Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation

Abstract

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

Keywords: HAI-2; HGF; MET; RCC; bone metastasis.

Figure 1

RT-qPCR analyses of MET, a c-met proto-oncogene product and receptor for hepatocyte growth factor (HGF), HGF activator inhibitor-1 (HAI-1), and HAI-2 and matriptase in six renal cell carcinoma cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the internal control.

Figure 2

Bioluminescent imaging of the mouse model. After intra-cardiac injection (A), six weeks from injection (B). Apparent bone metastasis is confirmed (B), red circle. Extracted specimens were used for RT-qPCR analyses. mRNA expression of MET, HAI-1, HAI-2, HGF and matriptase in bone metastasis and subcutaneous implantation of 786-O-Luc2 cells (C).

Figure 2

Bioluminescent imaging of the mouse model. After intra-cardiac injection (A), six weeks from injection (B). Apparent bone metastasis is confirmed (B), red circle. Extracted specimens were used for RT-qPCR analyses. mRNA expression of MET, HAI-1, HAI-2, HGF and matriptase in bone metastasis and subcutaneous implantation of 786-O-Luc2 cells (C).

Figure 3

Pathological and immunohisitochemical appearance of bone metastasis and subcutaneous implantation of 786-O-Luc2 cells using serial tissue sections. (A) Bone metastasis (bone), stained with hematoxylin and eosin (H&E); (B) subcutaneous implantation (SC), stained with H&E; (C) bone, phosphorylation of MET (p-MET) immunostaining; (D) SC, p-MET immunostaining; (E) bone, total-MET (t-MET) immunostaining; (F) SC, t-MET immunostaining. Scale bars = 100 μm.

Figure 4

(A) Expression of HAI-2 was confirmed by RT-qPCR analyses in HAI-2-knockdown 786-O-Luc2 (786-O-L2898) with and without 0.5 μg/mL of doxycycline (DOC). Effect of HAI-2-knockdown on expression of MET, HAI-1 and matriptase was also verified; (B) Expression of HAI-2, MET, HAI-1 and matriptase were confirmed in HAI-2-engineered expressed 786-O-Luc2 (786-O-L2853) cells with and without 0.5 μg/mL of doxycycline.

Figure 5

Effect of HAI-2 knockdown or engineered expression on motility and invasiveness of 786-O cells. Cell motility was evaluated by wound healing assay (A), and invasion assay (B) through Matigel with and without endogenous pro-HGF. Values are means ± standard deviation of triplicate experiments. * p < 0.05, Mann–Whitney U test.