Activation of hepatocyte growth factor-met autocrine loop enhances tumorigenicity in a human lung adenocarcinoma cell line

Abstract

Hepatocyte growth factor (HGF) is a multifunctional cytokine with effects on the proliferation, motility, and differentiation of cells that express its receptor Met. The co-expression of HGF and Met is common among nonsmall-cell lung cancers, especially adenocarcinoma. However, the biologic consequences of this putative HGF-Met autocrine signaling remain speculative. We have used retroviral gene transduction technique to express high levels of HGF in the NCI-H358 lung adenocarcinoma cells that have functionally active cell surface Met receptor. The activation of autocrine HGF-Met signaling was confirmed by the induction of spontaneous cell scattering activity. Compared to the parent and control cells transduced with the retroviral vector alone, HGF overexpressing H358 cells show enhanced capacity to colonize soft agar medium and to form xenograft tumors when implanted in the subcutaneous tissue of immune-deficient mice. These effects were not accompanied by changes in their growth rate in monolayer culture condition, or in the expression of vascular endothelial growth factor. The tumors formed by HGF overexpressing cells also showed more prominent glandular cell arrangement and functional activity. This report provides the direct in vivo evidence that autocrine HGF-Met signaling plays significant roles in the growth and differentiation of human lung adenocarcinoma cells.

Figure 1

Overexpression of HGF in NCI-H358 adenocarcinoma cells. (A) RT-PCR demonstrated high levels of HGF mRNA expression in clones of H358 cells transduced by the pLXSN-HGF retrovirus, but not in the parent or control PL cells transduced with pLXSN virus. The MGH-7 lung squamous cell carcinoma cell line that naturally expresses low levels of HGF was used as a positive control. (B) Western blot analyses of concentrated conditioned media showed the secretion of full length HGF by PSF but not by the parent H358 or PL control cells. The N-17 antibody detected both the 92-kDa unprocessed single chain and 60-kDa processed α-chain of HGF. (C). Northern blot analyses showing c-met and VEGF mRNA expression by parent H358, the control PL and HGF overexpressing PSF clones. Rehybridization with the β-actin cDNA demonstrated a relatively equal loading of RNA. (D) Met protein levels as detected by Western immunoblot with a Met antibody. The HGF/SF overexpressing cells tended to show reduced levels of Met receptor protein that is represented by the 145-kDa β-chain polypeptide.

Figure 2

The activation of autocrine HGF-Met signaling in HGF overexpressing H358-PSF cells. The phase contrast appearances of PL2 (A) and PSF24 (B) cells showed the presence of spontaneous scattering activity in the PSF cells only. Scattering activity was also quantified using the wound migration assay. The PL2 cells (C) demonstrated very sparse spontaneous movement into the “wound” area. In contrast, the PSF24 cells (D) showed a brisk cellular migration.

Figure 3

The effect of HGF overexpression on the proliferation of NCI-H358 cells in monolayer culture. (A) Control parent H358 and pL clones. (B) HGF overexpressing PSF clones. Data are represented by the means±S.E.M. of triplicate experiments.

Figure 4

The xenograft tumors formed by the HGF overexpressing H358 cells. (A) High levels of HGF protein was found in extracts of the tumors formed by the PSF cell lines but not in the tumor formed by the parent H358 cells. (B) The H358 tumor was a poorly differentiated adenocarcinoma with focal formation of glands with small lumens. (C) Focal area of tumour formed by HGF overexpressing H358 cells showing papillary and prominent alveolar gland structures. (D) HGF overexpressing tumors also consistently formed cysts and showed many cells with cytoplasmic vacuoles containing PAS positive and diastase resistant material (arrow). (B and C: H and E, x250; D: PASD, x400).