Predicting prostate cancer progression with a Multi-lncRNA expression-based risk score and nomogram integrating ISUP grading.

Prostate cancer is a highly heterogeneous disease; therefore, estimating patient prognosis accurately is challenging due to the lack of biomarkers with sufficient specificity and sensitivity. One of the current challenges lies in integrating genomic and transcriptomic data with clinico-pathological features and in incorporating their application in everyday clinical practice. Therefore, we aimed to model a risk score and nomogram containing long non-coding RNA (lncRNA) expression and clinico-pathological data to better predict the probability of prostate cancer progression. We performed bioinformatics analyses to identify lncRNAs differentially expressed across various prostate cancer stages and associated with progression-free survival. This information was further integrated into a prognostic risk score and nomogram containing transcriptomic and clinico-pathological features to estimate the risk of disease progression. We used RNA-seq data from 5 datasets from public repositories (total n = 178) comprising different stages of prostate cancer: pre-treatment primary prostate adenocarcinomas, post-treatment tumors and metastatic castration resistant prostate cancer. We found 30 lncRNAs with consistent differential expression in all comparisons made using two R-based packages. Multivariate progression-free survival analysis including the ISUP group as covariate, revealed that 7/30 lncRNAs were significantly associated with time-to-progression. Next, we combined the expression of these 7 lncRNAs into a multi-lncRNA score and dichotomized the patients into low- or high-score. Patients with a high-score showed a 4-fold risk of disease progression (HR = 4.30, 95 %CI = 2.66-6.97, p = 3.1e-9). Furthermore, we modelled a combined risk-score containing information on the multi-lncRNA score and ISUP group. We found that patients with a high-risk score had nearly 8-fold risk of progression (HR = 7.65, 95 %CI = 4.05-14.44, p = 3.4e-10). Finally, we created and validated a nomogram to help uro-oncologists to better predict patient's risk of progression at 3- and 5-years post-diagnosis. In conclusion, the integration of lncRNA expression data and clinico-pathological features of prostate tumors into predictive models might aid in tailored disease risk assessment and treatment for patients with prostate cancer.

Integrative Molecular Analyses of the MD Anderson Prostate Cancer Patient-derived Xenograft (MDA PCa PDX) Series.

PURPOSE: Develop and deploy a robust discovery platform that encompasses heterogeneity, clinical annotation, and molecular characterization and overcomes the limited availability of prostate cancer models. This initiative builds on the rich MD Anderson (MDA) prostate cancer (PCa) patient-derived xenograft (PDX) resource to complement existing publicly available databases by addressing gaps in clinically annotated models reflecting the heterogeneity of potentially lethal and lethal prostate cancer. EXPERIMENTAL DESIGN: We performed whole-genome, targeted, and RNA sequencing in representative samples of the same tumor from 44 PDXs derived from 38 patients linked to donor tumor metadata and corresponding organoids. The cohort includes models derived from different morphologic groups, disease states, and involved organ sites (including circulating tumor cells), as well as paired samples representing heterogeneity or stages before and after therapy. RESULTS: The cohort recapitulates clinically reported alterations in prostate cancer genes, providing a data resource for clinical and molecular interrogation of suitable experimental models. Paired samples displayed conserved molecular alteration profiles, suggesting the relevance of other regulatory mechanisms (e.g., epigenomic) influenced by the microenvironment and/or treatment. Transcriptomically, models were grouped on the basis of morphologic classification. DNA damage response-associated mechanisms emerged as differentially regulated between adenocarcinoma and neuroendocrine prostate cancer in a cross-interrogation of PDX/patient datasets. CONCLUSIONS: We addressed the gap in clinically relevant prostate cancer models through comprehensive molecular characterization of MDA PCa PDXs, providing a discovery platform that integrates with patient data and benchmarked to therapeutically relevant consensus clinical groupings. This unique resource supports robust hypothesis generation and testing from basic, translational, and clinical perspectives.

