Proteomic analysis of exosomes secreted during the epithelial-mesenchymal transition and potential biomarkers of mesenchymal high-grade serous ovarian carcinoma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) promotes cell signaling and morphology alterations, contributing to cancer progression. Exosomes, extracellular vesicles containing proteins involved in cell-cell communication, have emerged as a potential source of biomarkers for several diseases. METHODS: Our aim was to assess the proteome content of exosomes secreted after EMT-induction to identify potential biomarkers for ovarian cancer classification. EMT was induced in the ovarian cancer cell line CAOV3 by treating it with EGF (10 ng/mL) for 96 h following 24 h of serum deprivation. Subsequently, exosomes were isolated from the supernatant using selective centrifugation after decellularization, and their characteristics were determined. The proteins present in the exosomes were extracted, identified, and quantified using Label-Free-Quantification (LFQ) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). To identify potential biomarkers, the obtained proteomic data was integrated with the TGGA database for mRNA expression using principal component analysis and a conditional inference tree. RESULTS: The exosomes derived from CAOV3 cells exhibited similar diameter and morphology, measuring approximately 150 nm, regardless of whether they were subjected to EMT stimulation or not. The proteomic analysis of proteins from CAOV3-derived exosomes revealed significant differential regulation of 157 proteins, with 100 showing upregulation and 57 downregulation upon EMT induction. Further comparison of the upregulated proteins with the TCGA transcriptomic data identified PLAU, LAMB1, COL6A1, and TGFB1 as potential biomarkers of the mesenchymal HGSOC subtype. CONCLUSIONS: The induction of EMT, the isolation of exosomes, and the subsequent proteomic analysis highlight potential biomarkers for an aggressive ovarian cancer subtype. Further investigation into the role of these proteins is warranted to enhance our understanding of ovarian cancer outcomes.

Isolated Components From Spider Venom Targeting Human Glioblastoma Cells and Its Potential Combined Therapy With Rapamycin.

Glioblastomas (GBs) are responsible for a higher mortality rate among gliomas, corresponding to more than 50% of them and representing a challenge in terms of therapy and prognosis. Peptide-based antineoplastic therapy is a vast and promising field, and these molecules are one of the main classes present in spider venoms. Recently, our research group demonstrated the cytotoxic effects of Phoneutria nigriventer spider venom (PnV) in GBs. The present study aimed to select the purified PnV-components with potential antineoplastic effects, as well as to compare different metabolic conditions. Human GB (NG97) cells were treated with the PnV fractions: F1 (less than 3 kDa), F2 (between 3 and 10 kDa), and F3 (greater than 10 kDa). After treatments, viability (MTT), proliferation (CFSE), death (Annexin V/propidium iodide-PI), and cell cycle (PI) assays were performed. The F1 and F2 fractions in acute periods (1 and 5 h) and low concentrations (0.1 and 1 µg/ml) showed more relevant effects and were repurified in subfractions (SF1-SF11); from these, SF3 and SF4 showed the most significant effects. The previous inhibition of mTOR by rapamycin had a synergistic effect with SFs, reducing cell viability even more significantly than the untreated control. Taken together, the results point to components present in SF3 and SF4 as potential prototypes for the development of new drugs for GB treatment and stimulate studies to use these compounds in combination therapy with a rapamycin-like activity. Future studies will be conducted to characterize, synthesize the molecules, and to evaluate the efficacy and safety in preclinical models.

Schistosoma mansoni Heterochromatin Protein 1 (HP1) nuclear interactome in cercariae.

