Co-overexpression of Met and hepatocyte growth factor promotes systemic metastasis in NCI-H460 non-small cell lung carcinoma cells

Abstract

Complete resection of early-stage non-small cell lung cancer (NSCLC) is potentially curative, yet approximately 50% of patients are at risk for developing metastatic recurrence. Met, the receptor for hepatocyte growth factor (HGF) is a receptor tyrosine kinase with demonstrated roles in regulating cellular proliferation, motility, morphogenesis, and apoptosis. Met receptor and its ligand, HGF, are commonly overexpressed in NSCLC, and their overexpression has been associated with poor prognosis, which could potentially involve a paracrine and/or autocrine activation loop. However, there is as yet no direct evidence that HGF-Met signaling directly promotes metastasis in NSCLC cells. Using retroviral transduction, we overexpressed the human c-met and hgf complementary DNA, alone or in combination in the NCI-H460 human large cell carcinoma cell line. The HGF/Met co-overexpressing (H460-HGF/Met) cells demonstrated enhanced tumorigenicity in xenograft SCID mice. When these cells are implanted orthotopically into the lungs of nude rats, only the H460-HGF/Met cells showed higher spontaneous metastases to distant organs including bone, brain, and kidney. These results provide evidence that autocrine overactivation of the Met- HGF loop enhances systemic metastases in NSCLC. Targeted interference of this loop may potentially be an effective adjuvant therapy to improve survival of early-stage NSCLC patients.

Figure 1

Establishment of stable HGF/Met expressing H460 cell lines. (A) The messenger RNA (mRNA) expression of human hgf and c-met in HGF-, Met-, and Met/HGF-overexpressing H460 cell lines were evaluated using RT-qPCR (data were expressed as relative level of mRNA expression compared with pBMN control). (B and C) Met and HGF H460 cell lines with or without HGF stimulation (20 ng/ml) were analyzed by Western blot for the activated receptor using phospho-Met-specific antibody (pMet—top panel) and the total Met receptor levels using human Met antibody (hMet—bottom panel). (C) dnMet-H460 cell line was used as a negative control. The accuracy of dnMet-H460 cell line was measured using antibody specific for mouse c-Met. No phosphorylation of c-Met was observed in dnMet-H460 cell line both in the absence and in the presence of HGF.

Figure 2

HGF/Met-overexpressing H460 tumor growth rate in vivo. (A and B) Experiment conducted in duplicate, and data are represented as mean ± SEM (n = 8). (C) Primary tumor weight.

Figure 3

Expression of c-Met and hHGF in H460 tumors. (A and B) mRNA expression of human hgf and c-met was verified using RT-qPCR. (C) HGF and Met overexpression was detected in H460 tumors using Western blot for hHGF and c-Met and the activated Met receptor. H460-dnMet was used as a negative control. (D) Histogram shows the ratio between the two bands (phosphoprotein and total protein) relative to pBMN control. The intensity of the bands was measured using the microcomputer imaging device (MCID) program.

Figure 4

Constitutive activation of Met and HGF induced angiogenesis in HGF/Met-overexpressing H460 xenograft tumors. (A) Immunohistochemistry of xenografts formed by H460 cell lines without (pBMN) or with HGF and/or Met overexpression. (B) Number of mCD31-positive blood vessels in five fields of most vascular areas at 200x magnification. (C and D) Concentration of HGF and VEGF, respectively, in protein extracts of xenograft tumors by ELISA. Magnifications: x100 for HGF; x200 for h-Met and pMet. Asterisks (*) indicate statistical significance between groups.

Figure 5

Metastatic potential of H460 cells implanted orthotopically is dependent on both HGF and Met expression. Immunohistochemistry evaluation of HGF and Met in lymph nodes of representative tissues from HGF/Met overexpressing H460 cell line clone 4–9 and control H460 cell line harboring empty vector (H460-pBMN).