expression in prostate specimens to clinicopathological parameters of PC. We analyzed CT and CTR expression in cohorts of benign prostates and primary PCs with/without metastatic disease by immunohistochemistry. Furthermore, we correlated CT/CTR expression with several clinicopathological parameters. CT/CTR immunostaining in benign prostate acini was predominantly localized to basal epithelium. However, this spatial specificity was lost in malignant prostates. PC sections displayed a remarkable increase in cell populations expressing CT/CTR and their staining intensity. Tumors with higher CT/CTR expression consistently displayed metastatic disease and poor clinical outcome. High CT/CTR expression in primary prostate tumors may serve as a prognostic indicator of disease aggressiveness and poor clinical outcome.

The authors thank J. Bossenga for collecting urine and P.P. Eijk for their technical assistance in smallRNA sequencing library preparations., In many cancer types, the expression and function of ~22 nucleotide‑long microRNAs (miRNA) is deregulated. Mature miRNAs can be stably detected in extracellular vesicles (EVs) in biofluids, therefore they are considered to have great potential as biomarkers. In the present study, we investigated whether miRNAs have a distinct expression pattern in urine‑EVs of prostate cancer (PCa) patients compared to control males. By next generation sequencing, we determined the miRNA expression in a discovery cohort of 4 control men and 9 PCa patients. miRNAs were validated by using a stemloop RT‑PCR in an independent cohort of 74 patients (26 control and 48 PCa‑patients). Whereas standard mapping protocols identified > 10 PCa associated miRNAs in urinary EVs, miR‑21, miR‑375 and miR‑204 failed to robustly discriminate for disease in a validation study with RT‑PCR‑detection of mature miRNA sequences. In contrast, we observed that miRNA isoforms (isomiRs) with 3′ end modifications were highly discriminatory between samples from control men and PCa patients. Highly differentially expressed isomiRs of miR‑21, miR‑204 and miR‑375 were subsequently validated in an independent group of 74 patients. Receiver‑operating characteristic analysis was performed to evaluate the diagnostic performance of three isomiRs, resulting in a 72.9% sensitivity with a high (88%) specificity and an area under the curve (AUC) of 0.866. In comparison, prostate specific antigen had an AUC of 0.707 and measuring the mature form of these miRNAs yielded a lower 70.8% sensitivity and 72% specificity (AUC 0.766). We propose that isomiRs may carry discriminatory information which is useful to generate stronger biomarkers., Stichting Urologie 1973, VUmc-CCA, Worldwide Cancer Research (AICR) 15-1005, KWF-VU-5510, Worldwide Cancer Research 15-1005

BACKGROUND: It is important to identify markers that predict whether prostate cancer will metastasise. The adjacent noncancerous cells (influenced by the tumour cells) may also express potential markers. The objective of this study was to determine the influence of cancer cells on noncancerous cells and to assess the value of the cell-communication protein connexin-26 (Cx26) as a marker to predict the development of metastasis. METHODS: The effect of conditioned medium (CM) from PrCa cells on in vitro noncancerous cell proliferation, migration and invasion and Cx26 expression was determined. Connexin-26 expression was investigated in prostatectomy tissues from 51 PrCa patients by immunohistochemistry and compared with various clinicopathological parameters. RESULTS: Proliferation, migration and invasion of noncancerous cells were influenced by CM from the PrCa cell lines. Importantly, a clear relation was found between low Cx26 expression in the noncancerous tissue in prostatectomy sections and the risk of development of metastasis (Po0.0002). Kaplan–Meier analysis showed a relation between low Cx26 expression in noncancerous tissues and time to biochemical recurrence (P ¼0.0002). CONCLUSION: Measuring Cx26 expression in the adjacent noncancerous tissues (rather than cancer tissues) of prostatectomy sections could help to identify high-risk patients who may benefit from adjuvant therapy to decrease the risk of metastasis.

