Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-beta 1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.Peer reviewe

Typic: A Practical and Robust Tool to Rank Proteotypic Peptides for Targeted Proteomics

The selection of a suitable proteotypic peptide remains a challenge for designing a targeted quantitative proteomics assay. Although the criteria are well-established in the literature, the selection of these peptides is often performed in a subjective and time-consuming manner. Here, we have developed a practical and semiautomated workflow implemented in an open-source program named Typic. Typic is designed to run in a command line and a graphical interface to help selecting a list of proteotypic peptides for targeted quantitation. The tool combines the input data and downloads additional data from public repositories to produce a file per protein as output. Each output file includes relevant information to the selection of proteotypic peptides organized in a table, a colored ranking of peptides according to their potential value as targets for quantitation and auxiliary plots to assist users in the task of proteotypic peptides selection. Taken together, Typic leads to a practical and straightforward data extraction from multiple data sets, allowing the identification of most suitable proteotypic peptides based on established criteria, in an unbiased and standardized manner, ultimately leading to a more robust targeted proteomics assay

Typic: A Practical and Robust Tool to Rank Proteotypic Peptides for Targeted Proteomics

The selection of a suitable proteotypic peptide remains a challenge for designing a targeted quantitative proteomics assay. Although the criteria are well-established in the literature, the selection of these peptides is often performed in a subjective and time-consuming manner. Here, we have developed a practical and semiautomated workflow implemented in an open-source program named Typic. Typic is designed to run in a command line and a graphical interface to help selecting a list of proteotypic peptides for targeted quantitation. The tool combines the input data and downloads additional data from public repositories to produce a file per protein as output. Each output file includes relevant information to the selection of proteotypic peptides organized in a table, a colored ranking of peptides according to their potential value as targets for quantitation and auxiliary plots to assist users in the task of proteotypic peptides selection. Taken together, Typic leads to a practical and straightforward data extraction from multiple data sets, allowing the identification of most suitable proteotypic peptides based on established criteria, in an unbiased and standardized manner, ultimately leading to a more robust targeted proteomics assay

Different interactomes for p70‐S6K1 and p54‐S6K2 revealed by proteomic analysis

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70‐S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG‐tagged p70‐S6K1 and p54‐S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high‐scoring interactions. By a comparative approach, p70‐S6K1 interacting proteins were predominantly related to “cytoskeleton” and “stress response,” whereas p54‐S6K2 interactome was more associated to “transcription,” “splicing,” and “ribosome biogenesis.” Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway162026502666FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/13558-7; 2013/16848-9; 2013/22696-7; 2014/01386-

Periplasm-enriched fractions from Xanthomonas citri subsp. citri type A and X. fuscans subsp. aurantifolii type B present distinct proteomic profiles under in vitro pathogenicity induction.

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase β subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

<div><p>Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.</p></div

Treatment with chondroitinase ABC decreased SCC-9 LN-1 cell adhesion to extracellular matrix, but not SCC-9 LN-1 cell migration and increased sensibility of SCC-9 LN-1, SCC-9 and A431 cells to cisplatin.

<p>(A) SCC-9 LN-1 had a lower ability to adhere to extracellular matrix proteins (Matrigel) after treatment with 0.1 U/ml of chondroitinase for 4 h/37°C in serum free media (n = 3, triplicate, Student’s <i>t</i>-test, * indicates p<0.05). (B) SCC-9 LN-1 was treated with 0.1 U/ml for 4 h/37°C in serum free media were seeded in the upper chamber of 96-well transwell plates (n = 3, triplicate). RPMI media, which was supplemented with 1% FBS, was added in the lower chamber. (C) SCC-9 LN-1, SCC-9 and A431 cells treated with increasing concentrations of cisplatin (0–100 µM) for 48 h in the presence of 0.1 U/ml of chondroitinase showed increased sensibility to cisplatin, calculated by a non-linear regression of a dose-response curves (log[µM cisplatin] vs normalized response). Data are expressed as means ± SD from one independent experiment.</p

The role of agrin and perlecan in cell viability.

<p>The viability of SCC-9 (n = 2, A) and SCC-9 LN-1 (n = 3, B) was significantly reduced after siRNA-knockdown of agrin, but no difference in viability was observed in perlecan knockdown. The viability of A431 was not altered neither by agrin knockdown nor by perlecan knockdown (n = 3, C) (One-way ANOVA followed by Tukey’s test, different letters indicate statistically difference at p<0.05).</p