Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients.

BACKGROUND: SARS-CoV-2 infection portends a broad range of outcomes, from a majority of asymptomatic cases to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty, and co-morbidities such as obesity, diabetes, and cardiovascular disease. A precise knowledge of the molecular and biological mechanisms that may explain the association of severe disease with male sex is still lacking. Here, we analyzed the relationship of serum testosterone levels and the immune cell skewing with disease severity in male COVID-19 patients. METHODS: Biochemical and hematological parameters of admission samples in 497 hospitalized male and female COVID-19 patients, analyzed for associations with outcome and sex. Longitudinal (in-hospital course) analyses of a subcohort of 114 male patients were analyzed for associations with outcome. Longitudinal analyses of immune populations by flow cytometry in 24 male patients were studied for associations with outcome. RESULTS: We have found quantitative differences in biochemical predictors of disease outcome in male vs. female patients. Longitudinal analyses in a subcohort of male COVID-19 patients identified serum testosterone trajectories as the strongest predictor of survival (AUC of ROC = 92.8%, p < 0.0001) in these patients among all biochemical parameters studied, including single-point admission serum testosterone values. In lethal cases, longitudinal determinations of serum luteinizing hormone (LH) and androstenedione levels did not follow physiological feedback patterns. Failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and augmented circulating classical monocytes. CONCLUSIONS: Recovery or failure to reinstate testosterone levels is strongly associated with survival or death, respectively, from COVID-19 in male patients. Our data suggest an early inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by full recovery in survivors or a peripheral failure in lethal cases. These observations are suggestive of a significant role of testosterone status in the immune responses to COVID-19 and warrant future experimental explorations of mechanistic relationships between testosterone status and SARS-CoV-2 infection outcomes, with potential prophylactic or therapeutic implications.

C1 esterase inhibitor and the contact system in COVID-19.

Coronavirus disease 2019 (COVID-19) is frequently associated with severe systemic consequences, including vasculitis, a hyperinflammatory state and hypercoagulation. The mechanisms leading to these life-threatening abnormalities are multifactorial. Based on the analysis of publicly available interactomes, we propose that severe acute respiratory syndrome coronavirus-2 infection directly causes a deficiency in C1 esterase inhibitor, a pathogen-specific mechanism that may help explain significant systemic abnormalities in patients with COVID-19.

Autophagy inhibition as a promising therapeutic target for laryngeal cancer.

To identify the putative relevance of autophagy in laryngeal cancer, we performed an immunohistochemistry study to analyze the expression of the proteins involved in this process, namely, LC3, ATG5 and p62/SQSTM1. Additionally, Prostate tumor-overexpressed gene 1 protein (PTOV1) was included due to its potential relevance in laryngeal cancer. Moreover, as cancer resistance might involve autophagy in some circumstances, we studied the intrinsic drug resistance capacity of primary tumor cultures derived from 13 laryngeal cancer biopsies and their expression levels of LC3, ATG5, p62 and PTOV1. Overall, our results suggest that (i) cytoplasmic p62 and PTOV1 can be considered prognostic markers in laryngeal cancer, (ii) the acquisition of resistance seems to be related to PTOV1 and autophagy-related protein overexpression, (iii) by increasing autophagy, PTOV1 might contribute to resistance in this model and (iv) the expression of autophagy-related proteins could classify a subgroup of laryngeal cancer patients who will benefit from a therapy based upon autophagy inhibition. Our study suggests that autophagy inhibition with hydroxychloroquine could be a promising strategy for laryngeal cancer patients, particularly those patients with high resistance to the CDDP treatment that in addition have autophagy upregulation.

Expression patterns and bioinformatic analysis of miR-1260a and miR-1274a in Prostate Cancer Tunisian patients.

Currently, microRNAs (miRs) represent great biomarkers in cancer due to their stability and their potential role in diagnosis, prognosis and therapy. This study aims to evaluate the expression levels of miRs-1260 and -1274a in prostate cancer (PC) samples and to identify their eventual targets by using bioinformatic analysis. In this project, we evaluated the expression status of miRs-1260 and -1274a in 86 PC patients and 19 controls by using real-time quantitative PCR and 2-ΔΔCt method. Moreover, we retrieved validated and predicted targets of miRs from several datasets by using the "multiMir" R/Bioconductor package. We have found that miRs-1260 and -1274a were over-expressed in PC patients compared to controls (p < 1 × 10-5). Moreover ROC curve for miRs-1260 and 1274a showed a good performance to distinguish between controls group and PC samples with an area under the ROC curve of 0.897 and 0.784 respectively. However, no significant association could be shown between these two miRs and clinical parameters such as PSA levels, Gleason score, tumor stage, D'Amico classification, lymph node metastasis statues, tumor recurrence, metastasis status and progression after a minimum of 5 years follow-up. Finally, a bioinformatic analysis revealed the association between these two miRs and several targets implicated in prostate cancer initiation pathways.

