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

### **4.1 Cancer development**

316 Advances in Cancer Therapy

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

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

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

selected tyrosine residues (Y1234F, Y1235D, Y1349, Y1356) are in blue.

(Ponzetto et al., 1994; Sachs et al., 2000).

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 intrinsic resistance to EGFR tyrosine kinase inhibitors.

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

#### **4.2 Cancer invasion and metastasis**

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

Significance, Mechanisms, and Progress of Anticancer Drugs Targeting HGF-Met 319

Collectively, expression of HGF in cancer cells and/or host stromal cells closely participated in the resistance to EGFR tyrosine kinase inhibitors in NSCLC, even in NSCLC with Met

Fig. 4. Drug resistance of non-small-cell lung cancer (NSCLC) against EGFR tyrosine kinase inhibitors (EGFR-TKIs) through HGF-Met pathway. (**A**) Drug resistance through Met gene amplification (left) and HGF-dependent Met activation (right). Amplified Met associates with ErbB3 activates downstream signaling such as the PI3-Akt pathway, leading to the survival of

independent of EGFR and ErbB3. (**B**) Outline for the resistance of NSCLC against EGFR-TKI by an HGF-dependent mechanism. HGF acts through an autocrine and/or paracrine manner.

Clinical results of antiangiogenic therapy in human patients have not been as promising as expected earlier (Schmidt, 2009). Until recently there had been a question as to why tumors become resistant to antiangiogenic therapy. Experimental studies have suggested that hypoxia generated by angiogenesis inhibitor or the blockage of new blood vessels triggers signaling molecules that make tumors more aggressive and metastatic (Schmidt, 2009). In a model of pancreatic neuroendocrine cancer, inhibition of vascular endothelial cell growth factor receptor (VEGFR) tyrosine kinase shrank the primary tumor, but it also made the

cancer cells. HGF-dependent Met phosphorylation activates the PI3K-Akt pathway,

**4.4 Resistance to antiangiogenic therapy** 

gene amplification.

### **4.3 Resistance to EGFR tyrosine kinase inhibitors**

Gefitinib and erlotinib, selective inhibitors for EGFR tyrosine kinase, show favorable responses in NSCLC, especially those expressing activating mutations in EGFR (Lynch et al., 2004; Paez et al., 2004). Recent phase III clinical trials demonstrated that patients with EGFR mutant NSCLC had superior outcomes with gefitinib treatment, compared with standard first-line cytotoxic chemotherapy (Maemondo et al., 2010; Mitsudomi et al., 2010). However, almost without exception, the patients developed acquired resistance to EGFR tyrosine kinase inhibitors within several years (Morita et al., 2009). Furthermore, 20–25% of the patients with EGFR-activating mutations showed intrinsic resistance to EGFR tyrosine kinase inhibitors.

Three mechanisms have been noted to induce acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC with activating EGFR mutants. One is the T790M second mutation in *EGFR* (Kobayashi et al., 2005). Second is the amplification of the Met gene (Engelman et al., 2007) (Fig. 4A, left). The T790M second mutation occurs in about half of all patients with acquired resistance to gefitinib or erlotinib. Recent studies showed that Met gene amplification was detected in ~20% of patients with acquired resistance to gefitinib or erlotinib (Bean et al., 2007; Turke et al., 2010). As the third mechanism, HGF-dependent Met activation has been noted (Yano et al., 2008). HGF induces resistance to EGFR tyrosine kinase inhibitors in EGFR mutant lung cancer (Yano et al., 2008) (Fig. 4, right). In clinical specimens, HGF overexpression was detected in a population of specimens from EGFR mutant lung cancer patients who showed intrinsic or acquired resistance to EGFR tyrosine kinase inhibitors indicating the clinical relevance of this resistance mechanism in lung cancer (Yano et al., 2008; Turke et al., 2010; Onitsuka et al., 2010). HGF can be produced by both cancer cells and host stromal cells such as fibroblasts (Matsumoto et al., 1994; Khoury et al., 2005; Matsumoto & Nakamura, 2006) (Fig. 4B). Tumor-associated fibroblasts expressed HGF at high levels in tumors from a population of NSCLC patients, and co-injection of HGF-producing human lung fibroblast cells with gefitinib-sensitive EGFR mutant lung cancer cells caused gefitinib resistance, which could be reversed by anti-HGF antibody and NK4, an antagonist against HGF (Wang et al., 2009). These results indicated that HGF derived from host stromal cells and/or HGF secreted from cancer cells induced resistance to EGFR tyrosine kinase inhibitors through paracrine and/or autocrine actions (Fig. 4B).

