**2.2 Breakdown of basement membranes**

120 Hydrodynamics – Advanced Topics

and morphogenic activities in various types of cells via its receptor, MET (Bottaro *et al*., 1991; Higuchi *et al*., 1992). HGF is required for organogenesis in an embryonic stage and for tissue repair in adulthood during various diseases (Nakamura, 1991; Birchmeier & Gherardi, 1998; Nakamura & Mizuno, 2010). Several lines of *in vitro* studies indicate that HGF stimulates scattering and migration of cancer cells (Matsumoto *et al.,* 1994, 1996a; Nakamura *et al*., 1997). In malignant tumors, HGF is expressed by stromal cells, such as fibroblasts, while MET is over-expressed by cancer cells, thus suggesting in the mid-1990s that a paracrine signal from HGF-producing stroma cells to carcinomas may cause malignant behaviors,

NK4 is an intra-molecular fragment of HGF, which is generated by a chemical cleavage of mature form HGF (Date *et al*., 1997; Nakamura *et al*., 2010). NK4 includes an N-terminal hairpin domain and 4-kringle domains (K1-K4) of HGF α-chain, which binds to MET. Thus, NK4 antagonizes HGF activities as a competitive inhibitor. Using NK4 as an HGFantagonist in rodents with malignant tumors, we have accumulated evidence showing that endogenous HGF-MET cascade is a key conductor for tumor metastasis, while inhibition of MET signals leads to the arrests of tumor growth. Unexpectedly, NK4 prohibits tumor angiogenesis through a MET-independent mechanism. This review focuses on the roles of HGF in cancer biology and pathology. We also emphasize the effectiveness of NK4 in experimental cancer models where NK4 is supplemented via a "hydrodynamics-based"

In the mid-1980s, MET was identified as a mutated oncogene from carcinogen-induced osteosarcoma cells (MNNG-HOS) that transform NIH3T3 fibroblasts (Cooper *et al*., 1984). MET*-*encoding protein has a tyrosine kinase activity (Dean *et al*., 1985), suggesting that MET may be an orphan receptor of growth factors. In the early 1990s, MET-coding product was demonstrated to be a high-affinity receptor for HGF (Bottaro *et al*., 1991; Higuchi *et al*., 1992). Scatter factor (SF) stimulates tumor cell movement, as its name indicates, and is shown molecularly identical to HGF (Konishi *et al*., 1991; Weidner *et al*., 1991). HGF has several activities required for tumor cell invasion and metastasis, as described below. In this section, we summarize the direct effects of HGF on intra-tumor cells, including carcinoma, and on vascular and lymphatic cells prior to discussion of the contribution of HGF-MET cascades

Initial events for the metastatic spread of tumors involve loss of cell-cell contact within the primary tumor mass. The integrity and morphology of epithelial tumor cell colonies are maintained by cell-cell contact mediated by cadherins and its associated intracellular catenin molecules. Cancer cells must lose their tight cell-to-cell contact by down-regulation of cadherin-cadherin complex during invasion into adjacent tissues. HGF induces scattering (*i.e*., dispersion of cluster cells into single cells) via an endocytosis of E-cadherin from cell surface to cytoplasma (Watabe *et al*., 1993; Miura *et al*., 2001). During cell migration, HGF activates the Ras-Rab5 pathway for endocytosis of cadherins (Kimura *et al*., 2006), which triggers nuclear localization of β-catenin, a transcription factor of genes responsible for cell motility (Hiscox & Jiang, 1999). Stimulation of an Rho small G protein cascade and activation of cdc42, rac and PAK by HGF leads to the disassembly of stress fiber or focal adhesions, while lamellipodia

**2. Effects of HGF on intra-tumor cells during cancer progression** 

such as invasion and metastasis (Matsumoto *et al*., 1996b).

gene therapy.

during tumor malignancy.

**2.1 Scattering and migration of tumor cells** 

During cancer invasion, tumor cells must move across a basement membrane between epithelium and lamina propria (*i.e.,* sub-epithelium). HGF stimulates motility in a biphasic process: cells spread rapidly and form focal adhesions, and then they disassemble these condensations, followed by increased cell locomotion. In the early phase (*i.e*., within a few minutes post-stimulation), HGF induces phosphorylation of focal adhesion kinase (FAK) together with a tight bridge between the extra-cellular matrix (ECM) and integrins of cancer cells (Matsumoto *et al*., 1994; Parr *et al*., 2001). In the later phase, HGF-stimulated cancer cells invade into matrix-based gels *in vitro*, or across basement membrane ECM *in vivo* (Nakamura *et al*., 1997). In this process, HGF up-regulates several types of matrix metalloproteinase (MMP), such as MMP-1, -2, and -9, through activation of Ets, a transcriptional factor of MMPs (Li *et al*., 1998; Nagakawa *et al*., 2000; Jiang *et al*., 2001). Considering that MMP-inhibitors diminish HGF-mediated migration, the induction of MMP through HGF-Ets cascade is essential for tumor invasion into adjacent normal tissues.
