Integrated proteomics identified up-regulated focal adhesion-mediated proteins in human squamous cell carcinoma in an orthotopic murine model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by co95e98208FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/54067-3; 2010/19278-0; 2011/22421-2; 2009/53839-2; 2011/02267-9470567/2009-0; 470549/2011-4; 301702/2011-0; 470268/2013-1; 505413/2013-

Novel processed form of syndecan-1 shed from SCC-9 cells plays a role in cell migration.

The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines

Loss of membrane-bound syndecan-1 localization after PMA treatment in SCC-9 cells.

<p>SCC-9 cells were treated with PMA for 5 min, 30 min and 24 h, fixed, and labeled for syndecan-1 with goat anti-syndecan-1 antibody. (A) Immunofluorescence images revealed diminished syndecan-1 in membrane localization after 30 min and 24 h of PMA treatment. Green: anti-goat antibody conjugated with Alexa Fluor 488. Blue: DAPI. (B) Example of masks used for quantification of syndecan-1 in the cell surface membrane. (C) Quantification performed by the Operetta high content image system showed loss of cell membrane of syndecan-1 after 30 min and 24 h of PMA treatment. Three independent experiments were performed. Columns represent mean ± SD (n = 3) and * and ** indicate p<0.05 and p<0.01, respectively.</p

Synthetic syndecan-1-derived peptide promoted migration in SCC-9 cells by transwell migration assay.

<p>The migration of HaCaT and SCC-9 cells treated with synthetic syndecan-1-derived peptide (SYN-1) and its scrambled in a concentration of 10 µM in the absence of FBS was evaluated by transwell assay. The measurements were normalized with the control (scrambled peptide). SYN-1 did not induce migration in HaCaT cells (A). SCC-9 cell migration was statistically significant when compared to scrambled peptide (B). Three experiments were performed with triplicates. Columns represent mean ± SD (n = 3) and * indicate p<0.05.</p

Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value (normalized spectral counts), as indicated in <b>Table 1</b>.

<p>Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value (normalized spectral counts), as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043521#pone-0043521-t001" target="_blank"><b>Table 1</b></a>.</p

Synthetic syndecan-1-derived peptide promoted migration in HaCaT and SCC-9 cells by scratch assay.

<p>Migration of HaCaT and SCC-9 cells treated with synthetic syndecan-1 peptide (SYN-1) and its scrambled peptide (control) in the concentrations of 1 µM, 10 µM and 100 µM were evaluated by scratch assay. SYN-1 did not induce migration in HaCaT cells at 24 h (A), but 10 µM of SYN-1 promoted migration at 48 h in absence of FBS (B), 1 µM of SYN-1 induced migration in HaCaT cells at 24 h (C) and 48 h (D), both in the presence of 1% FBS. SCC-9 cell migration was statistically significant at the concentration of 1 µM of SYN-1 in the absence of FBS at 24 h (E) and 48 h (F), but it was not statistically significant when compared to the scrambled peptide in the presence of 1% FBS at 24 h (G) and 48 h (H). Three independent experiments were performed with duplicates. Columns represent mean ± SD (n = 3) and * and ** indicate p<0.05 and p<0.01, respectively.</p

Identification of proteins in the conditioned media by LC-MS/MS according to the ratio of quantitative value.

<p>The HaCaT and SCC-9 cells were treated with PMA for 24 h, the media were collected, the proteins were digested with trypsin and analyzed by LC-MS/MS. The data were submitted to Mascot search engine and the .dat files from Mascot output were analyzed in Scaffold Q+, which calculates the quantitative value by normalizing spectral counts across the experiments. The ratio of quantitative value of the PMA-stimulated cells/DMSO-treated cells for each protein is shown in the table. The proteins exclusively found in one condition are indicated as “only” and the proteins that are not present are indicated as “absent”.</p

Identification of endogenous peptides in the conditioned media by LC-MS/MS according to the total number of unique peptides and spectral counts.

<p>The HaCaT and SCC-9 cells were treated with PMA for 24 h, the media were collected and the endogenous peptides were analyzed by LC-MS/MS. The total numbers of the unique peptides and the number of spectral counts are shown in bold and the number of spectral counts is shown for each sequence. There is a statistically significant difference in the number of spectral counts of syndecan-1 fragments between SCC-9 cells and SCC-9 cells treated with PMA (Mann-Whitney test, p = 0.0273).</p

CID spectrum of a syndecan-1 peptide identified by LC-MS/MS.

<p>Endogenous peptides were identified by LC-MS/MS in the media after 24 h of PMA-treatment. The spectrum of syndecan-1 peptide (m/z 1019.5598, +2) was manually validated for b and y ion series.</p