**3.3 Inflammation-mediated HGF up-regulation mechanism**

In addition to stromal fibroblasts, tumor-associated macrophages (TAM) are known to highly produce HGF during non-small lung cancer invasion (Wang *et al*., 2011). It is reported that TAM isolated from 98 primary lung cancer tissues show the higher production of HGF, along with the concomitant increases in urokinase-type plasmin activator (uPA), cyclooxygenase-2 (Cox2) and MMP-9 (Wang *et al*., 2011). Anti-MMP-9 antibody largely diminishes TAM-induced invasion, while Cox2 and uPA are critical for HGF production and activation, respectively, suggesting that Cox2-uPA-HGF-MMP cascades in TAM participate in non-small lung cancer invasion. Likewise, HGF production is enhanced by neutrophils infiltrating bronchiolo-alveolar subtype pulmonary adenocarcinoma (Wislez *et al*., 2003).

Clinical studies demonstrate that serum levels of HGF are elevated in patients with recurrent malignant tumors (Wu *et al*., 1998; Osada *et al*., 2008), thus suggesting an

Endocrine Delivery System of NK4, an HGF-Antagonist and

invasion in a dose-dependent manner.

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 125

Fig. 2. Preparation of NK4 as an HGF-antagonist and its inhibitory effects on tumor invasion *in vitro*. (A) Preparation and structure of NK4. NK4 is generated via a cleavage of HGF between 478th Val and 479th Asn. (B) Inhibition of HGF-mediated MET tyrosine

phosphorylation by NK4 in lung carcinoma cells. (C) Biological activity of NK4. Cancer cell invasion (upper chamber) is induced across a Matrigel layer when fibroblasts (FB) are placed on a lower chamber. In this co-culture system, NK4 inhibits FB-induced tumor cell

growth factor (VEGF) (**Fig. 3A**) (Kuba *et al*., 2000). When a pellet containing b-FGF was implanted under the rabbit cornea, angiogenesis was rapidly induced. In this model, NK4 inhibited b-FGF-induced angiogenesis (**Fig. 3B**). *In vitro* models of EC proliferation, HGF and VEGF phosphorylate MET and KDR/VEGF receptor, respectively, whereas NK4 inhibits HGF-induced MET tyrosine phosphorylation, but not VEGF-induced KDR phosphorylation (Kuba *et al*., 2000). Nevertheless, NK4 inhibited the VEGF-mediated EC proliferation without modification of VEGF-mediated ERK1/2 (p44/42 mitogen-activated protein kinase) activation. These results suggest the presence of another mechanism

The fibronectin-integrin signal is essential for the spreading and proliferation of EC. Based on this background, we demonstrated that NK4-mediated growth arrest of EC is due to a loss of the fibronectin-integrin signal. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan (Sakai *et al*., 2009). Consistent with this result, NK4 was co-localized with perlecan in EC. Perlecan is a multi-domain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. Of interest, knockdown of perlecan expression by siRNA diminished the fibronectin assembly and EC spreading, indicating an essential role of fibronectin-perlecan interaction during EC movement. A recent report described that NK4-perlecan interaction suppressed the normal assembly of fibronectin by perlecan (Sakai *et al*., 2009). As a result, FAK activation became faint in EC after NK4 treatment. Under such a loss of fibronectin-integrin signaling by NK4, EC growth and motility were suppressed, even in the presence of b-FGF or VEGF. This is

whereby NK4 inhibits VEGF- and b-FGF-mediated angiogenesis.

the reason why NK4 arrests b-FGF- or VEGF-mediated angiogenesis (**Fig. 3C**).

endocrine mechanism of the HGF delivery system. In this regard, it is known that peripheral blood monocytes produce HGF, contributing to the increase in blood HGF levels via an endocrine mechanism (Beppu *et al*., 2001). Overall, production of HGF by inflammatory cells is involved in carcinoma invasion and metastasis (*i.e.*, local system), while peripheral blood monocytes seem to prevent tumor cell anoikis during metastasis, possibly by a release of HGF into blood (*i.e*., systemic system).