In some cases, a small fraction of cells with Met gene amplification pre-exists before exposure to EGFR tyrosine kinases, and HGF accelerates expansion of cells with Met gene amplification in the presence of EGFR tyrosine kinase inhibitors (Turke et al., 2010). HGF expression was higher in the drug-resistant specimens than in the pretreatment specimens (Turke et al., 2010). The results suggested that minor clones with Met gene amplification pre-existed before treatment with EGFR tyrosine kinase inhibitors, and that HGF accelerated expansion of a pre-existing minor population of tumor cells with Met gene amplification, which showed there is a relationship between HGF level and Met gene amplification. In recent studies, the EGFR-T790M second mutation and HGF expression were detected simultaneously in acquired resistant tumors in a considerable number of patients treated with gefitinib or erlotinib. EGFR-T790M second mutation was found in 7 of 10 NSCLC patients who acquired resistance to gefitinib, and 5 of 6 cases with EGFR-T790M second mutation showed high levels of HGF expression (Onitsuka et al., 2010). In 27 patients resistant to EGFR tyrosine kinase inhibitors, EGFR-T790M second mutation was seen in 15 of 27 cases, and 11 of these 15 tumors showed high-level HGF expression (Turke et al., 2010).

Gefitinib and erlotinib, selective inhibitors for EGFR tyrosine kinase, show favorable responses in NSCLC, especially those expressing activating mutations in EGFR (Lynch et al., 2004; Paez et al., 2004). Recent phase III clinical trials demonstrated that patients with EGFR mutant NSCLC had superior outcomes with gefitinib treatment, compared with standard first-line cytotoxic chemotherapy (Maemondo et al., 2010; Mitsudomi et al., 2010). However, almost without exception, the patients developed acquired resistance to EGFR tyrosine kinase inhibitors within several years (Morita et al., 2009). Furthermore, 20–25% of the patients with EGFR-activating mutations showed intrinsic resistance to EGFR tyrosine

Three mechanisms have been noted to induce acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC with activating EGFR mutants. One is the T790M second mutation in *EGFR* (Kobayashi et al., 2005). Second is the amplification of the Met gene (Engelman et al., 2007) (Fig. 4A, left). The T790M second mutation occurs in about half of all patients with acquired resistance to gefitinib or erlotinib. Recent studies showed that Met gene amplification was detected in ~20% of patients with acquired resistance to gefitinib or erlotinib (Bean et al., 2007; Turke et al., 2010). As the third mechanism, HGF-dependent Met activation has been noted (Yano et al., 2008). HGF induces resistance to EGFR tyrosine kinase inhibitors in EGFR mutant lung cancer (Yano et al., 2008) (Fig. 4, right). In clinical specimens, HGF overexpression was detected in a population of specimens from EGFR mutant lung cancer patients who showed intrinsic or acquired resistance to EGFR tyrosine kinase inhibitors indicating the clinical relevance of this resistance mechanism in lung cancer (Yano et al., 2008; Turke et al., 2010; Onitsuka et al., 2010). HGF can be produced by both cancer cells and host stromal cells such as fibroblasts (Matsumoto et al., 1994; Khoury et al., 2005; Matsumoto & Nakamura, 2006) (Fig. 4B). Tumor-associated fibroblasts expressed HGF at high levels in tumors from a population of NSCLC patients, and co-injection of HGF-producing human lung fibroblast cells with gefitinib-sensitive EGFR mutant lung cancer cells caused gefitinib resistance, which could be reversed by anti-HGF antibody and NK4, an antagonist against HGF (Wang et al., 2009). These results indicated that HGF derived from host stromal cells and/or HGF secreted from cancer cells induced resistance to EGFR tyrosine kinase inhibitors through paracrine and/or autocrine actions

In some cases, a small fraction of cells with Met gene amplification pre-exists before exposure to EGFR tyrosine kinases, and HGF accelerates expansion of cells with Met gene amplification in the presence of EGFR tyrosine kinase inhibitors (Turke et al., 2010). HGF expression was higher in the drug-resistant specimens than in the pretreatment specimens (Turke et al., 2010). The results suggested that minor clones with Met gene amplification pre-existed before treatment with EGFR tyrosine kinase inhibitors, and that HGF accelerated expansion of a pre-existing minor population of tumor cells with Met gene amplification, which showed there is a relationship between HGF level and Met gene amplification. In recent studies, the EGFR-T790M second mutation and HGF expression were detected simultaneously in acquired resistant tumors in a considerable number of patients treated with gefitinib or erlotinib. EGFR-T790M second mutation was found in 7 of 10 NSCLC patients who acquired resistance to gefitinib, and 5 of 6 cases with EGFR-T790M second mutation showed high levels of HGF expression (Onitsuka et al., 2010). In 27 patients resistant to EGFR tyrosine kinase inhibitors, EGFR-T790M second mutation was seen in 15 of 27 cases, and 11 of these 15 tumors showed high-level HGF expression (Turke et al., 2010).

**4.3 Resistance to EGFR tyrosine kinase inhibitors** 

kinase inhibitors.

(Fig. 4B).

Collectively, expression of HGF in cancer cells and/or host stromal cells closely participated in the resistance to EGFR tyrosine kinase inhibitors in NSCLC, even in NSCLC with Met gene amplification.

