**7. Summary and perspective**

132 Hydrodynamics – Advanced Topics

inhibited the HGF-mediated migration of MC-38 cells in a culture model of colon cancer invasion. This anti-invasive effect of NK4, obtained by *in vitro* studies, is demonstrated *in* 

An hepatic metastatic model was prepared by the injection of mouse MC-38 cells into the spleen. During the progression of colon cancer in hepatic tissues, HGF was over-produced by hepatic sinusoidal cells, while MET tyrosine phosphorylation became evident, particularly around the front lines of invasive zones. Supplementation of NK4 in blood and livers via a single injection of NK4-cDNA containing plasmid (pCAGGS-NK4) resulted in the loss of MET tyrosine phosphorylation (**Fig. 6**). Under such a MET-inactivated condition by NK4 treatment, hepatic invasion by colon carcinoma was strongly inhibited (Wen *et al*.,

Fig. 6. Successful outcome of hydrodynamics-based NK4 gene therapy in a mouse model of colon cancer. The hepatic invasion model is prepared by intra-splenic inoculation of MC-38 colon carcinoma in mice. In the control group, invasion of carcinoma cells into neighboring hepatic areas becomes evident, along with an induction of MET tyrosine phosphorylation (p-MET) and an increase in vessel numbers. In contrast, NK4 suppresses tumor invasion by inhibiting MET tyrosine phosphorylation and reducing angiogenesis. As a result, NK4 gene

Repeated administrations of NK4-containing plasmid DNA also inhibited the malignant behaviors of colon carcinoma (Wen *et al*., 2007). Actually, NK4 repetitive gene therapy potently inhibited the muscular invasion of MC-38 carcinoma cells. Furthermore, angiogenesis in the colon cancer was markedly suppressed by NK4 repetitive therapy, along with an increase in tumor apoptosis. Overall, the number of hepatic metastatic nodules was

therapy prolongs the survival of these mice (Wen *et al*., 2004).

*vivo* in the following two studies.

2004).

NK4-related studies provided a proof-of-concept that MET signaling from stroma-derived HGF plays a pivotal role in eliciting tumor invasion and metastasis (Matsumoto & Nakamura., 2005; Nakamura *et al*., 2010). Human genetic studies also strengthened the important role of MET activation for tumor malignancy. There is now ample evidence to demonstrate the role of MET mutations in tumor malignancy (Lengyel *et al*., 2007; Matsumoto *et al*., 2008a,b; Pao *et al*., 2011). Of interest, mutation of the von-Hippel-Lindau (VHL) gene leads to renal clear cell carcinoma through constitutive MET tyrosine phosphorylation (Nakaigawa *et al*., 2006), hence suggesting a critical role of wild-type VHL in inhibiting MET over-activation as a negative regulator.

During the progression of malignant tumors, soluble MET is producible by carcinoma cells through an ectodomain shedding cascade (Wader *et al*., 2011). Soluble MET inhibits the HGF-MET complex and signaling transduction. Thus, MET shedding system is considered as a self-defense response that minimizes tumor metastasis. Likewise, an NK4-like fragment of the HGF α-chain can be secreted from human breast carcinoma, which inhibits MET tyrosine phosphorylation (Wright *et al*., 2009). Thus, "endogenous" soluble MET and NK4 like variant appear to reduce HGF-MET signaling and delay tumor progression, but this response is insufficient, allowing for tumor metastasis. Thus, supplemental therapy with NK4 is a reasonable strategy to completely block tumor metastasis.

The hope is that angiogenesis inhibition might control tumor metastasis (Yancopoulos *et al*., 1998). However, long-term use of angiogenesis inhibitors, such as VEGF inhibitor, results in hypoxia-resistance (Fischer *et al*., 2007), possibly due to hypoxia-induced MET up-regulation by cancer (Bottaro & Liotta, 2003). NK4 is an angiogenesis inhibitor with the ability to inhibit MET activation, and discovery of this fragment opened up a new avenue for the development of freeze-and-dormancy therapy (**Fig. 7**). Thus, NK4 is now defined as "Malignostatin". In addition to NK4, several anti-metastatic drugs have been proposed, with a major focus on small molecules that inhibit the tyrosine kinase activity of MET; ribozyme; small-interfering RNA; anti-HGF antibodies; soluble MET; and HGF-variant decoys (Jiang *et al*., 2005; Benvenuti & Comoglio, 2007; Eder *et al*., 2009; Underiner *et al*., 2010; Cecchi *et al*., 2010). HGF-MET targeting research will shed more light on cancer biology, pathology and new technologies to overcome host death due to cancer metastasis.

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Fig. 7. Freeze-and-dormancy therapy of malignant tumors by NK4/malignostatin. NK4 blocks tumor invasion and metastasis through an inhibition of HGF-MET signals as an HGF-antagonist. Furthermore, NK4 inhibits tumor angiogenesis via a perlecan-dependent mechanism. Such a dual function of NK4 contributes to "freeze" and "dormancy" anticancer therapy.
