Keywords

7.1 Introduction

Historically, MET was discovered as the Trp-MET oncogene and the MET proto-oncogene, and molecular characterization was performed in 1986 [1]. One year later, a fibroblast-derived protein which caused the scattering of epithelial cells was found and named “scatter factor” (SF) [2]; it was later identified as a ligand of MET. In 1989, human hepatocyte growth factor (HGF) was first cloned [3], and its nucleotide sequence revealed that it was identical to SF and that both α- and β-chains were contained in a single open reading frame. In the middle of the 1990s, the relationship between HGF/SF and Met was clarified by using knockout (KO) mouse models. Using Met KO mice, Met was proved to have an essential role in the migration of myogenic precursor cells into the limb bud and diaphragm [4]. As a result, skeletal muscles of the limb and diaphragm did not form in the KO mice, and they died as embryos. Similarly, embryonic death with placental defects was observed in mice lacking HGF/SF [5], and HGF/SF was found to be essential for the development of important epithelial organs such as the liver [6]. Because the phenotypes are quite similar between Met KO mice and HGF/SF KO mice [7], HGF/SF is considered to be the only ligand for Met, and Met the only receptor for HGF/SF.

HGF/SF-MET signaling also regulates a wide range of cancer cell functions such as proliferation, cell cycle progression, and control of the expression of adhesion molecules that induce extracellular matrix activation, migration, invasion, and neovasculogenesis (Chap. 8).

To clarify the function of MET and its downstream signals, extensive experiments were performed using normal as well as tumor cells. Upon stimulation by HGF/SF, MET is phosphorylated, which initiates downstream signal. The phosphorylation of tyrosine 1234/1235 in the MET kinase domain is crucial to kinase activation. The phosphorylation of tyrosine 1349 and tyrosine 1356 in the C-terminal region provides a direct binding capability to GRB2 (growth factor receptor-bound protein 2) and GAB1 (GRB2-associated binder 1), which transduce subsequent downstream signals [8, 9]. Both the Gab1–Shp2–ERK/MAPK and Grb2–Ras–Raf–ERK/MAPK pathways stimulate cell cycle regulators to induce cell proliferation and cell cycle progression. The activation of extracellular matrix proteinases alters cytoskeletal functions that control migration, invasion, and proliferation. Ras–Rac1/Cdc42–PAK activation disrupts E-cadherin adhesion [10], which affects cell polarity and actin cytoskeleton remodeling and enhances cell motility [11]. Gab1–Crk–C3G–Rap1 activation regulates paxillin, focal adhesion kinase, and integrins, and it loosens cell junctions, which leads to cell migration and invasion [12]. Activation of GAB1–phosphatidylinositol 3-kinase (PI3K)–Akt/PKB pathway controls cell survival through the inhibition of apoptosis-related molecules such as Bad and caspase-9 [13, 14].

Ligand-dependent activation of the MET tyrosine kinase is crucial for downstream signaling that confers proliferation, cell cycle progression, migration, and motility on the cells under physiological settings. The Sema domain, which is the extracellular domain that bears structural similarity to other semaphorin family members, plays a critical role in MET activation by its ligand (see Fig. 8.1) [15,16,17]. Upon binding of HGF/SF with the MET Sema domain, the MET α-chain dimerizes, leading to signaling [18, 19].

Under physiological conditions, HGF/SF is secreted by fibroblasts and binds to heparan sulfate proteoglycans on cell surfaces and within the extracellular matrix [20]. The extracellular matrix often serves as a carrier of exogenous growth factors [21]. In response to inflammatory triggers, the pro-form of HGF/SF is proteolytically cleaved into an active α,β-heterodimer, stimulating the proliferation and migration of epithelial cells through MET activation [22]. It has been reported that sulfated oligosaccharides promote HGF/SF heterodimerization and govern its mitogenic activity [20], because heparin-like molecules stabilize HGF/SF oligomers, thereby facilitating MET receptor dimerization and activation.

In normal cells, the activation of MET following stimulation by HGF/SF is transient. In tumors such as breast cancer and prostate cancer, constitutive MET signaling induces an epithelial–mesenchymal transition (E–MT) (Fig. 7.1), ultimately leading to carcinoma [23].Various mechanisms can induce such MET pathway dysregulation, including ligand-dependent MET activation, MET mutation, MET amplification, and transactivation via other membrane receptors [23]. Viral or bacterial infection can also activate MET as an oncogene. In this chapter, we will mainly discuss the molecular basis of MET oncogenic activation that leads to carcinogenesis.

