Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation

Abstract

We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.

Keywords: Migration; Notch signaling; Osteosarcoma; Proliferation.