**2.4 Prevention of cancer cell anoikis**

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 HGF-mediated cell survival.

Endocrine Delivery System of NK4, an HGF-Antagonist and

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 123

(Matsumoto *et al*., 1996b). Herein, we will discuss the molecular basis whereby stromal HGF

The important roles of stroma during tumor progression are demonstrated through several independent studies. Carcinoma-associated fibroblasts, but not normal fibroblasts, stimulate tumor progression of initiated non-tumorigenic epithelial cells both in an *in vivo* tissue recombination and in an *in vitro* co-culture system (Olumi *et al*., 1999). Transforming growth factor (TGF)-β signaling is critical for down-regulating HGF production (Matsumoto *et al*., 1992). Of note, an inactivation of TGF-β type II receptor gene in stromal fibroblasts leads to the onset of epithelial growth and invasion (Bhowmick *et al*., 2004). In this process, activation of paracrine HGF is a key mechanism for stimulation of epithelial proliferation (Bhowmick *et al*., 2004). Thus, the suppression of HGF production by TGF-β seems to be

As repeated, a major source of HGF in tumors is stromal cells (including fibroblasts, endothelium, macrophages and neutrophils) (Wislez *et al*., 2003; Matsumoto & Nakamura, 2006; Grugan *et al*., 2010). Thus, how stromal HGF is up-regulated during tumor progression should be discussed. There is now ample evidence that numerous types of carcinoma cells secrete soluble factors that induce HGF production in stromal cells (*i.e*., HGF-inducers). For example, conditioned medium obtained from breast cancer cells enhances HGF production in fibroblasts, along with a raise in prostaglandin-E2 (Matsumoto-Taniura *et al*., 1999). Of note, suppression of prostaglandin-E2 production by indomethacin leads to downregulation of stromal HGF production and suppression of tumor migration *in vitro* (Matsumoto-Taniura *et al*., 1999), indicating that cancer-derived prostaglandins are important for up-regulating HGF in stromal cells (Matsumoto-Taniura *et al*., 1999; Pai *et al*., 2003). Other carcinoma-derived HGF-inducers are interleukin-1β (IL-1β), basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), and TGF-α (Hasina *et al*., 1999; Matsumoto & Nakamura, 2003). These results indicate a crosstalk between carcinoma and stroma, mediated via a paracrine loop of HGF-inducers produced by carcinoma and

production is up-regulated by tumor cells during cancer invasion and metastasis.

important for an escape from cancer metastasis (Matsumoto & Nakamura, 2006).

HGF secreted from stroma cells, such as fibroblasts (Matsumoto *et al*., 1996a).

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* 

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

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

*al*., 2003).

**3.2 Regulation of HGF production in stroma by tumor cells** 

**3.1 Stroma as a microenvironment to determine behaviors of tumors** 

Fig. 1. Various effect of HGF on cancer cells and endothelial cells (EC) during tumor progression. For example, sequential events during the lung metastasis of hepatic carcinoma are summarized as follows: (A) dissociation and scattering of hepatocellular cancer cells through an HGF-induced endocytosis of cadherins; (B) tumor migration into stromal areas across the basement membrane (BM) is mediated via MMP-dependent matrix degradation and Rho-dependent cell movement; (C) invasion of tumor cells into neighboring vessels (*i.e*., intravasation) where the tight junction between ECs is lost by HGF-MET signaling; (D) inhibition of tumor cell anoikisis by MET-AKT cascades during blood flow, and out-flux of tumor cells across vessel walls (*i.e*., extravasation); and (E) in the lung, HGF supports growth of metastatic nodules via providing vascular beds as an angiogenic factor.

Overall, HGF is shown to take direct action on carcinoma cells: (i) cell spreading via an endocytosis of cadherins; (ii) enhancement of invasion across basement membranes via Rhodependent and MMP-dependent pathways; and (iii) anti-anoikis activity during blood circulation. Toward tumor vessels, HGF elicits vascular and lymphatic EC proliferation and branching angiogenesis, while intravasation and extravasation are achieved through HGFinduced reduction of EC-EC integrity. These HGF-MET-mediated biological functions seem advantageous for invasion and metastasis of malignant tumors, including carcinoma and sarcoma (**Fig. 1**).

**[Note]** Long-term administration of recombinant HGF does not elicit tumor formation in healthy animals, and this result supports a rationale of HGF supplement therapy for treating chronic organ diseases, such as liver cirrhosis, at least in cancer-free patients.