Pin-Pointing the Key Hubs in the IFN-γ Pathway Responding to SARS-CoV-2 Infection.

Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1, and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient's viral load. Expression of MX1, MX2, ISG15, and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.

Bone Progenitors Pull the Strings on the Early Metabolic Rewiring Occurring in Prostate Cancer Cells.

Metastatic prostate cancer (PCa) cells soiling in the bone require a metabolic adaptation. Here, we identified the metabolic genes fueling the seeding of PCa in the bone niche. Using a transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3 or Raw264.7), we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. In a Principal Component Analysis using transcriptomic data from human PCa samples (GSE74685), the altered metabolic genes found in vitro were able to stratify PCa patients in two defined groups: primary PCa and bone metastasis, confirmed by an unsupervised clustering analysis. Thus, the early transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastatic samples. Further, the expression levels of five metabolic genes (VDR, PPARA, SLC16A1, GPX1 and PAPSS2) were independent risk-predictors of death in the SU2C-PCF dataset and a risk score model built using this lipid-associated signature was able to discriminate a subgroup of bone metastatic PCa patients with a 23-fold higher risk of death. This signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously, and appears to be under the regulatory control of the Protein Kinase A (PKA) signaling pathway. Secretome analyses of conditioned media showcased fibronectin and type-1 collagen as critical bone-secreted factors that could regulate tumoral PKA. Overall, we identified a novel lipid gene signature, driving PCa aggressive metastatic disease pointing to PKA as a potential hub to halt progression.

A Journey into the Clinical Relevance of Heme Oxygenase 1 for Human Inflammatory Disease and Viral Clearance: Why Does It Matter on the COVID-19 Scene?

Heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation, is involved in the maintenance of cellular homeostasis, exerting a cytoprotective role by its antioxidative and anti-inflammatory functions. HO-1 and its end products, biliverdin, carbon monoxide and free iron (Fe2+), confer cytoprotection against inflammatory and oxidative injury. Additionally, HO-1 exerts antiviral properties against a diverse range of viral infections by interfering with replication or activating the interferon (IFN) pathway. Severe cases of coronavirus disease 2019 (COVID-19), an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are characterized by systemic hyperinflammation, which, in some cases, leads to severe or fatal symptoms as a consequence of respiratory failure, lung and heart damage, kidney failure, and nervous system complications. This review summarizes the current research on the protective role of HO-1 in inflammatory diseases and against a wide range of viral infections, positioning HO-1 as an attractive target to ameliorate clinical manifestations during COVID-19.

Exploiting Interdata Relationships in Prostate Cancer Proteomes: Clinical Significance of HO-1 Interactors.

Prostate cancer (PCa) cells display abnormal expression of proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown the anti-tumoral role of heme oxygenase 1 (HO-1) in this disease. In this work, we undertook a mass spectrometry-based proteomics study to identify HO-1 molecular interactors that might collaborate with its modulatory function in PCa. Among the HO-1 interactors, we identified proteins with nuclear localization. Correlation analyses, using the PCa GSE70770 dataset, showed a significant and positive correlation between HMOX1 and 6 of those genes. Alternatively, HMOX1 and YWHAZ showed a negative correlation. Univariable analyses evidenced that high expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with increased relapse-free survival (RFS) in PCa patients. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, a decrease in RFS for patients with higher scores of this signature was observed using a prognostic risk score model. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. Multivariable analyses confirmed HSPB1, DDB1, and YWHAZ independence from PCa clinic-pathological parameters. In parallel, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (protein encoded by YWHAZ) interaction. Herein, we describe a novel protein interaction between HO-1 and 14-3-3ζ/δ in PCa and highlight these factors as potential therapeutic targets.

Fibroblast Growth Factor Receptor 1 Drives the Metastatic Progression of Prostate Cancer.

