Phosphoproteome analysis reveals differences in phosphosite profiles between tumorigenic and non-tumorigenic epithelial cells.

UNLABELLED: Oral cancer disease represents a significant fraction of all human cancer types and its poor early diagnosis contributes to reduced individual survival rate. The identification of proteins modulated in tumorigenic cells and its post-translational modifications may improve our understanding of tumor development in epithelial cells. We have analyzed the phosphoproteome of tumorigenic (SCC-9) and non-tumorigenic (HaCaT) cell lines using MS-based approach in order to identify phosphopeptides with differing patterns of modifications and/or abundance. Our results revealed the identity of 4,206 protein phosphorylation sites with sixty-two sites showing to be significantly modulated between the two cell lines. The phosphoproteome data showed an overrepresentation of proteins with a possible role in nuclear regulatory functions. Pathway analysis was further performed on the phosphoproteome dataset and differences and commonalities of the functional pathways present in tumorigenic and non-tumorigenic cells were identified. Phosphopeptides that belong to the proteins lamina-associated polypeptide 2 isoform alpha and serine-arginine repetitive matrix protein 2 were identified with differential abundance and they appear as promising tumor-related phosphopeptides. These two proteins may be related to the structural alterations generally found in the nucleus of tumorigenic cells. The identification of phosphorylation sites in tumorigenic cells may contribute to disclose novel signaling mechanisms associated with OSCC. SIGNIFICANCE: Oral Squamous Cell Carcinoma (OSCC) is an important cancer disease affecting thousands of people worldwide. Many cellular processes related to the development of oral cancer remain unknown; however, the studies performed in vitro with cancer cells have contributed to guide more specific research which may be further performed by using in vivo approaches or clinical samples. To our knowledge, only few studies have been published showing the results of phosphoproteome profiling of squamous cell carcinoma models, and many signaling proteins must be identified and functionally characterized in order to increase the knowledge available about the complexity of the signaling networks responsible for oral cancer development and its progression. Furthermore, our knowledge regarding proteins exclusive or very low abundant in cancer cells remains limited. A better understanding of the differences between signaling pathways present in epithelial cell lines may contribute to reveal the processes underlying the OSCC.

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.