Fig. 7.1
figure 1

MET signaling and cancer progression. HGF/SF-MET signaling is activated by either an HGF/SF paracrine route, an HGF/SF autocrine route, or a MET overexpression and constitutive activation, which induces an epithelial–mesenchymal transition (E–MT) and gives increased invasiveness to tumor cells

Full size image

7.2 HGF/SF-MET Signaling Dysregulation and Cancer

7.2.1 HGF/SF-Dependent MET Activation

7.2.1.1 Paracrine HGF/SF Production by Stromal Cells

HGF/SF secreted from fibroblasts is also an important paracrine factor which induces tumor invasiveness [24]. Cancer–stroma interactions play an important role in the invasive growth of cancer cells [25]. Cross talk between invasive cancers and host stromal fibroblasts (i.e., cancer-associated fibroblasts) is strongly involved in the promotion of tumorigenesis [26], malignant cell proliferation [27], and invasion into the extracellular matrix [28], which are all enhanced by HGF/SF secretion. Factors upregulating the production of HGF/SF include IL-1, TNF [29], prostaglandins [30], and others [31]. HGF/SF and IL-6 upregulate the expression of each other’s receptor, thereby promoting tumor malignancy [32]. By comparison the tumor expression studies of MET and HGF/SF were conducted with sarcoma tumor lines since MET expression in normal mesenchymal cells and tissues is low or nil, while carcinomas and normal epithelial tissues express abundant MET and HGF/SF expression.

7.2.1.2 HGF/SF-MET Autocrine Loop Activation

An HGF-MET autocrine loop significantly contributes to carcinogenesis. Experimentally, NIH/3T3 cells transformed by overexpression of human MET and HGF/SF were injected either subcutaneously or into the mammary fat pad of weanling athymic nude mice, and tumorigenicity was tested [33, 34]. Explants of tumors showed increased tumorigenicity as compared with primary transfectants. Histopathological examination revealed that these tumors were invasive. In addition, the tumor explants that showed increased levels of both HGF/SF and MET efficiently produced multifocal lung metastasis, suggesting the importance of the HGF/SF-MET autocrine signaling mechanism in tumorigenesis as well as the acquisition of metastatic potential. The most potent experimental HGF/SF and MET signaling has been observed in ligand and receptor which are co-expressed in the same cell and especially malignant when deficient in p53. Also, in a murine mammary carcinoma model, co-expression of HGF/SF and Met was proved to contribute in part to sustained tyrosine phosphorylation of several signaling molecules such as PI3 kinase, Src, focal adhesion kinase, and phospholipase C-γ and to the growth and development of an invasive phenotype [35].

The importance of the HGF/SF-MET autocrine loop has been reported for human cancers such as colon cancer, lung adenocarcinomas, malignant mesotheliomas, and ovarian and breast cancers. Molecular co-expression of HGF/SF and MET in primary colon cancer is considered to predict a metastatic phenotype and correlates well with an advanced stage and poor survival [36]. Xenografts of NCI-H358 human lung adenocarcinoma cells having an active MET receptor showed that the autocrine loop contributed to the prominent glandular cell arrangement, functional activity, and enhanced tumorigenicity [37]. Ovarian surface epithelium from families with a history of ovarian cancer showed much higher MET expression than epithelium from families having no such history, and exogenous HGF/SF was mitogenic in the ovarian surface epithelium and was required in tumorigenic transformation [38]. This suggests that the HGF/SF-Met autocrine loop takes part in an enhanced susceptibility to ovarian carcinogenesis.

In breast cancers, a Src/Stat3-dependent mechanism is involved in regulating HGF/SF promoter activity and is linked to the transformation of mammary epithelial cells by enhancing the HGF/SF-MET autocrine loop [39]. The existence of an autocrine loop might be a useful indicator for predicting the molecular stage of cancer cells. Disrupting this loop by using targeting antibodies or decoy molecules [40, 41], small chemical molecules [42], genetic tools, etc., could be a good therapeutic strategy against cancer.

7.2.2 MET Mutation and Carcinogenesis

MET mutation has been reported to contribute to carcinogenesis or enhanced malignancy in many cancers. The first recognized cases in humans were forms of papillary renal carcinoma (PRC) in which the MET mutation activated intracellular downstream signaling [43]. The introduction of mutant MET molecules into NIH 3T3 cells formed foci in vitro, and such cells injected into nude mice were tumorigenic, which showed that MET mutation was involved in a key step of carcinogenesis. This transforming ability of PRC mutant MET correlated with activation of the Ras pathway [44].

