**1. Introduction**

312 Advances in Cancer Therapy

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> Because growth factors and their receptors play critical roles in cancer development and progression, they are potential target molecules in molecular-targeted cancer therapy. Hepatocyte growth factor (HGF) was discovered and cloned as a mitogenic protein for hepatocytes (Nakamura et al., 1984; Nakamura et al., 1989; Miyazawa et al., 1989). These early studies implicated an important role for HGF in regeneration of the liver. The receptor for HGF was identified as the Met transmembrane receptor tyrosine kinase in 1991 (Bottaro et al., 1991; Naldini et al., 1991). The Met oncogene was first isolated as a fused transforming gene from a human osteosarcoma-derived cell line, wherein sequences from the TPR (translocated promoter region) were fused to the Met sequence (Tpr-Met) (Cooper et al., 1984). In 1991, the scatter factor, originally identified as a fibroblast-derived cell motility factor for epithelial cells (Stoker et al., 1987), was shown to be an identical molecule to HGF (Weidner et al., 1990). These findings implicated further biological and pathophysiological roles for HGF in epithelial wound healing, epithelial-mesenchymal interaction, and cancer development and invasion. Based on its close involvement — not only in tumor development, invasion, and metastasis but also in resistance to anticancer therapies — the HGF-Met pathway has become a hot target in anticancer drug development (Comoglio et al., 2008; Sattler & Salgia, 2009; Hanahan & Weinberg, 2011). In most cases in the relationship between growth factors and their receptor tyrosine kinases, a single growth factor activates multiple receptors that have structural similarities, while a single growth factor receptor has multiple ligands with structural and functional similarities. By contrast, the sole receptor of HGF is Met, while the sole ligand of Met is HGF; the relationship between HGF and Met is a "one-to-one relationship." This unique biochemical characteristic in the HGF-Met pathway promotes drug development by targeting HGF-Met through either the activation or the inhibition of the HGF-Met pathway.

### **2. Biochemical and biological characteristics**

Biologically active HGF, a protein composed of 697 or 692 amino acids, is a heterodimeric molecule composed of an -chain and a -chain (Fig. 1A). The -chain contains 4 kringle

Significance, Mechanisms, and Progress of Anticancer Drugs Targeting HGF-Met 315

al., 2004; Borowiak et al., 2004; Chmielowiec et al., 2007; Ma et al., 2009). Met-deficient epidermal keratinocytes were unable to contribute to re-epithelialization in skin wound healing, because of a disability in keratinocyte migration (Chmielowiec et al., 2007). HGF induces 3-dimensional (3-D) tubulogenesis in epithelial cells such as renal and mammary grand epithelial cells (Fig. 2) (Montesano et al., 1991). These approaches emphasize a particular role of the HGF-Met pathway in the migration of cells during development, morphogenesis, and regeneration. However, the dynamic actions of HGF in wound healing and tissue reconstruction — even in a 3-D spatial scaffold — remind us of the malignant behavior of tumors, i.e., invasion and metastasis (Fig. 2). Aberrant activation of the Met

receptor in tumor cells participates in the malignant progression of tumor cells.

Fig. 2. Outline for biological actions of HGF in tumor invasion-metastasis and tissue

HGF binds to Met through 2 different mechanisms: the -chain binds with high affinity while the -chain binds with low affinity. Among the -chain, NK1 (the N-terminal and first kringle domains) in the -chain of HGF provides a high-affinity binding site for Met. The -chain alone exhibits high-affinity binding to Met, whereas the binding of the -chain does not activate Met (Matsumoto et al., 1998). When Met is occupied by the -chain, the low-affinity binding of the -chain induces activation of Met and biological responses. Hence, the -chain is a high-affinity binding module to Met, while the -chain is an activation module for Met. The structure of the complex of HGF -chain and Sema was revealed by crystallographic analysis (Fig. 3A) (Stamos et al., 2004). The HGF -chain binds to a series of protruding polar side chains from Met, which originate from 3 separate loops: residues 124–128, residues 190–192, and residues 218–223. Although the -chain of HGF binds to Met with much higher affinity than that of the HGF -chain, the crystalline structure for the interaction between the HGF -chain and the extracellular region of Met is

regeneration.

**3. Met receptor activation** 

yet to be determined.

domains, while the -chain contains a serine protease-like structure (Nakamura et al., 1989; Miyazawa et al., 1989). HGF has a structural similarity to plasminogen, which is a heterodimeric serine protease containing 5 kringle domains. HGF is biosynthesized as a prepro-form of 728 amino acids, including a signal sequence and both - and -chains. After cleavage of a signal peptide of the first 31 amino acids, a single-chain HGF is further cleaved between Arg494 and Val495, and this processing is coupled to the conversion of biologically inactive pro-HGF to active HGF (Fig. 1A). Several proteases in the serum or cell membranes are involved in the activation of single-chain HGF, including HGF activator, urokinase-type plasminogen activator, plasma kallikrein, coagulation factors XII and XI, matriptase, and hepsin (Kataoka & Kawaguchi, 2010).

The Met receptor is composed of structural domains that include the extracellular Sema (the domain found in semaphorin receptors), PSI (the domain found in plexins, semaphorins and integrins) and IPT (the domain found in immunoglobulins, plexins, and transcription factors) domains, the transmembrane domain, and the intracellular juxtamembrane and tyrosine kinase domains (Fig. 1B) (Park et al., 1987). The Sema domain serves as a key element for ligand binding (Gherardi et al., 2006), while an involvement of IPT-3 and IPT-4 in the binding to HGF was demonstrated by another approach (Basilico et al., 2008).

Fig. 1. (**A**) Processing and structure of single-chain proHGF and mature HGF. (**B**) Domain structures of the Met receptor and representative signaling molecules that associate with Met.

HGF and Met genes are widely expressed, and HGF is expressed in mesenchymal/stromal cells, predominantly rather than exclusively. Deletion of either the HGF or Met gene in mice lethally disrupts embryogenesis, including impairing development of the placenta and liver, and disabling dynamic migration of myogenic precursor cells (Schmidt et al., 1995; Uehara et al., 1995; Bladt et al., 1995; Birchmeier et al., 2003). In adulthood, HGF and Met play important roles in protection and regeneration of various tissues following injuries and pathology (Nakamura et al., 2011). Tissue-specific deletion of the Met gene revealed that the HGF-Met pathway plays a critical role in regeneration of the liver, kidney, and skin (Huh et al., 2004; Borowiak et al., 2004; Chmielowiec et al., 2007; Ma et al., 2009). Met-deficient epidermal keratinocytes were unable to contribute to re-epithelialization in skin wound healing, because of a disability in keratinocyte migration (Chmielowiec et al., 2007). HGF induces 3-dimensional (3-D) tubulogenesis in epithelial cells such as renal and mammary grand epithelial cells (Fig. 2) (Montesano et al., 1991). These approaches emphasize a particular role of the HGF-Met pathway in the migration of cells during development, morphogenesis, and regeneration. However, the dynamic actions of HGF in wound healing and tissue reconstruction — even in a 3-D spatial scaffold — remind us of the malignant behavior of tumors, i.e., invasion and metastasis (Fig. 2). Aberrant activation of the Met receptor in tumor cells participates in the malignant progression of tumor cells.

Fig. 2. Outline for biological actions of HGF in tumor invasion-metastasis and tissue regeneration.