BACKGROUND: No curative therapy is currently available for metastatic prostate cancer (PCa). The diverse mechanisms of progression include fibroblast growth factor (FGF) axis activation. OBJECTIVE: To investigate the molecular and clinical implications of fibroblast growth factor receptor 1 (FGFR1) and its isoforms (α/ß) in the pathogenesis of PCa bone metastases. DESIGN, SETTING, AND PARTICIPANTS: In silico, in vitro, and in vivo preclinical approaches were used. RNA-sequencing and immunohistochemical (IHC) studies in human samples were conducted. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In mice, bone metastases (chi-square/Fisher's test) and survival (Mantel-Cox) were assessed. In human samples, FGFR1 and ladinin 1 (LAD1) analysis associated with PCa progression were evaluated (IHC studies, Fisher's test). RESULTS AND LIMITATIONS: FGFR1 isoform expression varied among PCa subtypes. Intracardiac injection of mice with FGFR1-expressing PC3 cells reduced mouse survival (α, p < 0.0001; ß, p = 0.032) and increased the incidence of bone metastases (α, p < 0.0001; ß, p = 0.02). Accordingly, IHC studies of human castration-resistant PCa (CRPC) bone metastases revealed significant enrichment of FGFR1 expression compared with treatment-naïve, nonmetastatic primary tumors (p = 0.0007). Expression of anchoring filament protein LAD1 increased in FGFR1-expressing PC3 cells and was enriched in human CRPC bone metastases (p = 0.005). CONCLUSIONS: FGFR1 expression induces bone metastases experimentally and is significantly enriched in human CRPC bone metastases, supporting its prometastatic effect in PCa. LAD1 expression, found in the prometastatic PCa cells expressing FGFR1, was also enriched in CRPC bone metastases. Our studies support and provide a roadmap for the development of FGFR blockade for advanced PCa. PATIENT SUMMARY: We studied the role of fibroblast growth factor receptor 1 (FGFR1) in prostate cancer (PCa) progression. We found that PCa cells with high FGFR1 expression increase metastases and that FGFR1 expression is increased in human PCa bone metastases, and identified genes that could participate in the metastases induced by FGFR1. These studies will help pinpoint PCa patients who use fibroblast growth factor to progress and will benefit by the inhibition of this pathway.

Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients.

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

Prostate cancer castrate resistant progression usage of non-canonical androgen receptor signaling and ketone body fuel.

Prostate cancer (PCa) that progresses after androgen deprivation therapy (ADT) remains incurable. The underlying mechanisms that account for the ultimate emergence of resistance to ADT, progressing to castrate-resistant prostate cancer (CRPC), include those that reactivate androgen receptor (AR), or those that are entirely independent or cooperate with androgen signaling to underlie PCa progression. The intricacy of metabolic pathways associated with PCa progression spurred us to develop a metabolism-centric analysis to assess the metabolic shift occurring in PCa that progresses with low AR expression. We used PCa patient-derived xenografts (PDXs) to assess the metabolic changes after castration of tumor-bearing mice and subsequently confirmed main findings in human donor tumor that progressed after ADT. We found that relapsed tumors had a significant increase in fatty acids and ketone body (KB) content compared with baseline. We confirmed that critical ketolytic enzymes (ACAT1, OXCT1, BDH1) were dysregulated after castrate-resistant progression. Further, these enzymes are increased in the human donor tissue after progressing to ADT. In an in silico approach, increased ACAT1, OXCT1, BDH1 expression was also observed for a subset of PCa patients that relapsed with low AR and ERG (ETS-related gene) expression. Further, expression of these factors was also associated with decreased time to biochemical relapse and decreased progression-free survival. Our studies reveal the key metabolites fueling castration resistant progression in the context of a partial or complete loss of AR dependence.

HO-1 Modulates Aerobic Glycolysis through LDH in Prostate Cancer Cells.