Prostate tumor OVerexpressed-1 (PTOV1) down-regulates HES1 and HEY1 notch targets genes and promotes prostate cancer progression.

BACKGROUND: PTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression. METHODS: Stable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis. RESULTS: Knockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression of HEY1 and HES1, and this correlation was significant in metastatic lesions. CONCLUSIONS: High levels of the adaptor protein PTOV1 counteract the transcriptional activity of Notch. Our evidences link the pro-oncogenic and pro-metastatic effects of PTOV1 in prostate cancer to its inhibitory activity on Notch signaling and are supportive of a tumor suppressor role of Notch in prostate cancer progression.

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations.

BACKGROUND: Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. METHODS: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer. RESULTS: Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases. CONCLUSIONS: The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.

Association of the rs1042522 SNP with prostate cancer risk: a study of cancer tissues, primary tumor cultures, and serum samples from a Spanish Caucasian population.

Background: Prostate cancer (PCa) is a leading cause of cancer-related deaths in European men, emphasizing the urgent need for effective risk assessment strategies. The TP53 gene, a tumor suppressor gene frequently mutated in cancer, commonly harbors the rs1042522 single nucleotide polymorphism (SNP), known as the P72R SNP, which may influence PCa susceptibility. This study investigated the prevalence of the P72R SNP in European Caucasian PCa samples and its association with PCa risk. Methods: Genotyping was conducted on 12 hormone-naïve aggressive PCa cultures (hnPCs) from untreated patients (Gleason ≥8), 11 radical prostatectomies (RP), and 94 serum samples using DNA Sanger sequencing and melting curve analysis. Comparative analysis utilized data from the GnomAD database's European Caucasian non-cancer population. Results: Our results demonstrate a significantly higher frequency of the P72R SNP in PCa samples and serums compared to the general European non-cancer population. A robust and statistically significant association (p < 0.0001) between the SNP and prostate cancer risk was identified, with an odds ratio of 7.937 (95% CI 5.37-11.00). Notably, the G allele (R72) showed a pronounced prevalence in high Gleason score (≥8) patients, although statistical significance was not reached. These results highlight a potential association with undifferentiated and malignant PCa lesions. Conclusion: The compelling association between the P72R SNP and prostate cancer risk underscores the potential utility of this marker for the early identification of patients at risk of aggressive metastatic prostate cancer. This insight could empower further research to intervene at an early stage by offering enhanced opportunities for timely and targeted interventions.

Rapid and accurate method for quantifying busulfan in plasma samples by isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Objectives: Administration of busulfan is extending rapidly as a part of a conditioning regimen in patients undergoing hematopoietic stem cell transplantation (HSCT). Monitoring blood plasma levels of busulfan is recommended for identifying the optimal dose in patients and for minimizing toxicity. The aim of this research was to validate a simple, rapid, and cost-effective analytical tool for measuring busulfan in human plasma that would be suitable for routine clinical use. This novel tool was based on liquid chromatography coupled to mass spectrometry. Methods: Human plasma samples were prepared using a one-step protein precipitation protocol. These samples were then resolved by isocratic elution in a C18 column. The mobile phase consisted 2 mM ammonium acetate and 0.1% formic acid dissolved in a 30:70 ratio of methanol/water. Busulfan-d8 was used as the internal standard. Results: The run time was optimized at 1.6 min. Standard curves were linear from 0.03 to 5 mg/L. The coefficient of variation (%CV) was less than 8%. The accuracy of this method had an acceptable bias that fell within 85-115% range. No interference between busulfan and the interfering compound hemoglobin, lipemia, or bilirubin not even at the highest concentrations of compound was tested. Neither carryover nor matrix effects were observed using this method. The area under the plasma drug concentration-time curves obtained for 15 pediatric patients who received busulfan therapy prior to HSCT were analyzed and correlated properly with the administered doses. Conclusions: This method was successfully validated and was found to be robust enough for therapeutic drug monitoring in a clinical setting.

Regulation of aurora B kinase by the lipid raft protein flotillin-1.

