Abstract
Background: Hepatocyte growth factor (HGF) was initially discovered as a mitogen for hepatocytes, but it is also known to be related to carcinogenesis in many other organs. However, the role of HGF in lung carcinogenesis is not fully-understood. In this study, we investigated the role of HGF in lung carcinogenesis using HGF transgenic mice. Materials and Methods: To elucidate the role of HGF in lung carcinogenesis, 5 μg/g body weight diethylnitrosamine (DEN) were administered intraperitoneally to HGF transgenic (TG) mice and wild-type (WT) mice at 15 days of age. The incidence and number of lung tumors, the expression of HGF and of its receptor (c-Met) were compared between HGF TG and WT mice. Results: HGF overexpression accelerated DEN-induced lung carcinogenesis. Seventy-six percent of TG mice (versus 50% of WT mice) developed malignant lung tumors by 48 weeks. The incidence of lung tumors was significantly higher in the TG mice in comparison with WT mice (p<0.05). Furthermore, the mean diameter and number of tumors in each mouse were significantly higher in the TG mice compared to the WT mice (p<0.01). The northern blotting analyses revealed that there was overexpression of the HGF transgene in the lung tumors of TG mice in comparison with the surrounding non-tumorous lesions. The western blotting analyses of the tumor lesions revealed increased phosphorylation of c-Met. Conclusion: Our results suggest that HGF promotes lung carcinogenesis through the autocrine activation of the HGF/c-Met signaling pathway. The HGF/c-Met signaling pathway appears to have vital roles in lung carcinogenesis.
Hepatocyte growth factor (HGF) is a polypeptide originally characterized as a highly potent hepatocyte mitogen (1, 2). HGF is a multifunctional cytokine and can elicit mitogenic, motogenic and morphogenic responses in cells expressing the transmembrane tyrosine kinase receptor, c-Met (3, 4). Robust expression of the c-Met proto-oncogene has been documented in diverse human and mouse tumors, including hepatocellular carcinoma (5, 6) and lung cancer (7, 8). The activation of HGF/c-Met signaling appears to be intimately associated with neoplastic transformation.
We previously reported that transgenic mice harboring full-length mouse HGF cDNA under the control of the mouse metallothionein (MT) gene promoter, developed liver tumors (9). Furthermore, we revealed that HGF overexpression accelerated diethylnitrosamine (DEN)-induced hepatocarcinogenesis using HGF transgenic mice (10). HGF promotes hepatocarcinogenesis through the autocrine activation of the HGF/c-Met signaling pathway, in association with the stimulation of angiogenesis, either by HGF itself and/or indirectly through the vascular endothelial growth factor (VEGF) (10).
Lung cancer is currently the leading cause of cancer mortality in Japan (11). HGF is expressed not only in the liver, but also in lung (12, 13). It has been reported that increased HGF, and c-Met expression is associated with a high tumor stage and poor prognosis in lung cancer (14, 15). Furthermore, HGF is related to the acquisition of drug resistance for targeted anticancer drugs used to treat lung cancer, such as gefitinib (16). Recently, it was revealed that HGF induces gefitinib resistance in lung adenocarcinoma cells by restoring the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via the phosphorylation of c-Met (16). High levels of HGF were also shown to be correlated with a poor prognosis of non-small cell lung cancer (17). As a result, the HGF/c-MET signaling pathway is highly-involved in lung carcinogenesis.
On the other hand, HGF prevents pulmonary injuries associated with acute respiratory distress syndrome via enhanced induction of heme oxygenase-1 in lung macrophages (18). Furthermore, HGF is reported to reduce bleomycin-induced lung injury and fibrosis (19, 20). The HGF/c-MET signaling network is complex, and a better understanding of HGF/c-MET signaling would lead to more effective targeting of this pathway for cancer therapy.
Stabile et al. (21) generated transgenic (TG) mice that overexpress HGF in the airway epithelium, and showed that lung carcinogenesis induced by a tobacco carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was enhanced by HGF overexpression. In this study, we examined lung carcinogenesis by HGF using a different strain of HGF TG mice and a different carcinogen, diethylnitrosamine (DEN), to induce lung carcinogenesis in metallothionein-HGF TG mice, and we herein provide a characterization of these lung tumors at the molecular level.
