**5. Anti-cancer therapy using NK4 in animal models**

Carcinoma and sarcoma show malignant phenotypes prompted by a stroma-derived HGF-MET signal at least *in vitro*. If NK4 could block MET signaling as an HGF-antagonist *in vivo*, supplemental therapy with NK4 would be a pathogenesis-based strategy to counteract

Fig. 3. Anti-angiogenic effects of NK4 via a perlecan-dependent mechanism. (A) NK4 suppresses HGF-, b-FGF-, and VEGF-induced proliferation of EC *in vitro* (Kuba *et al*., 2000). (B) Inhibition of b-FGF-induced corneal neovascularization by NK4 treatment in rabbits. (C) Involvement of perlecan (PC) in NK4-mediated growth arrest of EC. Left: Cell surface PC is required for the binding of fibronectin and α5β1-integrin, leading to FAK phosphorylation and crosstalk of VEGF-VEGF receptor (KDR) signaling. Right: NK4 binds to PC, and then the binding of integrin to fibronectin is impaired. As a result, VEGF fails to elicit G1/S

We have accumulated *in vitro* evidence showing that HGF-MET system may elicit cancer invasion via a paracrine loop of stroma-carcinoma interaction. This phenomenon is also demonstrated *in vivo*: anti-HGF antibody potently suppressed the tumor invasion in a mouse model of pancreas cancer (Tomiola *et al*., 2001). On the other hand, several investigators proposed, in the late-1990's, a new concept that tumor angiogenesis inhibition leads to the arrest of cancer growth and metastasis (Yancopoulos *et al*., 1998). Inhibition of tumor angiogenesis leads to local hypoxia, and then apoptotic death of cancer cells is associated with the arrests of tumor growth and metastasis (*i.e*., cytostatic therapy). In this regard, NK4 also elicits an anti-angiogenic effect via perlecan-dependent mechanism. Thus, bi-functional properties of NK4 as an HGF antagonist and angiogenesis inhibitor raise a

Carcinoma and sarcoma show malignant phenotypes prompted by a stroma-derived HGF-MET signal at least *in vitro*. If NK4 could block MET signaling as an HGF-antagonist *in vivo*, supplemental therapy with NK4 would be a pathogenesis-based strategy to counteract

progression of EC in the presence of NK4 (Sakai *et al*., 2009).

possibility that NK4 may prove therapeutic for cancer patients, as follows.

**5. Anti-cancer therapy using NK4 in animal models** 

tumor invasion and metastasis. This hypothesis is widely demonstrated through extensive studies using tumor-bearing animals, as described below.