The lipid raft protein Flotillin-1 was previously shown to be required for cell proliferation. Here we show that it is critical for the maintenance of the levels of the mitotic regulator Aurora B. Knockdown of Flotillin-1 induced aberrant mitotic events similar to those produced by Aurora B depletion and led to a marked decline in Aurora B levels and activity. Transfection of wild-type full-length Flotillin-1 or forms directed to the nucleus increased Aurora B levels and activity. Flotillin-1 interacted with Aurora B directly through its SPFH domain in a complex distinct from the chromosomal passenger protein complex, and the two proteins co-purified in nuclear, non-raft fractions. These observations are the first evidence for a function of Flotillin-1 outside of lipid rafts and suggest its critical role in the maintenance of a pool of active Aurora B.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population.

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

PTOV1 expression predicts prostate cancer in men with isolated high-grade prostatic intraepithelial neoplasia in needle biopsy.

PURPOSE: To analyze the expression of PTOV1 in high-grade prostatic intraepithelial neoplasia (HG-PIN) and to explore its usefulness to predict prostate cancer in patients with isolated HG-PIN in needle biopsy (prostate needle biopsy). EXPERIMENTAL DESIGN: PTOV1 expression in HG-PIN lesions from 140 patients was analyzed by immunohistochemistry in a semiquantitative manner (Histo-score). HG-PIN derived from 79 radical prostatectomies for prostate cancer and from 11 cistoprostatectomies for bladder cancer without prostate cancer were used as positive and negative controls, respectively. Fifty patients with HG-PIN without concomitant cancer at their first prostate needle biopsy were chosen as the study group. Patients were followed by a mean of 2.5 repeated prostate needle biopsies (1-5), during a mean period of 12.4 months (1-39). RESULTS: PTOV1 expression in HG-PIN from radical prostatectomies showed a significantly higher Histo-score (162.6) compared with specimens from cistoprostatectomies (67.0). In the study group, PTOV1 expression was significantly higher in samples with cancer in the follow-up (11 patients, 22%) compared with samples in which cancer was not detected (151.4 versus 94.6). PTOV1 expression was the only independent predictor of cancer in the multivariate analysis and the area under the curve was 0.803 (95% confidence interval, 0.728-0.878). A threshold of 100 for PTOV1 expression provided 90.9% sensitivity, 51.3% specificity, 34.5% positive predictive value, and 95.2% negative predictive value. CONCLUSIONS: PTOV1 is overexpressed in HG-PIN associated with cancer and is a potential marker for studying the carcinogenesis and progression of prostate cancer. Prostate needle biopsy with PTOV1 expression in HG-PIN above a threshold of 100 should be repeated immediately for the likely presence of undiagnosed cancer.

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts.

PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.

The role of prostate tumor overexpressed 1 in cancer progression.

Prostate-Tumor-Overexpressed-1 (PTOV1) is a conserved adaptor protein discovered as overexpressed in prostate cancer. Since its discovery, the number of binding partners and associated cellular functions has increased and helped to identify PTOV1 as regulator of gene expression at transcription and translation levels. Its overexpression is associated with increased tumor grade and proliferation in prostate cancer and other neoplasms, including breast, ovarian, nasopharyngeal, squamous laryngeal, hepatocellular and urothelial carcinomas. An important contribution to higher levels of PTOV1 in prostate tumors is given by the frequent rate of gene amplifications, also found in other tumor types. The recent resolution of the structure by NMR of the PTOV domain in PTOV2, also identified as Arc92/ACID1/MED25, has helped to shed light on the functions of PTOV1 as a transcription factor. In parallel, by studying its interaction with RACK1, we have discovered PTOV1 action in promoting mRNAs translation. Here, we will focus on the role of PTOV1 in cancer, re-examine its pro-oncogenic effects and re-evaluate the most relevant interactions and evidences of its cellular functions. The data are used to formulate a model for the mechanisms of action of PTOV1 in line with its recently described activities and cellular pathways modulated in cancer.

Prostate Tumor Overexpressed-1 (PTOV1) promotes docetaxel-resistance and survival of castration resistant prostate cancer cells.

Metastatic prostate cancer is presently incurable. The oncogenic protein PTOV1, first described in prostate cancer, was reported as overexpressed and significantly correlated with poor survival in numerous tumors. Here, we investigated the role of PTOV1 in prostate cancer survival to docetaxel and self-renewal ability. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis.

miR-99a reveals two novel oncogenic proteins E2F2 and EMR2 and represses stemness in lung cancer.

