Peptidomics Strategies to Evaluate Cancer Diagnosis, Prognosis, and Treatment.

Peptides have potential bioactive functions, and the peptidomics landscape has been broadly investigated for various diseases, including cancer. In this chapter, we reviewed the past four years of literature available and selected 16 peer-reviewed publications exploring peptidomics in diagnosis, prognosis, and treatment in cancer research. We highlighted their main aims, mass spectrometry-based peptidomics, multi-omics, data-driven and in silico strategies, functional assays, and clinical applications. Moreover, we underscored several levels of difficulties in translating the peptidomics findings to clinical practice, aiming to learn with the accumulated knowledge and guide upcoming studies. Finally, this review reinforces the peptidomics robustness in discovering potential candidates for monitoring the several stages of cancer disease and therapeutic treatment, leveraging the management of cancer patients in the future.

Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival.

While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumor tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumor tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumor tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides insight into the complex OSCC tissue N-glycoproteome, thereby forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glycomarkers for OSCC.

New Insights into the Impact of Human Papillomavirus on Oral Cancer in Young Patients: Proteomic Approach Reveals a Novel Role for S100A8.

Human papillomavirus (HPV) infection has recently been linked to a subset of cancers affecting the oral cavity. However, the molecular mechanisms underlying HPV-driven oral squamous cell carcinoma (OSCC) onset and progression are poorly understood. METHODS: We performed MS-based proteomics profiling based on HPV status in OSCC in young patients, following biological characterization and cell assays to explore the proteome functional landscape. RESULTS: Thirty-nine proteins are differentially abundant between HPV (+) and HPV (-) OSCC. Among them, COPS3, DYHC1, and S100A8 are unfavorable for tumor recurrence and survival, in contrast to A2M and Serpine1, low levels of which show an association with better DFS. Remarkably, S100A8 is considered an independent prognostic factor for lower survival rates, and at high levels, it alters tumor-associated immune profiling, showing a lower proportion of M1 macrophages and dendritic cells. HPV (+) OSCC also displayed the pathogen-associated patterns receptor that, when activated, triggered the S100A8 and NFκB inflammatory responses. CONCLUSION: HPV (+) OSCC has a peculiar microenvironment pattern distinctive from HPV (-), involving the expression of pathogen-associated pattern receptors, S100A8 overexpression, and NFκB activation and responses, which has important consequences in prognosis and may guide therapeutic decisions.

A meta-analysis reveals the protein profile associated with malignant transformation of oral leukoplakia.

The search for biomarkers associated with oral leukoplakia malignant transformation is critical for early diagnosis and improved prognosis of oral cancer patients. This systematic review and meta-analysis aimed to assess protein-based markers potentially associated with malignant transformation of oral leukoplakia. Five database and the grey literature were searched. In total, 142 studies were included for qualitative synthesis, where 173 proteins were investigated due to their potential role in malignant progression from oral leukoplakia (OL) to oral squamous cell carcinoma (OSCC). The abundance of these proteins was analyzed in fixed tissues and/or biofluid samples, mainly by immunohistochemistry and ELISA, and 12 were shared by both samples. Enrichment analysis revealed that the differential abundant proteins are mostly involved with regulation of cell death, regulation of cell proliferation, and regulation of apoptotic process. Also, these proteins are mainly expressed in the extracellular region (55.5%), cell surface (24.8%), and vesicles (49.1%). The meta-analysis revealed that the proteins related to tumor progression, PD-L1, Mdm2, and Mucin-4 were significantly associated with greater abundance in OSCC patients, with an Odds Ratio (OR) of 0.12 (95% CI: 0.04-0.40), 0.44 (95% CI: 0.24-0.81), and 0.18 (95% CI: 0.04-0.86), respectively, with a moderate certainty of evidence. The results indicate a set of proteins that have been investigated across OSCC initiation and progression, and whose transcriptional expression is associated with clinical characteristics relevant to the prognosis and aggressiveness. Further verification and validation of this biomarkers set are strongly recommended for future clinical application.

Discovery proteomics reveals potential protein signature associated with malignant phenotype acquisition in pleomorphic adenoma.