Fig. 4. Drug resistance of non-small-cell lung cancer (NSCLC) against EGFR tyrosine kinase inhibitors (EGFR-TKIs) through HGF-Met pathway. (**A**) Drug resistance through Met gene amplification (left) and HGF-dependent Met activation (right). Amplified Met associates with ErbB3 activates downstream signaling such as the PI3-Akt pathway, leading to the survival of cancer cells. HGF-dependent Met phosphorylation activates the PI3K-Akt pathway, independent of EGFR and ErbB3. (**B**) Outline for the resistance of NSCLC against EGFR-TKI by an HGF-dependent mechanism. HGF acts through an autocrine and/or paracrine manner.

#### **4.4 Resistance to antiangiogenic therapy**

Clinical results of antiangiogenic therapy in human patients have not been as promising as expected earlier (Schmidt, 2009). Until recently there had been a question as to why tumors become resistant to antiangiogenic therapy. Experimental studies have suggested that hypoxia generated by angiogenesis inhibitor or the blockage of new blood vessels triggers signaling molecules that make tumors more aggressive and metastatic (Schmidt, 2009). In a model of pancreatic neuroendocrine cancer, inhibition of vascular endothelial cell growth factor receptor (VEGFR) tyrosine kinase shrank the primary tumor, but it also made the

Significance, Mechanisms, and Progress of Anticancer Drugs Targeting HGF-Met 321

Biological inhibitors against HGF-Met include the following: 1) selected domains in HGF (NK4 and engineered NK1); 2) engineered single-chain HGF forms that are resistant to proteolytic processing; 3) truncated soluble forms of the Met extracellular region; and, 4)

Among the -chain of HGF, NK2 (the N-terminal, 1st kringle, and 2nd kringle domains), an alternative splicing variant, was first shown to competitively antagonize the growth stimulation by HGF (Chan et al., 1991). However, NK2 was later shown to stimulate cell motility and enhance HGF-driven metastasis in a mouse model (Stahl et al., 1997; Yu &

NK4 is the first identified HGF-Met inhibitor devoid of biological activity through its Met binding. NK4 is composed of the N terminal and 4 kringle domains (Date et al., 1997; Matsumoto et al., 1998; Matsumoto et al., 2008). NK4 inhibits biological responses triggered by activation of HGF-Met signaling, including the spreading and invasion of cancer cells (Fig. 6). It should be emphasized that NK4 inhibits angiogenesis in addition to its antagonistic action against HGF, and this angioinhibitory action of NK4 is independent of its antagonist action against HGF. NK4 inhibited proliferation, migration, and tube formation of vascular endothelial cells induced by basic fibroblast growth factor and VEGF as well as by HGF (Kuba et al., 2001; Sakai et al., 2009). NK4 binds to perlecan and inhibits the cell-associated assembly of fibronectin, and the impaired fibronectin assembly suppresses integrin-dependent endothelial cell proliferation and migration. Having two different biological activities through completely different mechanisms is unique to NK4.

humanized monoclonal antibodies (mAbs) against HGF or Met.

Combination therapy of NK4 with antiangiogenic drugs is expected.

Fig. 6. Inhibition of tumor invasion by NK4. Invasion of human gallbladder cancer cells through the Matrigel basement membrane was induced by co-culture with stromal

fibroblasts, and this aggressive invasion was inhibited by NK4 (**A**). 3-D invasion of human pleural malignant mesothelioma in collagen gel was enhanced by HGF, and was inhibited

The therapeutic effect of NK4 has been demonstrated in a variety of cancer models (Matsumoto et al., 2008). The inhibition of tumor growth by NK4 treatment was observed in a variety of tumors, and this inhibitory effect was associated with a reduction in blood vessels in tumor tissues. NK4 treatment inhibited *in situ* Met tyrosine phosphorylation, and

**5.1 Biologicals** 

Merlino, 2002).

by NK4 (**B**).

surviving cancer more aggressive with more metastatic behavior (Casanovas et al., 2005). Pathological and clinical studies indicate that the presence of hypoxic regions within neoplastic lesions correlates with poor prognosis and an increased risk of the development of distant metastases (Höckel & Vaupel, 2001). Importantly, a hypoxic condition induced the transcriptional activation of the Met receptor gene and subsequent amplification of HGF-Met signaling, thereby increasing the invasiveness of cancer cells (Penancchietti et al., 2004). A connection between hypoxia and the Met receptor seems to explain why hypoxia often correlates with invasive and metastatic behavior. Angiogenesis inhibition retards tumor growth by oxygen deprivation, at least in part. However, hypoxia caused by the inhibition of angiogenesis enhances HGF-Met signaling, thereby promoting tumor invasion and metastasis. The involvement of the HGF-Met pathway in the aggressive characteristics in the hypoxic regions of cancers, which includes tumors treated with antiangiogenic drugs, is considerable.