BACKGROUND: Bone metastasis is a complex process involving reciprocal interplay between cancer and bone cells. Metastatic prostate cancer (PCA) cells secrete extracellular vesicles (EVs), that contain microRNAs (miRNAs), and these can be found in blood. We hypothesize that precursor osteoclasts scavenge PCA EVs, leading to a manipulation of their behavior thereby driving metastasis. METHODS: To test how EV-miRNAs affect metastasis, we stably overexpressed two metastasis-related miRNAs in PCA cells. Bone metastases were induced by inoculating miRNA overexpressing PCA cells into nude mice via intracardiac route with or without the presence of subcutaneous tumors. Tumor progression was monitored by F-luciferase signal (IVIS) while bones were analyzed by microCT ex vivo. miRNAs expression was analyzed by qRT-PCR on precursor osteoclasts isolated from mice blood. Osteoclast differentiation assays were performed using human peripheral mononucleated blood cells on bone chips together with RANKL and M-CSF to drive differentiation. RESULTS: In vivo, intracardiac injection of PC3 cells that overexpress selected miRNAs accelerated the formation of bone metastases (0-85%) compared to control PC3 cells (43%). To determine whether EVs secreted by PCA cells affect osteoclasts, human monocytes were allowed to differentiate to osteoclasts while exposing them to PCA EVs. EVs secreted by control PCA cell lines increased osteoclast differentiation by 22.5% (p15% was used as indicator for metastasis induced bone loss. Mice that grew both a subcutaneous tumor and also received an intracardiac injection to drive metastasis formation led to an ~30% increased number of mice with >15% bone loss (p CONCLUSION: PCA miRNAs secreted via EVs stimulate osteoclast formation, potentially increasing the formation of overt metastases. Further investigation is needed to unravel novel pathways that can be targeted to prevent bone metastasis. Citation Format: L. Ayse Erozenci, Ning Wang, Albert A. Geldof, Jorn Mulder, Connie R. Jimenez, R. Jeroen van Moorselaar, Allison Gartland, Teun J. de Vries, Irene V. Bijnsdorp. Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1085.

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.

The objective of the present study is to determine whether uptake of [(18)F]fluoromethylcholine ([(18)F]FCH) in comparison with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) accurately reflects chemotherapy efficacy at the tumor cell level in prostate cancer (PC).The effects of docetaxel and cabazitaxel on viable tumor cell number were explored in four PC cell lines. Cellular uptake of [(18)F]FDG and [(18)F]FCH was compared with the effects measured using sulforhodamine B (SRB) assay, cell counting and colony formation assay (CFA), as proximators of viable tumor cell number. Agreement between uptake and cell numbers was assessed by Bland-Altman plots.[(18)F]FCH uptake in all PC cell lines significantly correlated to the cell numbers surviving the respective drug concentrations. Bland-Altman analysis showed that [(18)F]FDG uptake resulted in signal overestimation and higher variability after chemotherapy.[(18)F]FCH uptake correlates well with viable tumor cell numbers remaining after docetaxel and cabazitaxel exposure. Radiolabeled choline is a potential response monitoring biomarker after chemotherapy for PC.

Background : Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients. Method : Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS. Results : Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p

Prostate cells are dependent on androgens for growth and proliferation. Androgen deprivation therapy is the recommended treatment for advanced/metastatic prostate cancer. Under this therapy, prostate cancer will inevitably progress to castration resistant prostate cancer (CRPC). Despite putative castration resistance, testosterone might still play a crucial role in the progression of CRPC. The goal of this study was to determine the role of testosterone in the formation of metastases of CRPC in both in vitro and in vivo settings. In vitro, the effect of testosterone and the non-aromatizable androgen methyltrienolone on migration, invasion and proliferation of a castration-resistant prostate cancer rat cell line (Dunning R3327-MATLyLu) was assessed using a transwell assay and a sulforhodamine B assay and immunohistochemical detection of ki67. Androgen receptor status was determined using Western blot. In vivo, Copenhagen rats were divided in four groups (males, females, castrated males and females with testosterone suppletion) and inoculated with MATLyLu cells. Tumor size was assessed daily. Testosterone increased cell migration and invasion in a concentration-dependent manner in vitro. Testosterone did not affect in vitro cell proliferation. No difference was shown between the effect of testosterone and methyltrienolone. In vivo, in groups with higher levels of circulating testosterone, more rats had (micro)metastases compared with groups with low levels of testosterone. No effect was observed on primary tumor size/growth. Despite assumed castration resistance, progression of prostate cancer is still influenced by androgens. Therefore, continuous suppression of serum testosterone in patients who show disease progression during castration therapy is still warranted.

Prostate cancer is the second most common cause of death in males. Not all tumors will become metastatic within a patients life time, but when metastasized no curative therapy is available. It is therefore important to identify molecules that predict whether the tumor will become metastatic. Connexin-26 (Cx26) is a gap-junction protein that is involved in cell-cell contacts and in cell adhesion. Cx26 is involved as a tumor suppressor in various tumor types. The aim of the present study was to determine Cx26 expression in localized prostate cancer and relate the expression to the development of metastasis. Cx26 was stained by immunohistochemical staining in radical prostatectomy tissues of 43 patients. The expression levels were scored by a pathologist. Cx26 expression was observed in 95% of the patients. No relation was found to Cx26 expression in the tumor and the development of metastasis (p=0.2). Interestingly, a clear relation was found between low Cx26 expression in benign epithelial cells within the same sections and the development of metastasis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2011-LB-14