Schistosoma mansoni causes schistosomiasis, which affects 240 million people, and 700 million people are living at risk of infection. Epigenetic mechanisms are important for transcriptional control and are well-known conserved transcriptional co-regulators in evolution, already described in mammal, yeast, protozoa and S. mansoni, responsible for heterochromatization and gene silence mechanisms through the formation of complexes of transcriptional repression in chromatin. Previous results from another group have shown that HP1 (SmCBX) proteins form chromatin complexes with SmMDB2/3 proteins and regulate stem cells and oviposition in parasite adult worms. In addition, results from other groups have shown that cercariae are transcriptionally silent and epigenetic mechanisms are involved in the regulation of gene expression in this stage. In this work, our aim was to give insights into SmHP1 and proteins involved in transcriptional regulation in the cercariae stage. Using monoclonal anti-HP1 antibody for Western blotting, immunoprecipitation, and mass spectrometry, we preliminarily determined nuclear proteins that putatively interact with HP1 to form complexes to regulate gene expression, heterochromatin formation, and translational complexes in the cercariae stage. So far, our data is to give some insights into nuclear interactors in S. mansoni cercariae. SIGNIFICANCE: The significance of this original paper is the evidence for Heterochromatin Protein (HP1), interaction with nuclear proteins in the cercariae stage. Schistosoma mansoni cercariae are the infective stage of the human beings in endemic areas of schistosomiasis, a neglected disease, most prevalent in Brazil and Africa. While cercariae are waiting for a host, it does not feed, gene expression is silent and protein synthesis is stopped. These biochemical mechanisms are recovered when cercariae find a human host, but all proteins and mechanisms are not still elucidated. Until now, literature shows that these phenomena are regulated by epigenetics mechanisms, dependent of histone posttranslational modifications. But we have few pieces of evidence about the other proteins that participates in these processes and which are the co-regulators of expression.

Analysis of ovarian cancer cell secretome during epithelial to mesenchymal transition reveals a protein signature associated with advanced stages of ovarian tumors.

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production.

Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites.IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

One-Week High-Intensity Interval Training Increases Hippocampal Plasticity and Mitochondrial Content without Changes in Redox State.

Evidence suggests that physical exercise has effects on neuronal plasticity as well as overall brain health. This effect has been linked to exercise capacity in modulating the antioxidant status, when the oxidative stress is usually linked to the neuronal damage. Although high-intensity interval training (HIIT) is the training-trend worldwide, its effect on brain function is still unclear. Thus, we aimed to assess the neuroplasticity, mitochondrial, and redox status after one-week HIIT training. Male (C57Bl/6) mice were assigned to non-trained or HIIT groups. The HIIT protocol consisted of three days with short bouts at 130% of maximum speed (Vmax), intercalated with moderate-intensity continuous exercise sessions of 30 min at 60% Vmax. The mass spectrometry analyses showed that one-week of HIIT increased minichromosome maintenance complex component 2 (MCM2), brain derived neutrophic factor (BDNF), doublecortin (DCX) and voltage-dependent anion-selective channel protein 2 (VDAC), and decreased mitochondrial superoxide dismutase 2 (SOD 2) in the hippocampus. In addition, one-week of HIIT promoted no changes in H2O2 production and carbonylated protein concentration in the hippocampus as well as in superoxide anion production in the dentate gyrus. In conclusion, our one-week HIIT protocol increased neuroplasticity and mitochondrial content regardless of changes in redox status, adding new insights into the neuronal modulation induced by new training models.

Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGFß2 in breast cells.

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGFß and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGFß2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. BIOLOGICAL SIGNIFICANCE: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGFß2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages.

A proteomics outlook towards the elucidation of epithelial-mesenchymal transition molecular events.

The main cause of death in cancer is the spread, or metastasis, of cancer cells to distant organs with consequent tumor formation. Additionally, metastasis is a process that demands special attention, as the cellular transformations make cancer at this stage very difficult or occasionally even impossible to be cured. The main process that converts epithelial tumor cells to mesenchymal-like metastatic cells is the Epithelial to Mesenchymal Transition (EMT). This process allows stationary and polarized epithelial cells, which are connected laterally to several types of junctions as well as the basement membrane, to undergo multiple biochemical changes that enable disruption of cell-cell adherence and apical-basal polarity. Moreover, the cells undergo important reprogramming to remodel the cytoskeleton and acquire mesenchymal characteristics such as enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a large increase in the production of ECM components. As expected, the alterations of the protein complement are extensive and complex, and thus exploring this by proteomic approaches is of particular interest. Here we review the overall findings of proteome modifications during EMT, mainly focusing on molecular signatures observed in multiple proteomic studies as well as coordinated pathways, cellular processes and their clinical relevance for altered proteins. As a result, an interesting set of proteins is highlighted as potential targets to be further investigated in the context of EMT, metastasis and cancer progression.