7.2.2.1 MET Point Mutations and Hereditary Cancers

Some point mutations in the MET gene activate the MET tyrosine kinase, which can drive or facilitate the development of cancer. A series of reports have shown the relationship between MET mutation and human cancers (Table 7.1). The most common form of mutation in the MET tyrosine kinase receptor has been identified in both hereditary and sporadic forms of PRCs [43, 45,46,47]. Among those, most mutations of the MET proto-oncogene were found in hereditary PRCs, whereas non-inherited PRCs showed a low frequency of MET mutations [45]. In hereditary PRCs, therefore, MET point mutations seem to be strongly involved in their pathogenesis. Somatic missense mutations in the kinase domain of MET molecule are known to produce childhood hepatocellular carcinomas [48]; based on the early onset of this disease, mutations of that domain of MET might act to accelerate the carcinogenesis.

Table 7.1 MET mutation and cancers in humans
Full size table

MET-activating mutations also confer upon tumor cells invasive and metastatic properties [49, 50], resulting in poor clinical prognosis. Transgenic mice harboring mutationally activated MET developed metastatic mammary carcinomas in which the Ras-Raf-MEK-ERK signaling pathway was activated, enhancing cellular motility [51]. Different mutations near the signal transducer docking site of MET (Y1349 and Y1356) produce different phenotypes of transformation and invasive/metastatic activity [52]. Therefore, the main signal pathway used by a particular activating MET mutation is thought to be a key in determining whether the cancer cell is more proliferative or more invasive/metastatic.

A 2010 report indicated that aberrant HGF/SF-MET signaling that is often seen in human PRCs had an ability to induce centrosome amplification and chromosomal instability (CIN) via the PI3K-Akt pathway [53].

7.2.2.2 Other Types of MET Mutation and Cancers

While the ATP-binding site is the most important region for activating MET mutations [47, 48], other regions are also involved. For example, a germ line juxta-membrane missense MET mutation (P1009S) found in gastric cancer produces prolonged tyrosine phosphorylation in response to HGF/SF [54]. Somatic intronic mutations that lead to an alternatively spliced transcript are found in some lung cancers [55]. Such mutations cause a deletion of the juxta-membrane domain, resulting in the loss of Cbl E3-ligase binding, which ultimately delays the downregulation of MET because of decreased ubiquitination. Somatic splice-site alterations at MET exon 14 have been recently reported in 0.6% (221 out of 38,028) of tumor genomic profiles investigated [56].

In animal models, MET lacking the ectodomain but retaining the transmembrane and intracellular domains became an oncogenic driver [57]. Tumors developed in nude mice showed anchorage-independent growth and invasive activity.

7.2.3 MET Overexpression and Cancer Progression

The overexpression of receptor tyrosine kinases (RTKs) is the most common abnormality in human cancers. The overexpression of MET, which is observed in many cancers, leads to ligand-independent receptor dimerization and activation [15]. Non-autocrine, constitutive activation of MET is found in human anaplastic thyroid carcinoma cells [58]. MET protein expression is reported to correlate with survival in patients with late-stage nasopharyngeal carcinoma [59]. The dimerization of MET, which is enhanced by O-glycosylation of MET by core 1 β1,3-galactosyltransferase, is a key event in MET activation and subsequent signal transduction inside the cells [60]. Besides dimerization, overexpression of MET itself is a unique status. Overexpressed MET in tumor cells is hyperphosphorylated and can form dimers even in the absence of its ligand, HGF/SF [16, 41], thereby activating MET signaling and leading to oncogenic transformation. Transgenic mice that overexpress MET in hepatocytes developed hepatocellular carcinoma (HCC) [61], and MET was considered to be activated by cell attachment rather than by ligand.

MET expression often correlates strongly with poor prognosis in a variety of human cancers. For example, early-stage prostate cancers are reported to be generally androgen-sensitive and either Met-negative or weakly Met-positive (if expressed). As cancer cells change from androgen-sensitive to androgen-insensitive, however, the aberrant expression of HGF/SF and MET becomes obvious. Thus, the androgen receptor (AR) negatively regulates the expression of MET [62] in a ligand-dependent manner. AR interferes with the interaction between Sp1 and the functional Sp1 binding site within the MET promoter. Therefore, the combination of inhibiting the HGF/SF-MET signaling pathway plus androgen ablation is considered a good option for the treatment of prostate cancers. Suppressing Met expression by gene-targeting/modifying technologies is an effective way to decrease HGF/SF-Met signaling [63].

