RESUMO
Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.
Assuntos
Neoplasias Bucais , Proteoma , Saliva , Humanos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/diagnóstico , Saliva/química , Saliva/metabolismo , Proteoma/análise , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/diagnóstico , Feminino , Biomarcadores Tumorais/metabolismo , Masculino , Metástase Linfática , Conformação Proteica , Pessoa de Meia-Idade , Prognóstico , Proteômica/métodos , Transcetolase/metabolismo , Idoso , Espectrometria de Massas , Proteínas e Peptídeos Salivares/metabolismo , Proteínas e Peptídeos Salivares/análiseRESUMO
BACKGROUND AIMS: Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. METHODS: An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). RESULTS: No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen ß-chain were the most differentially expressed proteins between severity groups. CONCLUSION: Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.
Assuntos
COVID-19 , Vesículas Extracelulares , Proteômica , SARS-CoV-2 , Índice de Gravidade de Doença , Humanos , COVID-19/sangue , COVID-19/diagnóstico , COVID-19/imunologia , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Feminino , Masculino , Pessoa de Meia-Idade , Biomarcadores/sangue , Idoso , Adulto , Espectrometria de Massas em Tandem , Cromatografia LíquidaRESUMO
The poor prognosis of head and neck cancer (HNC) is associated with metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision-making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identify nodal metastasis signatures in blood and saliva. We present a proteomic characterization wiring multiple sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.
Assuntos
Neoplasias de Cabeça e Pescoço , Proteoma , Humanos , Metástase Linfática/patologia , Proteômica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Linfonodos/patologia , Microambiente TumoralRESUMO
Syndecans belong to the family of transmembrane heparan sulfate proteoglycans and are associated with many physiopathological processes, including oral cancer. As previously shown soluble syndecan-1 (SDC1) fragments and synthetic SDC1 peptide were able to induce cell migration in oral cancer cell lines. In order to explore the role of SDC1 in oral cancer, we have investigated SDC1 interacting partners and its functional role in oral cancer models. Here we have shown that SDC1 interacts with follistatin-related protein 1 (FSTL1) by its ectodomain (ectoSDC1) and extracellular juxtamembrane peptide (pepSDC1) and that their transcript levels can affect tumor events. Using orthotopic mouse model we identified that the knock-down for FSTL1 (shFSTL1) or for both FSTL1 and SDC1 (sh2KD) produced less aggressive and infiltrative tumors, with lower keratinization deposition, but with increased levels of epithelial-mesenchymal transition and proliferation compared to control and SDC1 knock-down. Based on cell culture assays, we suggest that the shFSTL1 effect on tumor tissues might be from significant increase of mRNA levels of Activin A (ActA) and its resceptors. This study shows for the first time two different complexes, SDC1 and FSTL1; pepSDC1 and FSTL1, exhibiting a close relationship in cell signaling events, as FSTL1 promotes a more aggressive phenotype. SIGNIFICANCE: This work contributes to the understanding of new SDC1 functions, based on the investigation of protein-protein complex formation in Oral Squamous cell carcinoma (OSCC) models. The FSTL1 identification, as an interacting partner of SDC1 ectodomain and of its derived peptide promotes molecular events that favors cancer development and progression, as highlighted by Activin A (ActA) and Epithelial-mesenchymal transition (EMT) gene expression and by changes in the phenotype of orthotopic OSCC mouse tumor tissues when SDC1-FSTL1 expression is modulated.
Assuntos
Carcinoma de Células Escamosas , Proteínas Relacionadas à Folistatina , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Animais , Proteínas Relacionadas à Folistatina/genética , Camundongos , Fenótipo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Sindecana-1/genética , Sindecana-1/metabolismoRESUMO
Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.
Assuntos
Saliva/microbiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/microbiologia , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Metagenômica/métodos , Microbiota/genética , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/microbiologia , Prognóstico , Proteômica/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismoRESUMO
The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.
Assuntos
Proteína ADAM17 , Cisteína , Tiorredoxinas , Cisteína/metabolismo , Células HEK293 , Humanos , Conformação Molecular , Oxirredução , Compostos de Sulfidrila , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/mortalidade , Neoplasias Bucais/mortalidade , Recidiva Local de Neoplasia/diagnóstico , Proteômica/métodos , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patologia , Tomada de Decisão Clínica , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Metástase Linfática , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/prevenção & controle , Peptídeos/análise , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Saliva/química , Taxa de SobrevidaRESUMO
AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.
Assuntos
Proteína ADAM17/metabolismo , Tiorredoxinas/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Oxirredução , Tiorredoxinas/análise , Tiorredoxinas/genética , Células Tumorais CultivadasRESUMO
ADAM17 (a disintegrin and metalloproteinase 17) is a plasma membrane metalloprotease involved in proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Through this process, ADAM17 is implicated in several aspects of tumor growth and metastasis in a broad range of tumors, including head and neck squamous cell carcinomas (HNSCC). In this study, mass spectrometry-based proteomics approaches revealed glypican-1 (GPC1) as a new substrate for ADAM17, and its shedding was confirmed to be metalloprotease-dependent, induced by a pleiotropic agent (PMA) and physiologic ligand (EGF), and inhibited by marimastat. In addition, immunoblotting analysis of GPC1 in the extracellular media from control and ADAM17shRNA pointed to a direct involvement of ADAM17 in the cleavage of GPC1. Moreover, mass spectrometry-based interactome analysis of GPC1 revealed biological functions and pathways related mainly to cellular movement, adhesion and proliferation, which were events also modulated by up regulation of full length and cleavage GPC1. Altogether, we showed that GPC1 is a novel ADAM17 substrate, thus the function of GPC1 may be modulated by proteolysis signaling. BIOLOGICAL SIGNIFICANCE: Inhibition of metalloproteases as a therapeutic approach has failed because there is limited knowledge of the degradome of individual proteases as well as the cellular function of cleaved substrates. Using different proteomic techniques, this study uncovered novel substrates that can be modulated by ADAM17 in oral squamous cell carcinoma cell line. Glypican-1 was validated as a novel substrate for ADAM17, with important function in adhesion, proliferation and migration of carcinoma cells. Therefore, this study opens new avenues regarding the proteolysis-mediated function of GPC1 by ADAM17.