Prostate cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer associated death in men worldwide. Dysregulation of cellular energetics has become a hallmark of cancer, evidenced by numerous connections between signaling pathways that include oncoproteins and key metabolic enzymes. We previously showed that heme oxygenase 1 (HO-1), a cellular homeostatic regulator counteracting oxidative and inflammatory damage, exhibits anti-tumoral activity in PCa cells, inhibiting cell proliferation, migration, tumor growth and angiogenesis. The aim of this study was to assess the role of HO-1 on the metabolic signature of PCa. After HO-1 pharmacological induction with hemin, PC3 and C4-2B cells exhibited a significantly impaired cellular metabolic rate, reflected by glucose uptake, ATP production, lactate dehydrogenase (LDH) activity and extracellular lactate levels. Further, we undertook a bioinformatics approach to assess the clinical significance of LDHA, LDHB and HMOX1 in PCa, identifying that high LDHA or low LDHB expression was associated with reduced relapse free survival (RFS). Interestingly, the shortest RFS was observed for PCa patients with low HMOX1 and high LDHA, while an improved prognosis was observed for those with high HMOX1 and LDHB. Thus, HO-1 induction causes a shift in the cellular metabolic profile of PCa, leading to a less aggressive phenotype of the disease.

Analysis workflow of publicly available RNA-sequencing datasets.

Differential gene expression analysis is widely used to study changes in gene expression profiles between two or more groups of samples (e.g., physiological versus pathological conditions, pre-treatment versus post-treatment, and infected versus non-infected tissues). This protocol aims to identify gene expression changes in a pre-selected set of genes associated with severe acute respiratory syndrome coronavirus 2 viral infection and host cell antiviral response, as well as subsequent gene expression association with phenotypic features using samples deposited in public repositories. For complete details on the use and outcome of this informatics analysis, please refer to Bizzotto et al. (2020).

The expression of YWHAZ and NDRG1 predicts aggressive outcome in human prostate cancer.

Some prostate cancers (PCas) are histo-pathologically grouped within the same Gleason Grade (GG), but can differ significantly in outcome. Herein, we aimed at identifying molecular biomarkers that could improve risk prediction in PCa. LC ESI-MS/MS was performed on human PCa and benign prostatic hyperplasia (BPH) tissues and peptide data was integrated with omic analyses. We identified high YWHAZ and NDRG1 expression to be associated with poor PCa prognosis considering all Gleason scores (GS). YWHAZ and NDRG1 defined two subpopulations of PCa patients with high and intermediate risk of death. Multivariable analyses confirmed their independence from GS. ROC analysis unveiled that YWHAZ outperformed GS beyond 60 months post-diagnosis. The genomic analysis of PCa patients with YWHAZ amplification, or increased mRNA or protein levels, revealed significant alterations in key DNA repair genes. We hereby state the relevance of YWHAZ in PCa, showcasing its role as an independent strong predictor of aggressiveness.

Following the footprints of variability during filopodial growth.

Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate tumor cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodial growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodial length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.

SARS-CoV-2 Infection Boosts MX1 Antiviral Effector in COVID-19 Patients.

In a published case-control study (GSE152075) from SARS-CoV-2-positive (n = 403) and -negative patients (n = 50), we analyzed the response to infection assessing gene expression of host cell receptors and antiviral proteins. The expression analysis associated with reported risk factors for COVID-19 was also assessed. SARS-CoV-2 cases had higher ACE2, but lower TMPRSS2, BSG/CD147, and CTSB expression compared with negative cases. COVID-19 patients' age negatively affected ACE2 expression. MX1 and MX2 were higher in COVID-19 patients. A negative trend for MX1 and MX2 was observed as patients' age increased. Principal-component analysis determined that ACE2, MX1, MX2, and BSG/CD147 expression was able to cluster non-COVID-19 and COVID-19 individuals. Multivariable regression showed that MX1 expression significantly increased for each unit of viral load increment. Altogether, these findings support differences in ACE2, MX1, MX2, and BSG/CD147 expression between COVID-19 and non-COVID-19 patients and point out to MX1 as a critical responder in SARS-CoV-2 infection.