OBJECTIVE: To analyze the proteomic profile of salivary pleomorphic adenoma (PA) and carcinoma ex pleomorphic adenoma (CXPA) samples and correlate them with the malignant transformation of the PA. MATERIALS AND METHODS: Thirty samples (10 PA, 16 CXPA, and 4 residual PA) were microdissected and submitted to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteomic data and protein identification were analyzed through LC-MS/MS spectra using the MaxQuant software. RESULTS: The proteomic analysis identified and quantified a total of 240 proteins in which 135 were found in PA, residual PA, and CXPA. The shared proteins were divided into six subgroups, and the proteins that showed statistically significant differences (p > 0.05) and fold-change > or <2.5 in one subgroup to another subgroup were included. Seven proteins (Apolipoprotein A-I-APOA1, haptoglobin-HP, protein of the synaptonemal complex 1-SYCP1, anion transport protein of band 3-SLC4A1, subunit µ1 of AP-1 complex-AP1M1, beta subunit of hemoglobin-HBB, and dermcidin-DCD) were classified as potential protein signatures, being HP, AP1M1, and HBB with higher abundance for PA to residual PA, APOA1 with higher abundance for PA to CXPA, SLC4A1 with lower abundance in the PA to CXPA, SYCP1with lower abundance for residual PA to CXPA, and DCD with higher abundance in the CXPA with epithelial differentiation to myoepithelial differentiation. CONCLUSIONS: In this work, we demonstrated the comparative proteomic profiling of PA, residual PA, and CXPA, and seven were proposed as protein signatures, some of which may be associated with the malignant phenotype acquisition.

Protein markers of primary salivary gland tumors: A systematic review of proteomic profiling studies.

OBJECTIVE: To integrate all the available data published in the English literature regarding the protein diagnostic and/or prognostic markers in salivary gland tumors identified by mass spectrometry (MS)-based discovery proteomics. DESIGN: An extensive search was carried out using MEDLINE/PubMed, EMBASE, Web of Science, and Scopus databases. Manual searching in Google Scholar and assessment of the reference list of the included articles also was performed. The risk of bias was assessed by the Joanna Briggs Institute Critical Appraisal tool for the specific type of study. RESULTS: A total of 1092 articles were initially retrieved within which 6 were used for data extraction, resulting in 145 cases of salivary gland tumors. The data was composted by eleven salivary gland tumor types. In total, 2136 proteins were detected by MS-based discovery proteomics in salivary gland tumors. Ninety-one proteins were proposed as potential diagnostic and/or prognostic markers. Of these, some have been identified in one or more studies, whereas fifteen were in common across studies and a total of seventy-six were non-repeat proteins. CONCLUSION: In summary, we compiled data about the proteomic profile of potential diagnostic and/or prognostic protein markers of the salivary gland tumors detected by MS-based discovery proteomics. The proteins ANXA1, ANXA5, CAPG, CRYAB, FGB, GNB2L1, IGHG1, PPIA, S100A9, and SOD1 were proposed as the most common potential diagnostic markers of salivary gland tumors.

Impact of head and neck radiotherapy on the longevity of dental adhesive restorations: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: Established restorative protocols for patients after head and neck radiotherapy are lacking, increasing the failure rates of dental adhesive restorations. PURPOSE: The purpose of this systematic review and meta-analysis was to analyze the evidence regarding the impact of head and neck radiotherapy on the longevity of dental adhesive restorations. MATERIAL AND METHODS: A search was performed using PubMed, Scopus, and Embase in May 2018 (updated in November 2020). Data extraction was performed regarding the percentage of restoration failure among dental adhesive materials, including glass ionomer cements, resin-modified glass ionomer cements, and composite resins. Risk of bias was assessed by the meta-analysis of statistics assessment and review instrument (MAStARI). Confidence in cumulative evidence was evaluated by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) protocol. RESULTS: Four studies met the inclusion criteria. All included studies were classified as having a moderate risk of bias and reported results regarding class V restorations. Overall, composite resins presented lower failure rates at 2 years (30%) when compared with resin-modified glass ionomer (41%) and glass ionomer cements (57%). Meta-analysis showed that the risk of failure with glass ionomer cements was greater than with resin-modified glass ionomer cements (RR: 1.71, P<.001). Composite resins presented lower risk of failure when compared with glass ionomer (RR: 2.29, P<.001) and resin-modified glass ionomer cements (RR: 1.30, P=.03). Three studies reported results regarding fluoride compliance, which had a negative effect on the survival rates of glass ionomer and resin-modified glass ionomer cements and a positive effect on composite resin restorations. CONCLUSIONS: The results suggest that composite resin restorations associated with fluoride gel compliance seems to be the best alternative for restoring class V lesions in patients after head and neck radiotherapy. However, the results showed moderate certainty of evidence, which justifies the need for more randomized clinical trials regarding this subject.

The role of osteopontin in oral cancer: A brief review with emphasis on clinical applications.

Osteopontin (OPN) is a calcium-binding glycol-phosphoprotein present in many physiologic and pathological processes. This protein can control bone cell adhesion, osteoclastic activity, and bone matrix mineralization. However, its participation in pathological processes such as atherosclerosis, sarcoidosis, tuberculosis, and cancer have been described. Some studies have shown that OPN may participate in the development and progression of oral cancer. Although the role of OPN in oral cancer is not fully understood, some studies have suggested that this protein may induce malignant phenotype of cells by activation of PI3K/AKT/mTOR pathway, which favors cell proliferation, invasion, metastasis, angiogenesis, and failure of treatment. This review discusses the possible mechanism of involvement of OPN in oral cancer and its potential clinical application in diagnosis and prognosis.

