**3. Met receptor activation**

314 Advances in Cancer Therapy

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

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

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

hepsin (Kataoka & Kawaguchi, 2010).

Met.

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 yet to be determined.

Significance, Mechanisms, and Progress of Anticancer Drugs Targeting HGF-Met 317

Ser985 is phosphorylated, HGF-induced activation of Met is suppressed. Therefore, activation of protein kinase-C, which occurs by different types of extracellular stimuli,

In normal tissues the activation of the Met receptor is tightly regulated, perhaps exclusively in a ligand-dependent manner. Aberrant activation of Met is associated with tumor development or progression to a tumor with malignant characteristics (Comoglio et al., 2004; Christensen et al., 2005; Matsumoto & Nakamura, 2006). Overexpression of Met through transcriptional upregulation has been noted in several cancers, including thyroid, ovarian, pancreatic, prostatic, renal, hepatocellular, breast, and colorectal cancers. Overexpression of Met through gene amplification was found in cancers with highly invasive and malignant characteristics, including gastric and esophageal carcinomas, medulloblastoma, and non-small-cell lung carcinomas (NSCLC) with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (see below). Autocrine and paracrine activation of Met through overexpression of HGF has been noted in breast cancer, glioblastoma, rhabdomyosarcoma, osteosarcoma, and in NSCLC with acquired and

The missense mutations in the Met gene are the causative genetic disorders in inherited, and in some sporadic, papillary renal carcinomas (Schmidt et al., 1997). Mutations found in papillary renal carcinomas are located in the tyrosine kinase domain of the Met receptor, and these Met mutations are likely to be gain-of-function mutations (Jeffers et al., 1997; Michieli et al., 1999). In addition to papillary renal carcinoma, missense mutations in the Met gene have been found in different types of cancers, including lung cancer, hepatocellular carcinoma, and gastric cancer in the Sema, IPT, juxtamembrane, and tyrosine kinase

The biological programs regulated by the HGF-Met pathway are adopted in cancer tissues, particularly for their invasive and metastatic behavior (Birchmeier et al., 2003; Matsumoto & Nakamura, 2006): 1) the dissociation of cancer cells at the primary site; 2) invasion, i.e., detachment from the primary site and migration through the basement membrane and stroma; and, 3) escape from apoptosis in anchorage-independent conditions during circulation. In a unique 3-D invasion in collagen gel, HGF was identified as a fibroblast-derived factor that definitively induces invasiveness of oral carcinoma cells (Matsumoto et al., 1989; Matsumoto et al., 1994). HGF increases extracellular protease expression coupled with the dissociation of cancer cells and their motility by which HGF promotes invasion in 3-D extracellular matrices and subsequent metastasis. HGF-Met signaling participates in the transition of epithelial to mesenchymal cell types (Birchmeier et al., 2003). Angiogenic and lymphangiogenic activities of HGF may facilitate cancer metastasis (Jiang et al., 2005). Collectively, the HGF-Met pathway has become a hot target in research and development of molecular targeted therapy for cancer, particularly to inhibit

regulates HGF-dependent Met inactivation/activation.

intrinsic resistance to EGFR tyrosine kinase inhibitors.

domains (Christensen et al., 2005; Cipriani et al., 2009).

cancer invasion and metastasis (Hanahan & Weinberg, 2011).

**4.2 Cancer invasion and metastasis** 

**4.1 Cancer development** 

**4. HGF-Met in cancer development and progression** 

The Met tyrosine kinase domain follows a conserved bilobal protein kinase architecture mainly with an N-terminal, -sheet-containing domain linked through a hinge segment mainly to the -helical C lobe (Fig. 3B) (Schiering et al., 2003; Wang et al., 2006). The characteristic feature of Met is the presence of the C-terminal tail that contains tyrosine residues (1349YVHVNAT1356YVNV). Binding of HGF to the extracellular region of Met results in receptor dimerization and phosphorylation of multiple tyrosine residues within the cytoplasmic region. Phosphorylation of Tyr1234 and Tyr1235 within the tyrosine kinase domain positively regulates the catalytic activity of tyrosine kinase (Fig. 3B). The staurosporine analog K-252a inhibits Met tyrosine kinase through its binding in the ATP pocket (Schiering et al., 2003). The phosphorylation of C-terminal tyrosine residues Tyr1349 and Tyr1356 recruits intracellular signaling molecules, including PI3K (phosphatidylinositol 3-kinase), Grb2 (growth-factor-receptor-bound protein 2), Gab1 (Grb2-associated binder 1), PLC (phospholipase C), and Shp2 (SH2-domain-containing protein tyrosine phosphatase 2). Direct interaction of Gab1 with tyrosine phosphorylated Met is mediated by the Met-binding site in Gab1, and Gab1 is the most crucial substrate for the HGF-Met pathway (Ponzetto et al., 1994; Sachs et al., 2000).

Fig. 3. Crystal structures for the complex of HGF -chain and the Met Sema domain (**A**) and the Met tyrosine kinase domain (**B**). The crystal structures for the complex of HGF -chain and the Met Sema domain were reported by Stamos et al. (2003) (PDB number: 1SHY). The crystal structure for Met tyrosine kinase was reported by Schiering et al. (2003) (PDB number 1ROP). In **B**, the activation loop (A-loop) is shown in yellow, K-252a in green, and selected tyrosine residues (Y1234F, Y1235D, Y1349, Y1356) are in blue.

The cytoplasmic juxtamembrane domain, which is composed of 47 highly conserved amino acids, acts as a negative regulator in terms of Met-dependent signal transduction. Cbl, an E3 ubiquitin ligase, binds phosphorylated Y1003 of Met, and this Cbl binding results in Met ubiquitination, endocytosis and transport to the endosomal compartment, then degradation (Peschard et al., 2001). Cbl-mediated degradation of the activated Met provides a mechanism that attenuates or terminates Met-mediated signaling. Phosphorylation of Ser985 in the juxtamembrane domain regulates the activation status of Met upon HGF stimulation. Ser985 is phosphorylated by protein kinase-C and is dephosphorylated by protein phosphatase-2A (Gandino et al., 1994; Hashigasako et al., 2004). In cells in which Ser985 is phosphorylated, HGF-induced activation of Met is suppressed. Therefore, activation of protein kinase-C, which occurs by different types of extracellular stimuli, regulates HGF-dependent Met inactivation/activation.