Myxovirus Resistance Protein 1 (MX1), a Novel HO-1 Interactor, Tilts the Balance of Endoplasmic Reticulum Stress towards Pro-Death Events in Prostate Cancer.

The inflammatory tumor microenvironment is a fertile niche accelerating prostate cancer (PCa). We have reported that heme-oxygenase (HO-1) had a strong anti-tumoral effect in PCa. We previously undertook an in-depth proteomics study to build the HO-1 interactome in PCa. In this work, we used a bioinformatics approach to address the biological significance of HO-1 interactors. Open-access PCa datasets were mined to address the clinical significance of the HO-1 interactome in human samples. HO-1 interactors were clustered into groups according to their expression profile in PCa patients. We focused on the myxovirus resistance gene (MX1) as: (1) it was significantly upregulated under HO-1 induction; (2) it was the most consistently downregulated gene in PCa vs. normal prostate; (3) its loss was associated with decreased relapse-free survival in PCa; and (4) there was a significant positive correlation between MX1 and HMOX1 in PCa patients. Further, MX1 was upregulated in response to endoplasmic reticulum stress (ERS), and this stress triggered apoptosis and autophagy in PCa cells. Strikingly, MX1 silencing reversed ERS. Altogether, we showcase MX1 as a novel HO-1 interactor and downstream target, associated with ERS in PCa and having a high impact in the clinical setting.

HO-1 Interactors Involved in the Colonization of the Bone Niche: Role of ANXA2 in Prostate Cancer Progression.

BACKGROUND: Prostate cancer (PCa) dissemination shows a tendency to develop in the bone, where heme oxygenase 1 (HO-1) plays a critical role in bone remodeling. Previously by LC/ESI-MSMS, we screened for HO-1 interacting proteins and identified annexin 2 (ANXA2). The aim of this study was to analyze the relevance of ANXA2/HO-1 in PCa and bone metastasis. METHODS: We assessed ANXA2 levels using a co-culture transwell system of PC3 cells (pre-treated or not with hemin, an HO-1 specific inducer) and the pre-osteoclastic Raw264.7 cell line. RESULTS: Under co-culture conditions, ANXA2 mRNA levels were significantly modulated in both cell lines. Immunofluorescence analysis unveiled a clear ANXA2 reduction in cell membrane immunostaining for Raw264.7 under the same conditions. This effect was supported by the detection of a decrease in Ca2+ concentration in the conditioned medium. HO-1 induction in tumor cells prevented both, the ANXA2 intracellular relocation and the decrease in Ca2+ concentration. Further, secretome analysis revealed urokinase (uPA) as a key player in the communication between osteoclast progenitors and PC3 cells. To assess the clinical significance of ANXA2/HO-1, we performed a bioinformatics analysis and identified that low expression of each gene strongly associated with poor prognosis in PCa regardless of the clinico-pathological parameters assessed. Further, these genes appear to behave in a dependent manner. CONCLUSIONS: ANXA2/HO-1 rises as a critical axis in PCa.

Heme Oxygenase-1 Is a Pivotal Modulator of Bone Turnover and Remodeling: Molecular Implications for Prostate Cancer Bone Metastasis.