Proteomic Identification and Time-Course Monitoring of Secreted Proteins During Expansion of Human Mesenchymal Stem/Stromal in Stirred-Tank Bioreactor.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) is widely recognized for the treatment of several diseases, including acute graft-vs.-host disease (GVHD), hematological malignancies, cardiovascular, bone, and cartilage diseases. More recently, this therapeutic efficacy has been attributed to the bioactive molecules that these cells secrete (secretome), now being referred as medicinal signaling cells. This fact raises the opportunity of therapeutically using MSC-derived soluble factors rather than cells themselves, enabling their translation into the clinic. Indeed, many clinical trials are now studying the effects of MSC-secretome in the context of cell-free therapy. MSC secretome profile varies between donors, source, and culture conditions, making their therapeutic use very challenging. Therefore, identifying these soluble proteins and evaluating their production in a reproducible and scalable manner is even more relevant. In this work, we analyzed the global profile of proteins secreted by umbilical cord matrix (UCM) derived-MSC in static conditions by using mass spectrometry, enabling the identification of thousands of proteins. Afterwards, relevant proteins were chosen and monitored in the supernatant of a fully-controllable, closed and scalable system (bioreactor) by using multiple reaction monitoring (MRM) mass spectrometric technique in a time-dependent manner. The results showed that the majority of interesting proteins were enriched through time in culture, with the last day of culture being the ideal time for supernatant collection. The use of this regenerative "soup," which is frequently discarded, could represent a step toward a safe, robust and reproducible cell-free product to be used in the medical therapeutic field. The future use of chemically defined culture-media will certainly facilitate secretome production according to Good Manufacturing Practice (GMP) standards.

The highly efficient powerhouse in the Wistar audiogenic rat, an epileptic rat strain.

The Wistar audiogenic rat (WAR) is an animal model of tonic-clonic epileptic seizures, developed after genetic selection by sister × brother inbreeding of Wistar rats susceptible to sound stimuli. Although metabolic changes have been described in this strain, nothing is known about its mitochondrial metabolism. Here, we addressed mitochondrial aspects of oxidative phosphorylation, oxidative stress, biogenesis, and dynamics in liver, skeletal muscle, and heart of male WARs and correlating them with physiological aspects of body metabolism. The results showed higher mitochondrial content, respiration rates in phosphorylation and noncoupled states, and H2O2 production in WARs. Liver presented higher content of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and mammalian target of rapamycin, proteins related to mitochondrial biogenesis. In agreement, isolated liver mitochondria from WARs showed higher respiration rates in phosphorylation state and ADP-to-O ratio, as well as higher content of proteins related to electron transport chain ATP synthase, TCA cycle, and mitochondrial fusion and fission compared with their Wistar counterparts. Mitochondria with higher area and perimeter and more variable shapes were found in liver and soleus from WARs in addition to lower reduced-to-oxidized glutathione ratio. In vivo, WARs demonstrated lower body mass and energy expenditure but higher food and water intake and amino acid oxidation. When exposed to a running test, WARs reached higher speed and resisted for a longer time and distance than their Wistar controls. In conclusion, the WAR strain has mitochondrial changes in liver, skeletal muscle, and heart that improve its mitochondrial capacity of ATP production, making it an excellent rat model to study PGC1α overexpression and mitochondrial function in different physiological conditions or facing pathological challenges.

Quantification of Coagulation Factor VIII by Selective Reaction Monitoring.

Coagulation factor VIII (FVIII) is an important glycoprotein involved in the extrinsic coagulation cascade. Mutations in FVIII gene results in hemophilia A, a recessive coagulation disorder that is clinically managed by administration of purified FVIII from blood donors or recombinant FVIII. Because of its fundamental therapeutic application, biotechnological production of FVIII requires rigid quality control and monitoring in patients and clinical trials. Here, we describe a protocol for a mass spectrometry based approach termed selective reaction monitoring (SRM) as an important alternative tool for accurate and sensitive quantitation of purified or recombinant FVIII.

Selective Reaction Monitoring Mass Spectrometry for Quantitation of Glycolytic Enzymes in Postmortem Brain Samples.

Patients with psychiatric disorders exhibit dysfunctions in peripheral and central metabolism. This may be a root cause of impaired neuronal function, manifested as changes in mood, behavior, and cognitive capabilities in patients suffering with these conditions. Here we describe a selective reaction monitoring mass spectrometry (SRM-MS)-based targeted proteomic protocol for precise simultaneous quantitation of three glycolytic enzymes in postmortem brain tissue extracts. The SRM-MS approach has several advantages in terms of sensitivity, reproducibility, and reduced sample consumption, compared to traditional MS methods.

