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.

Follistatin-related protein 1 interacting partner of Syndecan-1 promotes an aggressive phenotype on Oral Squamous cell carcinoma (OSCC) models.

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.

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.

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.

Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer.

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.

NEK1 kinase domain structure and its dynamic protein interactome after exposure to Cisplatin.

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.

Mass spectrometry-based proteomics revealed Glypican-1 as a novel ADAM17 substrate.

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.

Mycobacterium tuberculosis dihydrofolate reductase reveals two conformational states and a possible low affinity mechanism to antifolate drugs.

Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR.