**6.2 Inhibition of colon cancer metastasis by NK4 gene delivery**

Colon cancer is one of the most common cancers in the world, with a high propensity to metastasize: 30-40% of patients have metastatic disease at the initial diagnosis. The liver is the most frequent site of metastasis, and hepatic failure is a lethal event during colon cancer. Thus, direct inhibition of the dissociation, spreading and invasion of cancer cells is expected to become efficient treatment. With regard to this, HGF stimulates the invasion of MC-38 mouse colon cancer cells across MatriGel (Parr *et al*., 2000), which is composed of laminin and other matrices and mimics the basement membrane *in vivo*. In this model, NK4 has

Endocrine Delivery System of NK4, an HGF-Antagonist and

based gene therapy (Wen *et al*., 2007).

**7. Summary and perspective** 

in inhibiting MET over-activation as a negative regulator.

NK4 is a reasonable strategy to completely block tumor metastasis.

Anti-Angiogenic Regulator, for Inhibitions of Tumor Growth, Invasion and Metastasis 133

dramatically decreased by the repeated injections of NK4-cDNA containing plasmid. This study provides an anti-tumor model where NK4 is supplemented via a hydrpdynamics-

Recently, hydrodynamic gene delivery using a rapid injection of a relatively large volume of DNA solution has facilitated experimental gene therapy studies, particularly in rodents (Suda *et al*., 2007). This method is superior to the existing delivery systems because of its simplicity, efficiency, and versatility. Hydrodynamic gene delivery is also useful for supplementation of HGF, an intrinsic repair factor, for the inhibition of, or recovery from, intractable organ diseases, such as acute renal failure (Dai *et al*., 2002) or pulmonary airway hyper-responsiveness during asthma (Okunishi *et al*., 2005). In these experiments, plasma HGF levels were sustained within a pharmacological range (3-30 ng/ml). Wide success in applying hydrodynamic principles to delivery of NK4- or HGF-related DNA, RNA, proteins, and synthetic compounds, into the cells in various tissues of small animals, has inspired the recent attempts at establishing a hydrodynamic procedure for clinical use.

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

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

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.

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 vivo* in the following two studies.

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*., 2004).

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

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 dramatically decreased by the repeated injections of NK4-cDNA containing plasmid. This study provides an anti-tumor model where NK4 is supplemented via a hydrpdynamicsbased gene therapy (Wen *et al*., 2007).

Recently, hydrodynamic gene delivery using a rapid injection of a relatively large volume of DNA solution has facilitated experimental gene therapy studies, particularly in rodents (Suda *et al*., 2007). This method is superior to the existing delivery systems because of its simplicity, efficiency, and versatility. Hydrodynamic gene delivery is also useful for supplementation of HGF, an intrinsic repair factor, for the inhibition of, or recovery from, intractable organ diseases, such as acute renal failure (Dai *et al*., 2002) or pulmonary airway hyper-responsiveness during asthma (Okunishi *et al*., 2005). In these experiments, plasma HGF levels were sustained within a pharmacological range (3-30 ng/ml). Wide success in applying hydrodynamic principles to delivery of NK4- or HGF-related DNA, RNA, proteins, and synthetic compounds, into the cells in various tissues of small animals, has inspired the recent attempts at establishing a hydrodynamic procedure for clinical use.