Another example comes from the use of B16 melanoma cells for evaluating the role of MET expression in cancer malignancies [64, 65]. On the one hand, high MET expression in metastatic melanoma cells is ascribed to induction of the gene rather than preferential selection of tumor cells expressing high levels of MET [66]. On the other hand, the liver metastasis melanoma cell line B16-LS9 shows a dramatic overexpression of MET, with the gene constitutively active and more responsive to HGF/SF stimulation than in B16-F1, the parental line [67]. The relationship between MET expression and the metastatic potential of melanoma cells was further verified by testing three melanoma lines, B16-BL6, B16-F1, and B16-F10, for their metastatic potential (Fig. 7.2). The strength of MET expression was B16-F10 > B16-BL6 > B16-F1, and MET phosphorylation paralleled that order. After intravenous injection of mice with melanoma cells, the MET phosphorylation order correlated well with the lung weight (= amount of metastasized melanoma cells) as expected, and the mouse death rate also showed the same order, which shows the importance of MET activation in metastasis [68].

Fig. 7.2
figure 2

Met expression in B16 melanoma cells and cancer malignancy. (a) Three B6 melanoma cell lines of common origin were used. The order of Met expression was F10 > BL6 > F1, and Met phosphorylation was the same order. (b) Mice were injected intravenously with 1 × 106 B6 melanoma cells of three lines (BL6, F1, F10) and were sacrificed at Day 16. Mice injected with F10 cells showed numerous black metastatic nodules in the lungs; mice injected with F1 cells showed the lowest frequency of lung metastasis. (c) The duration of mouse survival after IV injection with 1 × 106 B6 melanoma cells was F1 > BL6 > F10, suggesting a close relationship between Met expression/phosphorylation and cancer malignancy

Full size image

7.2.4 MET Amplification and Cancers

The relationship between amplification and carcinogenesis was first recognized when NIH 3T3 mouse fibroblasts became spontaneously transformed and showed Met amplification [69, 70]. Clinically, amplification of the human MET gene is frequent in many types of cancers, including scirrhous-type stomach cancer [71] and other types of gastric cancer [72], ovarian clear-cell adenocarcinoma [73, 74], glioblastoma [75], hepatocellular carcinoma [76], and giant cell tumor of the bone [77]. Also, a significant correlation between the amount of MET protein and an increased gene copy number has been shown in esophageal cancer [78], gastric cancer [79], and PRC [80].

MKN45 is a human gastric cancer cell line in which MET is amplified [81]. This line is often used for analyzing ligand-independent MET activation mechanisms (Fig. 7.3) and for the development of MET-targeting tools [82]. There is accumulating evidence about the relationship between MET expression and the aggressiveness of human carcinomas (https://resources.vai.org/Met/Index.aspx).

Fig. 7.3
figure 3

MKN45 is a ligand-independent cell line. (a) In MKN45 cells from a poorly differentiated gastric cancer, Met is constitutively active, and Met phosphorylation is independent of growth factors (serum) or HGF/SF stimulation. SK-LMS-1, leiomyosarcoma; DU-145, prostate cancer; PC3, prostate cancer; DBTRG, glioblastoma; DA3, mouse mammary carcinoma. Akt and p44/42MAPK are Met downstream signals and are activated when stimulated with HGF/SF. (b) RT-PCR confirms that MKN45 does not produce HGF/SF. DBTRG, MKN45, and PC3 are HGF/SF-negative; SK-LMS-1 and hHGF-Tg mouse liver express HGS/SF

Full size image

7.2.5 MET Transactivation Via Other Membrane Receptors

Transactivation of MET by other membrane receptors can produce cancer initiation or progression. It has been reported that integrins such as LFA-1 (lymphocyte function-associated antigen 1) and VLA4 (very late antigen 4) regulate cancer cell adhesion to the endothelium and the subsequent invasion into tissues. CD44 stimulates the integrin-induced adherence of colon cancer cells to the endothelial cells. CD44 stimulation also induces the expression of MET on cancer cells [83]. In this system, HGF/SF further amplifies the LFA-1-mediated adhesion of cells stimulated by CD44 signaling. In pancreatic cancer cells, α6,β4 integrin is known to upregulate several genes in the epidermal growth factor receptor (EGFR) pathway and cooperates with MET [84]. Fibronectin and vitronectin modulate the responses of endothelial cells to HGF/SF and work as an important pro-angiogenic mediator [85]. Fibronectin and vitronectin can bind to HGF/SF and form complexes that strongly promote MET–integrin association and lead to enhanced cell migration via a Ras-dependent mechanism. Extracellular matrix adhesion-dependent activation of Met is reported to be mediated by Src and the focal adhesion kinase (FAK) signaling pathway during transformation of breast epithelial cells [86].