Lung cancer is one of the most aggressive tumours with very low life expectancy. Altered microRNA expression is found in human tumours because it is involved in tumour growth, progression and metastasis. In this study, we analysed microRNA expression in 47 lung cancer biopsies. Among the most downregulated microRNAs we focussed on the miR-99a characterisation. In vitro experiments showed that miR-99a expression decreases the proliferation of H1650, H1975 and H1299 lung cancer cells causing cell cycle arrest and apoptosis. We identified two novel proteins, E2F2 (E2F transcription factor 2) and EMR2 (EGF-like module-containing, mucin-like, hormone receptor-like 2), downregulated by miR-99a by its direct binding to their 3'-UTR. Moreover, miR-99a expression prevented cancer cell epithelial-to-mesenchymal transition (EMT) and repressed the tumourigenic potential of the cancer stem cell (CSC) population in both these cell lines and mice tumours originated from H1975 cells. The expression of E2F2 and EMR2 at protein level was studied in 119 lung cancer biopsies. E2F2 and EMR2 are preferentially expressed in adenocarcinomas subtypes versus other tumour types (squamous and others). Interestingly, the expression of E2F2 correlates with the presence of vimentin and both E2F2 and EMR2 correlate with the presence of ß-catenin. Moreover, miR-99a expression correlates inversely with E2F2 and directly with ß-catenin expression in lung cancer biopsies. In conclusion, miR-99a reveals two novel targets E2F2 and EMR2 that play a key role in lung tumourigenesis. By inhibiting E2F2 and EMR2, miR-99a represses in vivo the transition of epithelial cells through an EMT process concomitantly with the inhibition of stemness features and consequently decreasing the CSC population.

Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer.

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.

Intracellular clusterin induces G2-M phase arrest and cell death in PC-3 prostate cancer cells1.

Enhanced clusterin gene expression has been related frequently to organ remodeling, tissue involution, and cell death. Whether clusterin represents a leading cause or a consequence of apoptosis induction is still a matter of debate. Clusterin is known as an extracellular secreted glycoprotein in the mature form. However, truncated isoforms of the protein and nuclear localization of clusterin have been described recently in association to cell death. Here, we show the biological effects triggered in PC-3 androgen-independent prostate cancer cells by overexpression of an intracellular, not secreted form of clusterin (intracellular-clusterin). Transient transfection of PC-3 cells with intracellular-clusterin resulted in nuclear localization signal-independent massive nuclear localization of the protein leading to G2-M phase blockade followed by caspase-dependent apoptosis. Constitutive expression of intracellular-clusterin (pFLAG- intracellular-clusterin) in recombinant PC-3 cells caused clonogenic toxicity. The rare pFLAG-intracellular clusterin surviving clones showed inhibition of the proliferation rate and altered phenotype with impaired mitosis and endoreduplication. In these cells, caspase-independent cell death was induced. Impaired cell cycle progression in pFLAG-intracellular-clusterin clones was associated to arrest at the G2-M checkpoint by down-regulation of the mitotic complex cyclin B1/cyclin-dependent kinase 1. Intriguingly, intracellular-clusterin was localized exclusively in the cytoplasm in stably transfected cells, suggesting a negative correlation between nuclear clusterin accumulation and cell survival. These findings may possibly explain the conflicting results obtained in different laboratories, suggesting that clusterin might be a proapoptotic or a survival gene, also opening new perspectives for the characterization of androgen-independent and apoptosis-resistant prostate cancer cells.

Oxidative stress and cancer: an overview.

Reactive species, which mainly include reactive oxygen species (ROS), are products generated as a consequence of metabolic reactions in the mitochondria of eukaryotic cells. In normal cells, low-level concentrations of these compounds are required for signal transduction before their elimination. However, cancer cells, which exhibit an accelerated metabolism, demand high ROS concentrations to maintain their high proliferation rate. Different ways of developing ROS resistance include the execution of alternative pathways, which can avoid large amounts of ROS accumulation without compromising the energy demand required by cancer cells. Examples of these processes include the guidance of the glycolytic pathway into the pentose phosphate pathway (PPP) and/or the generation of lactate instead of employing aerobic respiration in the mitochondria. Importantly, ROS levels can be used as a thermostat to monitor the damage that cells can bear. The implications for ROS regulation are highly significant for cancer therapy because commonly used radio- and chemotherapeutic drugs influence tumor outcome through ROS modulation. Moreover, the discovery of novel biomarkers that are able to predict the clinical response to pro-oxidant therapies is a crucial challenge to overcome to allow for the personalization of cancer therapies.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.