Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway

• Published in: Carcinogenesis (New York) Vol. 35; no. 2; pp. 324 - 332
• Main Authors: Radziwon-Balicka, Aneta, Santos-Martinez, Maria J, Corbalan, J Jose, O'Sullivan, Shane, Treumann, Achim, Gilmer, John F, Radomski, Marek W, Medina, Carlos
• Format: Journal Article
• Language: English
• Published: England 01.02.2014
• Subjects: Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - genetics; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - metabolism; Blood Platelets - metabolism; Blotting, Western; Caco-2 Cells; Cell Movement; Cell Proliferation; Chromatography, Liquid; Clusterin - genetics; Clusterin - metabolism; Colonic Neoplasms - genetics; Colonic Neoplasms - metabolism; Colonic Neoplasms - pathology; Flow Cytometry; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Matrix Metalloproteinase 9 - genetics; Matrix Metalloproteinase 9 - metabolism; Neoplasm Invasiveness; p38 Mitogen-Activated Protein Kinases - genetics; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphorylation; Proteomics; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Signal Transduction; Tandem Mass Spectrometry
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/bgt332

Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.