A Reductionist Approach Using Primary and Metastatic Cell-Derived Extracellular Vesicles Reveals Hub Proteins Associated with Oral Cancer Prognosis.

Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell- and SCC-9 cell-derived EVs. A multi-omics integration identified 11 'hub proteins' significantly decreased at the metastatic site compared with primary tumor-derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven 'hub proteins' in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC.

Epigenetic modulation of the tumor microenvironment in head and neck cancer: Challenges and opportunities.

Head and neck cancer is globally challenging due to the resistance to therapy and aggressive behavior leading to high rates of mortality. Recent findings show that the tumor microenvironment plays a role in the maintenance and progression of many solid tumors, including head and neck cancer. The mechanisms involved in the modulation and regulation of the tumor microenvironment remain poorly understood. Increasing evidence suggests that epigenetic events can modulate the crosstalk between neoplastic and non-neoplastic cells during tumor progression. In this review, we explore the current understanding of the involvement of epigenetic events in the modulation of the tumor microenvironment and its impact on head and neck cancer behavior. We also explore the latest therapeutic strategies that use epigenetic-modulating drugs to manage tumor growth and progression.

Proteomic approaches to assist in diagnosis and prognosis of oral cancer.

Introduction: Oral squamous cell carcinoma (OSCC) ranks among the top 10 leading causes of cancer worldwide, with 5-year survival rate of about 50%, high lymph node metastasis, and relapse rates. The OSCC diagnosis, prognosis, and treatment are mostly based on the clinical TNM classification. There is an urgent need for the discovery of biomarkers and therapeutic targets to assist in the clinical decision-making process.Areas covered: We summarize proteomic studies of the OSCC tumor, immune microenvironment, potential liquid biopsy sites, and post-translational modifications trying to retrieve information in the discovery and verification or (pre)validation phases. The search strategy was based on the combination of MeSH terms and expert refinement.Expert opinion: Untargeted combined with targeted proteomics are strategies that provide reliable and reproducible quantitation of proteins and are the methods of choice of many groups worldwide. Undoubtedly, proteomics has been contributing to the understanding of OSCC progression and uncovers potential candidates as biomarker or therapeutic targets. Nevertheless, none of these targets are available in the clinical practice yet. The scientific community needs to overcome the limitations by investing in robust experimental designs to strengthen the value of the findings, leveraging the translation of knowledge, and further supporting clinical decisions.

Metformin impairs cisplatin resistance effects in A549 lung cancer cells through mTOR signaling and other metabolic pathways.

Lung cancer is the leading cause of cancer­associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin­resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription­quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL­12­mediated signaling. In CIS­resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

Ancient enamel peptides recovered from the South American Pleistocene species Notiomastodon platensis and Myocastor cf. coypus.

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SIGNIFICANCE: This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

Extracellular vesicles produced by immunomodulatory cells harboring OX40 ligand and 4-1BB ligand enhance antitumor immunity.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

Impact of Clustering Oral Symptoms in the Pathogenesis of Radiation Caries: A Systematic Review.

Radiation-related caries (RRC) is a disease with a high potential for destruction of the dentition, which impairs quality of life in head-and-neck (HN) cancer (HNC) patients who undergo radiotherapy. In light of the recently described "clustering of oral symptoms theory," the present systematic review (PROSPERO CRD42019132709) aims to assess HN and gastrointestinal (GI) symptom clusters among HNC patients and discusses how these indirect effects of cancer therapy play a pivotal role in the pathophysiology of RRC. The search was performed at PubMed, Scopus, and Embase and resulted in 11 studies that met the inclusion criteria. Data extraction was performed with respect to the presence of HN/GI symptom clusters among HNC patients. The methodological data of the studies included were assessed using the MAStARI and GRADE instruments. The most prevalent reported HN symptoms were dysphagia, xerostomia, and pain. Taste alterations and fatigue were also commonly reported by the patients. Loss of appetite and weight loss were regularly reported in the studies, as well as nausea and vomiting. The results of the present study suggest that HNC treatment generates clusters of oral symptoms, leading to dietary changes, impaired oral hygiene, enamel fragility, and a highly cariogenic oral environment, which may impact the risk for RRC. A better understanding of oral symptom clustering could be of considerable clinical significance for the oral health and quality of life of HNC patients. Therefore, contemporary protocols of RRC prevention must take this broader treatment scenario of symptom clusters such as oral side effects into account.

Complex Formation between Mur Enzymes from Streptococcus pneumoniae.

Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

Extracellular vesicles derived from cancer-associated fibroblasts induce the migration and invasion of oral squamous cell carcinoma.

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

Interactome analysis of the human Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (hMTr1) protein.

In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.