Cell–matrix adhesion of the cancer cells is also reported to be correlated with constitutive activation of MET [87]. Fibronectin is a unique molecule for the invasive and metastatic capacity of ovarian cancers [88], and through an α5,β1-integrin/MET/FAK/Src-dependent signaling pathway, Met downstream signaling is upregulated in an HGF/SF-independent manner. Also, cellular adherence is proved to be an important event in eliciting ligand-independent activation of MET [89]. The tyrosine phosphorylation of Met in mouse melanoma cells was compared before and after attachment to substrata, and the results showed the involvement of mechanical stimuli but not biochemical stimuli. This ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells.

Because co-activation of MET and EGFR mediated by cross talk between these two molecules is thought to be involved in cancer progression, blocking both the HGF/SF-Met and EGFR signaling cascades for cancer treatment may be a good strategy for overcoming cross talk-related resistance to EGFR inhibitors [90, 91]. From this viewpoint, genomic profiling to see whether other genes are amplified in MET-activated tumors is effective in predicting the effectiveness of molecular targeting drugs [92]. Treating lung cancer with an EGFR inhibitor often induces resistant tumors which have MET amplification [93], suggesting that MET amplification could also be involved in resistance to other RTK inhibitors. The cross talk between MET and other signaling pathways and its implications for therapeutics are discussed in Chap. 8 [91].

7.2.6 Infectious Disease-Mediated Activation of the MET Pathway in Cancer

Many pathogens are thought to use the host HGF/SF-MET system to establish a comfortable environment for infection [94]. The inflammatory process caused by infection, in combination with the effect of viral/bacterial proteins, induces an HGF/SF-dependent MET activation and pushes the cell cycle into S phase. MET activation and its subsequent biological effects are often mediated by an autocrine HGF/SF circuit; for example, in viral-related carcinogenesis of human malignant mesothelioma [95]. In gastric cancers, EBV infection is reported to be associated with abnormal MET expression [96].

The agent that is closely involved in gastric cancers is Helicobacter pylori. The H. pylori virulence factor CagA associates with MET, activates intracellular signaling, and induces the proliferation of gastric epithelial cells [97]. H. pylori stimulates the Wnt/β-catenin pathway by activating MET and EGFR [98], which presumably play a key role in the development of gastric cancers. The HGF/SF-MET pathway has also been suggested to contribute to lymphomagenesis in MALT (mucosa-associated lymphoid tissue) lymphoma after H. heilmannii infection [99]. There is also accumulating evidence that hepatocellular carcinomas caused by hepatitis B virus (HBV) or hepatitis C virus (HCV) infection are often associated with increased amounts of HGF/SF or increased MET activation [100, 101].

7.2.6.1 Helicobacter pylori and MET Stimulation in Gastric Cancer

H. pylori infection is considered to be involved in the carcinogenesis of gastric cancers by interacting with gastric epithelial cells and activating important oncogenic signaling pathways. Recently we have shown that high MET expression is closely related to a poor prognosis of gastric cancers with H. pylori infection, but this is not the case without H. pylori infection [102]. The activity of H. pylori in the growth of gastric cancers is ascribed to its lipopolysaccharide, which stimulates the Toll-like receptor 4 pathway in cancer cells, causing proliferation, and attenuates the antitumor activities of human mononuclear cells [103].

The relation of H. pylori to MET activation was recognized when it was found that the H. pylori effector protein CagA [104] targets MET and promotes cellular processes leading to a forceful motogenic response. Via a type IV secretion system, CagA is translocated into epithelial cells and modulates intracellular MET activity [105]. Gastric epithelial cell invasion after CagA stimulation is mediated through a MET-dependent signaling pathway and an increase in MMP-2 and MMP-9 activity [106]. The CRPIA motif in non-phosphorylated CagA is thought to interact with activated MET, which leads to sustained activation of the PI3K/Akt pathway and ultimately to the activation of β-catenin and NF-κB signaling [107]. CagA is also involved in H. pylori-induced loss of gastric epithelial cell adhesion [108].

7.2.6.2 Hepatitis Viruses and MET Activation in Liver Cancer

The MET activation pathway is considered one of the most important pathways closely involved in hepatocarcinogenesis [109]. Significant increases in HGF/SF and EGF in patients with active HBV infection have been reported. The activation of those liver-regeneration factors may be a risk factor for establishing viral persistence [110], thus contributing to the progression of chronic disease and ultimately to hepatocellular carcinoma (HCC). Serum HGF/SF in patients with chronic hepatitis B was significantly correlated with serum alanine aminotransferase (ALT) and HBV DNA [111], suggesting that HGF/SF promotes viral replication and is involved in the destruction of hepatocytes. In a model of HBV-associated HCC, HGF/SF produced by cells in the inflammatory and cirrhotic lesions of a precancerous liver plays a key role in hepatic oncogenesis by stimulating the production of liver regeneration nodules [112]. In the analysis of human HCC samples, the processed form of p145 β-MET was significantly greater in tumor tissue than in non-tumor areas [113]. This also suggests that processing of the MET pro-receptor is closely associated with regeneration and carcinogenesis of the liver.

