RESUMO
Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions.
Assuntos
Carcinoma de Células Escamosas/irrigação sanguínea , Colágeno/fisiologia , Neoplasias Bucais/irrigação sanguínea , Idoso , Proteínas Angiogênicas/fisiologia , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/sangue , Neoplasias Bucais/genética , Neovascularização Patológica/metabolismo , Estrutura Terciária de Proteína , Regulação para Cima , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Collagen XVI belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). It is overexpressed during the progression of oral squamous cell carcinoma (OSCC). The present data show a strong collagen XVI-dependent induction of MMP9 and an increase in OSCC cell invasion. We found activated integrin-linked kinase (ILK) in a complex with kindlin-1 and activation of protein kinase B (PKB/Akt) to be responsible for MMP9 induction. Inhibition of the formation of focal adhesions reduced MMP9 expression. Moreover, collagen XVI overexpressing OSCC cell clones (COLXVI cell clones) transfected with vectors containing different MMP9 promoter fragments adjacent to a luciferase reporter revealed an increase in luciferase signal dependent on AP-1 binding sites. Deletion of the AP-1 binding site 98 bp upstream of the reported transcription start site and inhibition of AP-1 with Tanshinone IIA resulted in decreased MMP9 expression. The AP-1 subunit JunB showed differential expression between COLXVI cell clones and mock control cells. Additionally, mass spectrometric analysis of immunoprecipitates revealed that c-Fos interacted strongly with dyskerin in COLXVI cell clones compared to mock controls.