Aims: Bone is the most frequent site of prostate cancer (PCa) metastasis. Tumor cells interact with the bone microenvironment interrupting tissue balance. Heme oxygenase-1 (HO-1; encoded by Hmox1) appears as a potential target in PCa maintaining the cellular homeostasis. Our hypothesis is that HO-1 is implicated in bone physiology and modulates the communication with PCa cells. Here we aimed at (i) assessing the physiological impact of Hmox1 gene knockout (KO) on bone metabolism in vivo and (ii) determining the alterations of the transcriptional landscape associated with tumorigenesis and bone remodeling in cells growing in coculture (PCa cells with primary mouse osteoblasts [PMOs] from BALB/c Hmox1+/+, Hmox1+/-, and Hmox1-/- mice). Results: Histomorphometric analysis of Hmox1-/- mice bones exhibited significantly decreased bone density with reduced remodeling parameters. A positive correlation between Hmox1 expression and Runx2, Col1a1, Csf1, and Opg genes was observed in PMOs. Flow cytometry studies revealed two populations of PMOs with different reactive oxygen species (ROS) levels. The high ROS population was increased in PMOs Hmox1+/- compared with Hmox1+/+, but was significantly reduced in PMOs Hmox1-/-, suggesting restrained ROS tolerance in KO cells. Gene expression was altered in PMOs upon coculture with PCa cells, showing a pro-osteoclastic profile. Moreover, HO-1 induction in PCa cells growing in coculture with PMOs resulted in a significant modulation of key bone markers such as PTHrP and OPG. Innovation and Conclusion: We here demonstrate the direct implications of HO-1 expression in bone remodeling and how it participates in the alterations in the communication between bone and prostate tumor cells.

CTBP1 depletion on prostate tumors deregulates miRNA/mRNA expression and impairs cancer progression in metabolic syndrome mice.

About 20% of prostate cancer (PCa) patients progress to metastatic disease. Metabolic syndrome (MeS) is a pathophysiological disorder that increases PCa risk and aggressiveness. C-terminal binding protein (CTBP1) is a transcriptional corepressor that is activated by high-fat diet (HFD). Previously, our group established a MeS/PCa mice model that identified CTBP1 as a novel link associating both diseases. Here, we integrated in vitro (prostate tumor cell lines) and in vivo (MeS/PCa NSG mice) models with molecular and cell biology techniques to investigate MeS/CTBP1 impact over PCa progression, particularly over cell adhesion, mRNA/miRNA expression and PCa spontaneous metastasis development. We found that CTBP1/MeS regulated expression of genes relevant to cell adhesion and PCa progression, such as cadherins, integrins, connexins, and miRNAs in PC3 xenografts. CTBP1 diminished PCa cell adhesion, membrane attachment to substrate and increased filopodia number by modulating gene expression to favor a mesenchymal phenotype. NSG mice fed with HFD and inoculated with CTBP1-depleted PC3 cells, showed a decreased number and size of lung metastases compared to control. Finally, CTBP1 and HFD reduce hsa-mir-30b-5p plasma levels in mice. This study uncovers for the first time the role of CTBP1/MeS in PCa progression and its molecular targets.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer.

Lysine-specific demethylase 6A (KDM6A) and members of the Switch/Sucrose Non-Fermentable (SWI/SNF) family are known to counteract the activity of Enhancer of Zeste Homolog 2 (EZH2), which is often overexpressed and is associated with poor prognosis in muscle-invasive bladder cancer. Here we provide evidence that alterations in chromatin modifying enzymes, including KDM6A and members of the SWI/SNF complex, are frequent in muscle-invasive bladder cancer. We exploit the loss of function mutations in KDM6A and SWI/SNF complex to make bladder cancer cells susceptible to EZH2-based epigenetic therapy that activates an immune response to drive tumor cell differentiation and death. We reveal a novel mechanism of action of EZH2 inhibition, alone and in combination with cisplatin, which induces immune signaling with the largest changes observed in interferon gamma (IFN-γ). This upregulation is a result of activated natural killer (NK) signaling as demonstrated by the increase in NK cell-associated genes MIP-1α, ICAM1, ICAM2, and CD86 in xenografts treated with EZH2 inhibitors. Conversely, EZH2 inhibition results in decreased expression of pluripotency markers, ALDH2 and CK5, and increased cell death. Our results reveal a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF mutations to EZH2 inhibition alone and in combination with cisplatin. This sensitivity is mediated through increased NK cell-related signaling resulting in tumor cell differentiation and cell death.