The pathogenesis of liver tumors in mice expressing conditional transgenes of MET in their hepatocytes has been studied [114]; the genotypes of the resulting hyperplasia and benign and malignant tumors resembled those of the human counterparts. This strongly supports an indispensable role for MET in the genesis of human liver tumors caused by HBV and HCV infection, because hepatitis induces the cycles of hepatocyte destruction and regeneration and HGF/SF-MET signaling is thought to be strongly involved in those steps. Another study using MET transgenic (Tg) mice showed that the prognostic significance of gene expression signatures between mouse models and human samples was parallel and could be used as biomarkers for HCC. Especially, mouse liver tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway [100]. Mouse models showing overexpression of HGF/SF have been reported to strongly promote HBV-induced HCC progression [101]. The analysis of molecular signatures showed that the patterns were similar to human HCC cases, with overall shorter survival in both Myc/TGF-α-Tg and HGF/SF-Tg animals, suggesting the importance of these genes in HBV/HCV-induced HCC.

7.3 Carcinogenesis and Mouse Models Targeting MET

Genetically modified mouse models have been powerful tools for studying the roles of HGF and MET in cancer initiation and progression. In knock-in animals, the HGF or MET genes are replaced with a mutated functional gene. Since the inserted mutant gene is located exactly in the same place as the original and its expression is controlled under the original promoter, the natural course of the effect of mutant gene can be investigated. MET knockout models were produced for the analysis of its biological function during embryogenesis. Because knocking out MET causes embryonic death, conditional knockouts are used to eliminate MET expression in adult mice. Human HGF transgenic mouse models are used to study the role of HGF/SF-paracrine-dependent tumor growth and for preclinical evaluation of MET-targeted therapeutics.

7.3.1 MET Knock-in Mouse Models and Carcinogenesis

Since MET-activating mutations were identified in human carcinomas, experimental approaches to clarify their transforming potential have been conducted both in vitro and in animal models. Experiments using NIH 3T3 cells stably transfected with murine MetD1246N or MetM1268T mutations revealed a direct link between Met endocytosis and tumorigenicity [115]. Those Met mutants exhibited increased endocytosis or recycling activity and decreased degradation, leading to the accumulation of Met molecules on endosomes, the activation of Rac1 GTPase, and ultimately to loss of actin stress fibers and increased cell migration. Subcutaneous grafting of the cells into nude mice showed a rapid formation of tumors.

Targeted mutations in the murine Met locus were used to create five knock-in (KI) mouse lines (WT, D1226N, Y1228C, M1248 T, and M1248 T/L1193 V) on a C57BL/6 J;129/SV background [116, 117]. Each mutant line developed a unique profile of tumors (Table 7.2). Sarcomas developed in MetD1226N, MetY1228C, and MetM1248T/L1193V but not in MetM1248T, whereas carcinomas developed in MetM1248T but not in other lines. Lymphomas were found in most lines but not in MetD1226N. Further, MetD1226N and MetY1228C showed a higher incidence of tumor formation than others, suggesting they carry a higher tumorigenic potential. Glomerulonephritis and hydronephrosis were observed in some activating Met-KI mice, but no renal carcinomas were detected in those animals. Thus, activating mutations of Met were proved to be a driving force for carcinogenesis, though the effects of such mutations in mice seems to act differently from those in humans. Interestingly, nonrandom duplication of mutant Met alleles was observed in the Met-KI animals. This may suggest that secondary events beyond Met mutation are required for tumor progression, which has been observed in human hereditary papillary renal carcinoma cases [118, 119].

Table 7.2 Met-activating mutations and carcinogenesis in mice
Full size table

Similar animal models on different genetic backgrounds developed different tumor types. For example, murine lines with Met D1226N, M1248 T, or Y1228C on the FVB/N background developed a high incidence of mammary carcinomas with diverse histopathologies [120]. The MetM1248T/L1193V-KI line developed the most aggressive type of mammary tumor, in which Met is highly expressed and progesterone receptor (PR) and ErbB2 are negative [121]. The tissue microarray analysis of human breast cancers confirmed the importance of high MET expression: it significantly correlated with the gene expression patterns of PR-negative/ErbB2-negative tumors and with basal breast cancers.