Materials and Methods
Animals. Metallothionein-HGF TG mice were generated on an albino FVB genetic background, as described previously (22). The transgenic and control mice used in this study were typically produced from the mating of HGF heterozygote TG males with FVB females (22). All mouse work was performed in accordance with the guidelines for animal care and use established by Gunma University School of Medicine.
Carcinogen-induced non-small cell lung tumors. DEN (Sigma Chemical Co., St. Louis, MO, USA) was injected intraperitoneally into 15-day-old mice at a dose of 5 μg/g of body weight. At 21 days of age, the male mice were separated and their genotype was determined. The total duration of the study was 48 weeks, with interim sacrifices at 16, 24, 32 and 40 weeks (n=4-8 in each time point, total number of mice: WT 36 mice; TG 33 mice). Mice found to be moribund or dead were also examined for tumor development. Tissues were fixed in either 10% buffered-formalin or 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for a histopathological analysis. A portion of each tissue or tumor was snap frozen in liquid nitrogen and stored at −80°C for a molecular analysis.
Analysis of RNA transcripts. The HGF and c-Met transcripts were detected by northern blot hybridization. The mouse HGF cDNA probe and mouse c-Met cDNA probe were synthesized by polymerase chain reaction, as described previously (22). The total RNA was isolated using Isogen (Wako Pure Chemical Industries, Osaka, Japan), and 20 μg of RNA were loaded per lane onto 1% agarose/formaldehyde gels and transferred to nylon membranes after electrophoresis.
Analysis of c-Met and c-Met activation. The quantification of c-Met and c-Met tyrosine phosphorylation was performed as described previously (23). For immunoprecipitation, 500 μg of lysate were incubated with an antibody to c-Met (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h on ice. After the addition of Gamma-Bind G Sepharose (Boehringer Mannheim, Mannheim, Germany) and washing in radioimmunoprecipitation assay (RIPA) buffer, the immunoprecipitates were fractionated on 10% polyacrylamide gels. After electrophoretic transfer to nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA, USA), the filters were blocked and then incubated with an antibody to c-Met or phosphotyrosine (Upstate Biotechnology, Lake Placid, NY, USA) overnight. c-Met was visualized by incubation with anti-goat antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) by using enhanced chemiluminescence reagents. Phosphotyrosine was visualized as above, except that an anti-mouse antibody conjugated to horseradish peroxidase was used.
Data analysis. All data are expressed as the mean−S.D. Differences in tumor incidence were examined for statistical significance using the Fischer's exact probability test. The distributions of continuous variables were analyzed by the Mann–Whitney U-test with Bonferroni's correction, when a significant difference was obtained by the Kruskal-Wallis analysis. A p-value less than 0.05 denoted the presence of a statistically significant difference.
Results
Chemical induction of lung tumors in HGF TG mice. A significant difference in the development of lung tumors was observed between HGF TG mice and WT mice treated with DEN (Table I). Seventy-six percent (25/33) of HGF TG mice developed grossly visible lung tumors, although only 50% (28/36) of WT mice had such tumors (p<0.05). Although all HGF TG and WT mice developed lung tumors by 48 weeks of age, the development of lung tumors was observed earlier in the HGF TG mice in comparison with the WT mice. The lung tumors in HGF TG mice were also larger in size (Table II) and greater in number (Table III) compared to those arising in WT mice (p<0.01). The typical macroscopic appearance of lung tumors is shown in Figure 1. Lungs from DEN-treated TG mice contained larger tumors than did those from WT mice at 32 weeks. The typical microscopic appearance of the lung tumors at 24 weeks is shown in Figure 2A. At 48 weeks of age, the tumors were developed into adenocarcinomas in the HGF TG mice (Figure 2B).
Analysis of transcripts of HGF and its receptor (c-Met) in the lungs of DEN-treated TG and WT mice. Our previous data showed that HGF transgene expression was not elevated by DEN administration (10). To determine if lung carcinogenesis in HGF TG mice consistently correlated with a further enhancement in HGF transgene expression, HGF expression was analyzed in tumors and adjacent non-tumorous tissues of TG mice. The northern blot analyses showed that the HGF transgene transcript levels of the tumors were higher than those found in adjacent non-tumorous tissues (Figure 3). Endogenous c-Met transcripts were detected, but no apparent differences were noted between tumors and adjacent lung tissues (Figure 3).