Proteomic analysis of ovarian cancer cells during epithelial-mesenchymal transition (EMT) induced by epidermal growth factor (EGF) reveals mechanisms of cell cycle control.

Epithelial to mesenchymal transition (EMT) is a well-orchestrated process that culminates with loss of epithelial phenotype and gain of a mesenchymal and migratory phenotype. EMT enhances cancer cell invasiveness and drug resistance, favoring metastasis. Dysregulation of transcription factors, signaling pathways, miRNAs and growth factors including EGF, TGF-beta and HGF can trigger EMT. In ovarian cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior. Here, the ovarian adenocarcinoma cell line Caov-3 was induced to EMT with EGF in order to identify specific mechanisms controlled by this process. Caov-3 cells induced to EMT were thoroughly validated and a combination of subcellular proteome enrichment, GEL-LC-MS/MS and SILAC strategy allowed consistent proteome identification and quantitation. Protein network analysis of differentially expressed proteins highlighted regulation of metabolism and cell cycle. Activation of relevant signaling pathways, such as PI3K/Akt/mTOR and Ras/Erk MAPK, in response to EGF-induced EMT was validated. Also, EMT did not affected the proliferation rate of Caov-3 cells, but led to cell cycle arrest in G1 phase regulated by increased levels of p21Waf1/Cip1, independently of p53. Furthermore, a decrease in G1 and G2 checkpoint proteins was observed, supporting the involvement of EGF-induced EMT in cell cycle control. BIOLOGICAL SIGNIFICANCE: Cancer is a complex multistep process characterized by accumulation of several hallmarks including epithelial to mesenchymal transition (EMT), which promotes cellular and microenvironmental changes resulting in invasion and migration to distant sites, favoring metastasis. EMT can be triggered by different extracellular stimuli, including growth factors such as EGF. In ovarian cancer, the most lethal gynecological cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior, increasing mortality rate caused by metastasis. Our proteomic data, together with specific validation of specific cellular mechanisms demonstrated that EGF-induced EMT in Caov-3 cells leads to important alterations in metabolic process (protein synthesis) and cell cycle control, supporting the implication of EGF/EMT in cancer metastasis, cancer stem cell generation and, therefore, poor prognosis for the disease.

A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics.

UNLABELLED: Tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in the context of proteomics studies. To uncover differences among benign and malignant ovarian samples, we performed a quantitative proteomic study consisting of albumin immunodepletion, isotope labeling with acrylamide and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histological type. 1135 proteins were identified, corresponding to 505 gene products. 223 proteins presented associated quantification and the comparative analysis of histological types revealed 75 differentially abundant proteins. Based on this, we developed a panel for targeted proteomic analysis using the multiple reaction monitoring (MRM) method for validation of 51 proteins in individual samples of high-grade serous ovarian tumor fluids (malignant) and benign serous cystadenoma tumor fluids. This analysis showed concordant results in terms of average amounts of proteins, and APOE, SERPINF2, SERPING1, ADAM17, CD44 and OVGP1 were statistically significant between benign and malignant group. The results observed in the MRM for APOE were confirmed by western blotting, where APOE was more abundant in malignant samples. This molecular signature can contribute to improve tumor stratification and shall be investigated in combination with current biomarkers in larger cohorts to improve ovarian cancer diagnosis. BIOLOGICAL SIGNIFICANCE: Despite advances in cancer research, ovarian cancer has a high mortality and remains a major challenge due to a number of particularities of the disease, especially late diagnosis caused by vague clinical symptoms, the cellular and molecular heterogeneity of tumors, and the lack of effective treatment. Thus, efforts are directed to better understand this neoplasia, its origin, development and, particularly the identification and validation of biomarkers for early detection of the disease in asymptomatic stage. In the present work, we confirmed by MRM method in individual ovarian tumor fluid samples the regulation of 27 proteins out of 33 identified in a highthroughput study. We speculate that the presence and/or differential abundance observed in tumor fluid is a cooperation primarily of high rates of secretion of such tumor proteins to extra tumor environment that will at the end accumulate in plasma, and also the accumulation of acute-phase proteins throughout the entire body. On top of that, consideration of physiological influences in the interpretation of expression observed, including age, menopause status, route-of-elimination kinetics and metabolism of the tumor marker, coexisting disease, hormonal imbalances, life-style influences (smoking, alcoholism, obesity), among others, are mandatory to enable the selection of good protein tumor marker candidates for extensive validation.