7.3.2 HGF/SF-Tg Mouse Models and HGF/SF-Dependent Tumor Growth

The importance of the HGF/SF-MET signaling in carcinogenesis has been proved in several HGF/SF transgenic animal models, which develop malignant melanomas [122, 123] and multifocal invasive ductal carcinomas of the mammary gland with lung metastasis [124]. Livers of HGF/SF-Tg mice exhibit a significant increase in the number of hepatocytes and in liver mass [125]. This proliferative stimulus is considered to trigger the formation of hepatocellular adenomas and/or carcinomas in most Tg mice. HGF/SF may also play a critical role in lymphangiogenesis, thereby contributing to lymphatic metastasis [126]. Mice expressing the HGF transgene only in the lung were used to test tobacco-induced lung carcinogenesis; the results showed that lung cancers were preferentially induced and enhanced in those mice [127].

HGF/SF-Tg mice are also used to evaluate the growth and metastatic capability of cancer cells, especially when they behave ligand dependently. Transplantation of non-autocrine melanoma cells into HGF/SF-Tg mice revealed that activation of Met was a key signal that enhanced metastatic colonization [128]. The authors generated a mouse strain transgenic for human HGF/SF on a severe combined immunodeficiency (SCID) background [129] (Fig. 7.4). Because xenogeneic tumor cells can be easily transplanted into this model and because they grow much faster than in regular SCID mice if tumor cells express Met on their surface, it is a good tool for evaluating the effectiveness of anticancer drugs and diagnostic agents.

Fig. 7.4
figure 4

hHGF-Tg SCID mouse is a good model for predicting HGF/SF-dependent tumor growth. (a) RT-PCR analysis showing that hHGF mRNA is produced in the liver of hHGF-Tg SCID mice. (b) Heparin bead pulldown assay showing hHGF is found in the serum of hHGF-Tg SCID mice. (c) SK-LMS-1 is a MET-positive tumor cell and grows rapidly when xenografted into hHGF-Tg SCID mice. Figures are from Zhang et al. [129]

Full size image

7.3.3 Conditional Met-KO Mouse Models and Hepatocellular Carcinoma

Because Met knockouts are embryonic lethal [4], the indispensable role of Met in adult mice was not clear in early studies, but conditional KO technology provided new insights. Liver-specific Met-KO mice had significant impairment of liver regeneration after partial hepatectomy [130]. In that study, the activation of ERK1/2 kinase during liver regeneration depended exclusively on Met; the cell cycle halted and could not enter into S phase. In another model using a challenge with a necrogenic dose of CCl4, Met-KO mice exhibited impaired recovery from centrolobular lesions [131]; in this case, the scattering/migration of hepatocytes into diseased areas (rather than hepatocyte proliferation) was impaired. Also, hepatocyte-specific Met deletion disrupted redox homeostasis, and the mice showed a hypersensitive reaction to Fas-induced liver injury [132]. Thus liver-specific Met KO models showed dysfunction of hepatocytes under stressful conditions, but those mice lived a normal life span unless they received obvious stresses to the liver [130].

A mouse model with conditional inactivation of Met in cardiomyocytes was found to be prone to cardiomyocyte hypertrophy associated with interstitial fibrosis. This shows a physiological cardioprotective role of Met in adult mice by acting as an endogenous regulator of heart function through oxidative stress control [133]. Mice with pancreatic deletion of Met showed significantly diminished β-cell mass, loss of regeneration, and decreased glucose tolerance, suggesting the crucial role of HGF/SF-Met signaling in pancreatic function [134]. Thus, Met function in adult mice appears to be involved in the proliferative/regenerative pathways of damaged organs.

From the oncogenic viewpoint, the partial deletion of Met, such as deletion of the ectodomain, upregulates Met phosphorylation and activates its downstream signaling pathways [57]. In an experiment of chemically induced tumor initiation, conditional Met-KO mice injected with N-nitrosodiethylamine showed a higher prevalence of visible liver tumors and of glutamine synthetase-positive and glucose-6-phosphatase-deficient liver lesions than did wild-type mice [135]. Glutamine synthetase is a transcriptional target of β-catenin and is therefore overexpressed in liver tumors; glucose-6-phosphatase is usually absent or lower in activity in tumor hepatocytes.

