Disruption of IGF-1R signaling by a novel quinazoline derivative, HMJ-30, inhibits invasiveness and reverses epithelial-mesenchymal transition in osteosarcoma U-2 OS cells

Abstract

Osteosarcoma is the most common primary malignancy of the bone and is characterized by local invasion and distant metastasis. Over the past 20 years, long-term outcomes have reached a plateau even with aggressive therapy. Overexpression of insulin-like growth factor 1 receptor (IGF-1R) is associated with tumor proliferation, invasion and migration in osteosarcoma. In the present study, our group developed a novel quinazoline derivative, 6-fluoro-2-(3-fluorophenyl)- 4-(cyanoanilino)quinazoline (HMJ-30), in order to disrupt IGF-1R signaling and tumor invasiveness in osteosarcoma U-2 OS cells. Molecular modeling, immune-precipitation, western blotting and phosphorylated protein kinase sandwich ELISA assays were used to confirm this hypothesis. The results demonstrated that HMJ-30 selectively targeted the ATP-binding site of IGF-1R and inhibited its downstream phosphoinositide 3-kinase/protein kinase B, Ras/mitogenactivated protein kinase, and I?K/nuclear factor-κB signaling pathways in U-2 OS cells. HMJ-30 inhibited U-2 OS cell invasion and migration and downregulated protein levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9. An increase in protein levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was also observed. Furthermore, HMJ-30 caused U-2 OS cells to aggregate and form tight clusters, and these cells were flattened, less elongated and displayed cobblestone-like shapes. There was an increase in epithelial markers and a decrease in mesenchymal markers, indicating that the cells underwent the reverse epithelial-mesenchymal transition (EMT) process. Overall, these results demonstrated the potential molecular mechanisms underlying the effects of HMJ-30 on invasiveness and EMT in U-2 OS cells, suggesting that this compound deserves further investigation as a potential anti-osteosarcoma drug.