Mechanism Researches Of Inhibiting EGFR Signaling Pathway To Treat Giant Cell Tumor Of Bone

Background Giant cell tumor of bone(GCTB) is generally benign but an aggressive, expansile, osteolytic bone tumor, and accounts for approximately 5% of all primary bone tumors. GCTB is composed of three cell types: osteoclast like multinucleated giant cells(MGCs), mononucleated spindle-like stromal cells(SCs), and round-shaped monocyte/macrophage origin mononuclear cells. Its etiology is still unknown to date and the biological behavior is unpredictable. SCs that attract and activate monocyte/macrophage mononuclear cells to differentiate into osteoclast-like MGCs are considered as the neoplastic component of the tumor. At present stage, the preferred treatment of primary GCTB is definitely surgical resection, include intralesional, segmental, and en-bloc resection. Although complete resection combines with adjuvant treatment could significantly reduce the local recurrences, the rates of 27% to 65% is still not desirable. However, adjuvant treatment options are limited when the tumors are unresectable due to multiple lesions, distant metastases, functional requirements, or inaccessible anatomical location. Nowadays, no effective chemotherapy has been explored, the pharmacotherapy including Bisphosphonates and Denosumab only aimed at inhibiting bone resorption of MGCs. Both of them have showed little benefit on suppressing the proliferation or apoptosis of SCs, so the local recurrence is difficult to avoid completely. Moreover, GCTB is usually rich vascularized, about 10-14% of GCTB accompany with secondary aneurysmal bone cysts(ABCs) formation. Therefore, to exploring a new treatment that can either inhibit the osteoclastogenesis of MGCs and the proliferation of SCs, or suppress the angiogenesis, might be a significant improvement on curing GCTB.EGFR is a transmembrane glycoprotein containing an extracellular ligand binding domain and an intracellular receptor tyrosine kinase domain. It belongs to the human epidermal growth factor receptor(HER)-erb B family. The EGFR can be specially activated through the binding with ligands belonging to the EGF family of peptide growth factors. Subsequently, the downstream signaling pathways of EGFR such as: Ras-MAPK, PI3K-AKT-m TO, Src-JAK-STATs, PLC?-PKC are activated via phosphorylation to regulate the cell migration, proliferation, apoptosis and cycle. Dysregulation of EGFR signaling pathway by overexpression, mutations, or excessive activation can promote tumor processes including angiogenesis and metastasis. Peter Balla and Linda Moskovszky, et al, had revealed that EGFR signaling pathway contributes to the regulation of the proliferation of mononucleated spindle-like SCs, osteoclastogenesis and disease progression in GCTB. This does suggests that EGFR could be the more precise and effective therapeutic target for GCTB.Objective(1) We compared the expression of key signaling components between GCTB cells and bone marrow mesenchymal stem cells(BMSCs),tumor specimens and normal cancellous bone to evaluate the activation of the EGFR signaling pathway in GCTB.(2) We next identified whether inhibition of the EGFR pathway restrained the formation and differentiation of osteoclast-like multinucleated giant cells.(3) We ascertained Whether targeted blocking EGFR signal pathway could inhibit the proliferation of stromal cells or induce the apoptosis.(4) To identify whether blocking EGFR signaling could inhibit the osteoclastogenesis of BMMs, how SCs regulated the differentiation of osteoclast and at which stage EGFR signaling regulated osteoclastogenesis.(5) We examined whether SCs induced migration of human umbilical vein endothelial cells(HUVECs), and determined whether blockade of the EGFR signaling had any effect on HUVECs proliferation, migration and angiogenesis.(6) We explored the in vivo therapeutic effect of inhibiting EGFR signaling via mouse models and chicken embryo chorioallantoic membrane(CAM) models.Methods(1) We compared the expression of key signaling components between GCTB cells and BMSCs, tumor specimens and normal cancellous bone via Western blotting and immunohistochemistry.(2) We employed in vitro differentiation of osteoclast-like MGCs, TRAP, PCR and pits assays identified whether inhibition of the EGFR pathway restrained the formation and differentiation of osteoclast-like MGCs.(3) Cells growth inhibition assays and detection of apoptosis via flow cytometry were undertaken to explored whether blocking EGFR signal pathway inhibits the proliferation of SCs or induced apoptosis.(4) We stimulated BMMs with M-CSF, RANKL or conditioned medium of GCTB as an in vitro osteoclastogenesis model, to identify how SCs regulated osteoclast maturation and at which stage EGFR signaling regulated osteoclastogenesis. PCR assays were applied to explore the biological mechanisms of osteoclastogenesis which was induced by SCs.(5) We verified whether blocking EGFR signal pathway can inhibit the proliferation, migration and angiogenesis of HUVECs through Cells growth inhibition assays, Western blotting, Transwell assays and in vitro tube formation assays of HUVECs.(6) We created the in vivo mouse models of GCTB by intra-tibial injection. Gefinitib was employed to block EGFR signaling and Bisphosphonate was applied as control group. The therapeutic effect was estimated through imaging examination. Subsequently, we established a CAM model for GCTB and observed the effects of Gefitinib on inhibiting tumorigenesis.Results(1) Significantly elevated activation of EGFR signaling, as evidenced by increased expression of P-EGFR, P-AKT and P-ERK in GCTB cells and samples.(2) The formation and differentiation of MGCs was suppressed in dose-dependent manners. At 5?M of Gefitinib or Afatinib and 10?M Erlotinib, the differentiation was vastly inhibited. With pits assay, the inhibitory effect of bone resorption was found to be associated with the concentration. PCR assays manifested the expression of RANKL decreased while OPG increased along with the rise of drug concentration.(3) EGF-induced proliferation of SCs was significantly inhibited by Gefitinib in dose-dependent manners. Flow cytometrc analysis revealed that blocking EGFR signaling pathway had a weak effect on the apoptosis of SCs.(4) Targeted blocking EGFR signaling inhibits the osteoclastogenesis of BMMs in dose-dependent manners. Conditioned medium of GCTB with low dose RANKL induced osteoclastogenesis of BMM more effectively than RANKL itself. This provided evidence that SCs attract and activate BMMs to differentiate into osteoclasts. Moreover, EGFR signaling modulates osteoclastogenesis only at early stages but not later stages. The expression of NFATc1, TRAP, MMP9, M-CSF, MCP-1 and SDF-1 significantly reduced in dose-dependent manners while EGFR pathway was blocked.(5) Blocking EGFR signaling pathway inhibits the proliferation, migration and SCs-induced angiogenesis of HUVECs in dose-dependent manners.(6) The results of in vivo assay indicated that blocking EGFR signaling could significantly reduced the osteolytic destruction and tumorigenesis.Conclusion In this study, we employed western blotting and immunohistochemistry to confirm the excessive expression of EGFR signaling and the activation of related protein in GCTB. Tyrosine kinase inhibitors(TKIs) were applied to block the EGFR pathway specially. The results demonstrated not only the osteoclastogenesis of MGCs and the proliferation of SCs were suppressed effectively, but also the proliferation, migration and angiogenesis of HUVECs were restrained. Furthermore, the in vivo assay demonstrated that inhibiting EGFR signal pathway was more effectively on preventing the bone destruction and tumorigenesis. These data strongly suggest the EGFR signaling as a promising therapeutic target in GCTBs.