STAG2 expression is associated with adverse survival outcomes and regulates cell phenotype in muscle-invasive bladder cancer.

STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.

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.

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.

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.

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.

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy.