Many tumor cells are generated in epithelial tissues (for example, cells covering the inside of the stomach or intestines). The tumor cells migrate from the primary site break down the basal membrane and then enter into the surrounding tissues. This process is referred to as tumor invasion.
The tumor cells systemically move to distant sites via blood or lymph vessel, and proliferate to form a new colony (metastatic focus). This is the mechanism involved in tumor invasion and metastasis, andultimately results in cancer being among the most common causes of death in developed countries (Fig.1).
Fig.1: Click for large view

Multistep mutagenesis in oncogenes and tumor-suppressor genes form the genetic background to the development of cancer. However, the malignant behaviors of a variety of tumor cells, such as invasion, metastasis, and angiogenesis, are regulated by their interactions with host stromal cells, as tumor-stromal interactions (Fig.2). For instance, when human gallbladder cancer cells are cultured on a collagen gel, they do not invade the gel. However, when stromal fibroblasts from gallbladder stroma are co-cultured with cancer cells, cancer cells actively invade the collagen gel.
This result indicates that fibroblasts secrete a factor that induces the invasion of cancer cells. Similar results have also been obtained in various other types of cancer cells. Drs. Nakamura and Matsumoto identified that this factor was the protein HGF, hepatocyte growth factor. A number of studies indicate that HGF is a highly potent stimulant for invasion and metastasis in a variety of cancer cells. Based on this background, it is thought that the blockage of the HGF/cMet receptor interaction may function as a novel therapeutic strategy to inhibit tumor invasion and metastasis.
Fig.2: Click for large view

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors. Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, and therefore supply nutrients that allow the tumor to grow, Therefore, angiogenesis is a necessary step for the transition from a small harmless cluster of cells, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis. Therefore, the inhibition of angiogenesis has the potential to form the basis of new anti-cancer strategies.

After several attempts to prepare an HGF-antagonist, we were able to isolate an HGF-antagonist from elastase-digested HGF molecules(Fig.3). The elastase-treatment produces two intermolecular fragments. Amino acid sequence analysis indicated that the fragment derived from the a-chain contains an N-terminal hairpin domain (N-domain) and four kringle domains (four K-domains; thus we named the molecule NK4). NK4 contains the cMet receptor binding domain, but NK4 has no biological activity. NK4 competitively displaces HGF to bind to the cMet receptor, but NK4 itself does not activate the cMet receptor (Fig.3).
Therefore, NK4 is a competitive antagonist of HGF/cMet coupling. Although the smaller variants, NK1, NK2, and NK3, can bind the cMet receptor, these variants have agonistic activities, including stimulation of tumor invasion. Furthermore, NK4 was subsequently shown to be act as a potent angiogenesis inhibitor. NK4 inhibits the angiogenic responses induced by bFGF, VEGF and HGF. This angioinhibitory activity is independent of its action as an HGF-antagonist (Fig.4).

Fig. 3: Click for large view

Fig. 4: Click for large view

NK4 is bifunctional. NK4 is an HGF-antagonist that inhibits tumor invasion and metastasis mediated by tumor-stromal interactions. Furthermore, NK4 is an angiogenesis inhibitor that inhibits angiogenic responses via interaction with various distinct angiogenic growth factors. The bifunctional characteristic of NK4 as both an angiogenesis inhibitor and an HGF-antagonist is highly unique and provides a therapeutic basis to suppress the malignant behavior of cancer, and treat it as though it were dormant or static (Fig.5).

Fig. 5: Click for large view

Although many pharmaceutical companies have been developing angiogenesis inhibitors as cancer drugs, none of them have unique bifunctionality like NK4( HGF-antagonist and anti-angiogenesis activity). Therefore, NK4, as a novel anti-cancer drug, harbors enormous potential to contribute to global healthcare in the 21st century. In mice experimental models, NK4 treatment via either by gene or protein yielded dramatic therapeutic effects on a wide variety of solid tumors, as summarized in Table 1.

Therapeutic Effects of NK4 on Cancers in Mice:
CancersNK4 treatmentTherapeutic effects
Gene therapy
Inhibition of tumor growth, metastasis angiogenesis
BreastProteinInhibition of tumor growth, metastasis angiogenesis
Gene therapy
Inhibition of invasion, tumor growth
Gene therapy
Inhibition of tumor growth, angiogenesis, peritoneal dissemination, ascites accumulation, prolonged survival
StomachGene therapyInhibition of peritoneal dissemination
ProstateProteinInhibition of tumor growth,angiogenesis
OvaryGene therapyInhibition of peritoneal dissemination, ascites accumulation, prolonged survival
ColonGene therapyInhibition of growth, metastasis, angiogenesis
MelanomaGene therapyInhibition of growth, metastasis, angiogenesis
GlioblastomaProteinInhibition of growth, metastasis, angiogenesis
LymphomaProteinInhibition of growth, metastasis, angiogenesis

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