The authors have explored the role of HGF/SF-MET signaling in hepatocarcinogenesis by using genetically engineered mouse models [101]. Because transgenic mice carrying hepatitis B virus surface antigen (HBsAg-Tg mice) are reported to be a good model for developing hepatocellular carcinoma (HCC) [136], we crossed HBsAg-Tg mice with HGF/SF-Tg mice or with liver-specific Met-KO mice and monitored the incidence of HCC (Table 7.3). B6 mice with liver-specific Met-KO showed a higher incidence of HCC than B6 wild-type (WT) mice (30.0% vs. 8.3%) but a much lower incidence than B6 HBsAg-Tg mice (94.6%). There was no substantial difference in the HCC incidence between B6 HBsAg-Tg mice and those with liver-specific Met-KO (94.6% vs. 96.4%). Therefore, the effect of losing Met on the development of HCC is considered to be limited. In contrast, HGF/SF-Tg (C3H hHGF-Tg) mice showed a high HCC incidence (92.3%), and mice with both C3H hHGF-Tg and HBsAg-Tg showed 100% incidence. The C3H strain was less sensitive to HBsAg transgene-induced development of HCC than B6 (76.0% vs. 94.6%), so the effect of hHGF-Tg on hepatocarcinogenesis was remarkable. The short average survival of hHGF-Tg animals also supports the importance of constitutive/sustained activation of HGF/SF-Met signaling in the progression of HCC.

Table 7.3 Genetically engineered mouse models and HCC incidencea
Full size table

7.4 MET Therapeutics and Drug Resistance

Since MET became a promising anticancer target, many approaches have been developed to target it. The authors have targeted the MET molecule directly using siRNAs, which are effective against regular cancers and cancers in which MET is activated ligand independently [82]. Currently, a wide variety of small molecules that inhibit MET phosphorylation are under clinical trials [137, 138]. Other approaches against ligand-independent MET activation include a MET therapeutic protein antagonist [139] and an engineered, chemically modified antibody [140]. A list of MET inhibitors and the status of clinical trials can be found in Chap. 8.

Because activating mutations and amplification of MET are involved in cancer and because the behavior of cancer cells often results from an addiction to MET signaling, MET is an attractive candidate for targeted therapies. However, cancer cells often develop resistance to small-molecule inhibitors of MET during the treatment course. Such resistance may be partly explained by an increase of a certain type of mutated allele; for example, a M1268 T mutated allele in the time-of-progression sample relative to the pretreatment sample was found in a patient with papillary renal carcinoma [141]. Because drugs that inhibit certain mutant MET variants are being developed [142], it seems important to investigate the relationship between the type of mutation and its specific sensitivity to MET inhibitors. Also, a resistance screen using specific inhibitors is important to predict resistance mutations that could emerge during use of the inhibitor in patient treatment [143].

Changes in the gene copy number may also contribute to resistance to MET-targeting drugs. Retrospective studies in non-small-cell lung cancer have shown that a higher MET copy number is a negative prognostic factor, and MET amplification has been considered as one of the crucial events for acquired resistance in EGFR-mutated lung adenocarcinomas that are resistant to EGFR tyrosine kinase inhibitors [144]. This is because of intracellular cross talk between the MET and EGFR receptors or their signal transduction pathways [90, 145]. Thus, co-inhibition of MET and EGFR may be an effective strategy for overcoming the resistance of cancer cells to tyrosine kinase inhibitors. Further, overexpression of MET is expected to be a predictive marker for some metastatic colorectal cancer patients who might benefit from anti-EGFR therapy [146]. Because patients with MET overexpression showed less disease control and shorter progression-free survival than those with normal MET expression, the use of MET overexpression as a biomarker in combination with other markers (such as BRAF and PIK3CA mutations) would be more effective than existing methods.

7.5 Conclusion and the Way Forward

Because the activation of HGF/SF-MET signaling—whether due to MET mutation, MET overexpression, or strong activation of HGF/SF-MET autocrine/paracrine loop—plays an important role for carcinogenesis, disruption of this pathway appears as a good option for targeted therapies against specific cancers. Approaches to evaluate the molecular determinants that control MET signaling activation are being developed as biomarkers to predict the effectiveness of such therapies. For example, the monitoring of MET by IHC and FISH, of MET mutations, and of tissue HGF/SF should provide good data for selecting suitable drugs [147]. The presence of activating MET mutations or amplifications are key events that predict cancer malignancy and sensitivity to MET-targeting therapies [92, 101]. Resistance to MET small-molecule inhibitors [148] and unexpected side effects from some small molecules [149] have been reported, which will prompt efforts to develop more effective and less toxic drugs. From the clinical viewpoint, how can we overcome resistance to MET-targeting drugs [150] is an important issue and a long-term goal.