Analysis of c-Met protein level and activity. To quantify the total expression level and phosphorylation of the c-Met protein, extracts of lung tumors were subjected to immunoprecipitation using an antibody against c-Met, followed by a western blot analysis using an antibody against either c-Met or phosphotyrosine. Figure 4 shows that the levels of the c-Met protein were generally in agreement with the c-Met transcript data. c-Met tyrosine phosphorylation was then examined in extracts obtained from transgenic tumors and adjacent non-tumorous tissue. c-Met tyrosine phosphorylation was enhanced in the tumors, thus suggesting that the activation of c-Met is related to lung carcinogenesis.
Discussion
In the present study, we demonstrated that HGF overexpression in vivo appears to promote lung carcinogenesis in TG mice when initiated with DEN, a well-characterized genetic mutagen. We also demonstrated that lung carcinogenesis in HGF TG mice was driven by the creation of HGF/c-Met autocrine loop, as evidenced by the elevation of both HGF expression and c-Met phosphorylation in tumors relative to that of the adjacent tissues. The acquisition of autonomous growth has been strongly implicated in the development and progression of lung cancer. Several growth factor signals, including transforming growth factor-α (TGF-α)/ epidermal growth factor receptor (EGFR), HGF/c-Met, and VEGF/VEGF receptor, are reported to participate in this autonomous growth (24-26).
A multitude of human cell lines and tumors, particularly sarcomas, overexpress both c-Met and its ligand (29, 30). In fact, diverse tumors developed in our HGF TG mice, including melanoma and rhabdomyosarcoma, demonstrating the formation of HGF/c-Met autocrine loops as a result of the forced expression of the transgene and overexpression of the endogenous receptor (29). In the lung tumors of this model, c-Met kinase activity was enhanced in the tumorous tissue, but not in adjacent tissues, in accordance with HGF transgene expression. These results suggest that lung carcinogenesis requires the selection of cells capable of expressing the HGF transgene in a strong, constitutive fashion, providing a mechanism for autonomous cellular proliferation through autocrine signal transduction. The anti-apoptotic effects of HGF may facilitate lung carcinogenesis, as it was documented that HGF blocked massive Fas-mediated apoptosis (30). HGF/c-Met signaling may, therefore, encourage the survival of initiated cells that would otherwise succumb to mutation-induced death.
Stabile et al. (21) reported that HGF overexpression in airway epithelium enhanced the lung carcinogenesis induced by NKK, a tobacco carcinogen. In their model, the main difference in tumor formation between TG and WT mice was an increase in tumor multiplicity. Tumors arising after NNK exposure expressed c-Met, and therefore had the capacity to respond to HGF (21). These results suggest that lung tumors induced by a tobacco carcinogen are promoted by the expression of the HGF transgene, possibly through both the increased proliferation of individually-mutated cells and by the migration of tumor cells to other areas of the lung where additional tumors form. In our model, the tumor multiplicity also increased in HGF TG mice treated by DEN.
Increased HGF, and c-Met expression by human tumor cells has been associated with a high tumor stage and poor prognosis in lung cancer (14, 15). The strong correlation between HGF and c-Met expression and patients' survival clearly supports the hypothesis that the HGF/c-Met pathway plays an important role in the pathogenesis of human lung cancer. It was recently revealed that HGF induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations (16). Because the HGF/c-Met pathway is involved in the pathogenesis of human lung cancer, its pathway may represent a good therapeutic target for lung cancer. Our animal model of lung carcinogenesis can also be used to test such inhibitors of the HGF/c-Met pathway.
We herein presented data demonstrating that environmental carcinogens can synergize with, and dramatically accelerate, HGF-mediated carcinogenesis in the lung, further supporting the oncogenic risk associated with persistent in vivo exposure to high levels of HGF.
- Received December 18, 2012.
- Revision received February 3, 2013.
- Accepted February 4, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved