**1. Introduction**

118 Hydrodynamics – Advanced Topics

Yusupov V.I., Chudnovskii V.M., Kortunov I.V., Bagratashvili V.N. (2011b). Laser-induced

No. 3, (March 2011), pp. 214–218, ISSN 1612-2011

self-organization of filaments from Ag nanoparticles. *Laser Physics Letters*, Vol. 8,

Estimates of the worldwide incidence and mortality from 27 cancers in 2008 have been prepared for 182 countries by the International Agency for Research on Cancer (Ferlay *et al*., 2010). Overall, an estimated 12.7 million new cancer cases and 7.6 million cancer deaths occur in 2008, with 56% of new cancer cases and 63% of the cancer deaths occurring in the less developed regions of the world. The most commonly diagnosed cancers worldwide are lung (1.61 million, 12.7% of the total), breast (1.38 million, 10.9%) and colorectal cancers (1.23 million, 9.7%). Cancer is neither rare anywhere in the world, nor mainly confined to highresource countries. Many cancer subjects die from cancer as a result of organ failure due to "metastasis" (Geiger & Peeper, 2009), thus indicating that medical control of tumor metastasis leads to a marked improvement in cancer prognosis.

The acquisition of the metastatic phenotype is not simply the result of oncogene mutations, but instead is achieved through an interstitial stepwise selection process (Mueller & Fusenig, 2004). The dissociation and migration of cancer cells, together with a breakdown of basement membranes between the parenchyme and stroma, are a prerequisite for tumor invasion. The next sequential events involved in cancer metastasis include the following: (i) penetration of cancer cells to adjacent vessels (*i.e*., intravasation); (ii) suppressed anoikis (*i.e*., suspension-induced apoptosis) of cancer cells in blood flow; and (iii) an extravascular migration and re-growth of metastatic cells in the secondary organ. For an establishment of anti-metastasis therapy, it is important to elucidate the basic mechanism(s) whereby tumor metastasis is achieved through a molecular event(s).

Hepatocyte growth factor (HGF) was discovered and cloned as a potent mitogen of rat hepatocytes in a primary culture system (Nakamura *et al*., 1984, 1989; Nakamura, 1991). Beyond its name, HGF is now recognized as an essential organotrophic regulator in almost all tissues (Nakamura, 1991; Rubin *et al*., 1993; Zarnegar & Michalopoulos, 1995; Birchmeier & Gherardi, 1998; Nakamura & Mizuno, 2010). Actually, HGF induces mitogenic, motogenic

Endocrine Delivery System of NK4, an HGF-Antagonist and

**2.2 Breakdown of basement membranes** 

down-stream mechanism of MET-mediated cancer invasion.

**2.3 Endothelial attachment and extravasation of cancer cells** 

tumors and extravasation in metastatic organs) (**Fig. 1**).

**2.4 Prevention of cancer cell anoikis** 

HGF-mediated cell survival.

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 121

formation and cell spreading are enhanced by HGF (Royal *et al*., 2000). These changes confer a

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.

Needless to say, tumor angiogenesis as well as lymphatic vessel formation are important for delivery of cancer cells from the primary tumor to secondary organs. HGF enhances angiogenesis via induction of the proliferation and morphogenesis of endothelial cells (EC) (Bussolino *et al*., 1992; Nakamura *et al*., 1996). Actually, HGF supplementation leads to the enhancement of tumor angiogenesis *in vivo* (Laterra *et al*., 1997). Recent studies delineated the capacity of HGF to induce lymphatic morphogenesis (Kajiya *et al*., 2005; Saito *et al*., 2006). Thus, HGF is considered to facilitate cancer metastasis via neo-induction of vascular or lymphatic vessel beds. HGF has a direct effect on EC for enhancing tight adhesion of tumor cells on endothelium via FAK phosphorylation (Kubota *et al*., 2009a). Furthermore, HGF decreases endothelial occludin, a cell-cell adhesion molecule (Jiang *et al*., 1999a). Under such a loss of EC-EC integrity, HGF decreases the trans-endothelial resistance of tumor vessels and enhances cancer invasion across an EC barrier (*i.e.,* intravasation in primary

Anoikis, also known as suspension-induced apoptosis, is a term used to describe programmed cell death (apoptosis) of epithelial cells induced by loss of matrix attachment. In addition to gaining functions of invasion and angiogenesis, cell resistance to anoikis also appears to play an important role in tumor progression and metastasis as tumor cells lose matrix attachment during metastasis. However, it is unknown how cancer cells escape from anoikis-like death during metastasis. It was demonstrated, in a non-adherent culture models, that HGF is a key molecule inhibiting suspension-induced anoikis, and this effect is mediated via a crosstalk that is, in turn, mediated by phosphatidyl-inositol 3-kinase (PI-3K) and extracellular signal-regulated kinase (ERK)-1/2 (Zeng *et al*., 2002; Kanayama *et al*., 2008). A recent report described that tetraspanin CD151-knockdown abolishes preventive effect of HGF on tumor anoikis (Franco *et al*., 2010). Thus, it is likely that cell surface tetraspanins are important for signaling complexes between MET and integrin-β4, a known amplifier of

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

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" gene therapy.
