The objective of our research is to elucidate the mechanisms of how cancer cells acquire the malignant characteristics involved in tumor progression, by analyzing the changes in cellular signal transduction. The involvement of tyrosine kinases and phosphotyrosine-containing substrates of tyrosine kinases in cancer metastasis, invasion, and anchorage-independent growth is being intensively studied. This information will be further utilized to propose novel targets for phenotype-specific cancer chemotherapy.

High expression of ephrin-B1, a ligand of the EphB family of tyrosine kinases, is observed in various cancer cells and is correlated with tumor invasion. We demonstrated that ephrin-B1- mediated cellular signaling regulates the secretion of matrix metalloproteinase-8 (MMP-8). In our study, the ephrin-B1 ectodomain was processed by MMP-8 and secreted into the culture medium of the human pancreatic cancer cell lines. The activity of MMP-8 was enhanced by stimulation of the EphB2 receptor, which did not require de novo protein synthesis, but depended on signaling through the carboxyl terminus of ephrin-B1. Stimulation of ephrin-B1 accelerated the process of intracellular transport and secretion of MMP-8. Moreover, the invasiveness of tumor cells expressing epherin-B1 was promoted by the stimulation with the EphB2 receptor, through MMP-8 activation. Our results suggest the possibility that the interaction of ephrin-B1-expressing cancer cells with the surrounding stromal cells expressing EphB receptors promotes the invasiveness of cancer cells by activating a specific set of metalloproteinases ⁽⁵⁹⁾. Reduction of ephrin-B1 expression by RNA interference (RNAi) or overexpression of mutant ephrin-B1 lacking the phosphorylation sites suppressed migration and invasion of scirrhous gastric cancer cells in vitro, without affecting the proliferative or apoptotic activity of the tumor cells. Blocking of tyrosine phosphorylation of ephrin-B1 attenuated not only dissemination of cancer cells injected intraperitoneally, but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice ⁽⁶⁰⁾. Our results suggest that tyrosine phosphorylation of ephrin-B1 may promote invasion of cancer cells in vivo, and is a potential therapeutic target in some types of gastrointestinal cancers.

Malignant tumor cells frequently exhibit resistance to anoikis, a form of apoptosis induced by detachment from the extracellular matrix, which results in anchorage-independent growth of the cells. This property of cancer cells is thought to be essential for the process of distant metastasis of tumors. Several tyrosine-phosphorylated proteins characteristic to a subset of anchorage-independent lung cancer cell lines were associated with Fyn kinase, a member of the Src family kinases (SFK), in the suspension culture condition. Two of the major Fyn-associated phosphoproteins turned out to be a single protein CUB-domain containing protein 1 (CDCP1). Using RNAi suppression and overexpression of CDCP1 mutants in lung cancer cells, we demonstrated that tyrosine- phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-associated molecule, PKCd, was found to be phosphorylated by the CDCP1-SFK complex and was essential for the resistance to anoikis downstream of CDCP1. Loss of CDCP1 by stable expression of CDCP1 siRNA also inhibited the metastatic potential of the A549 cells in vivo ⁽⁶¹⁾.

Expression and phosphorylation levels of CDCP1 were also correlated with the highly invasive potential of scirrhous gastric cancers. Expression and phosphorylation of CDCP1 was also detected by immunohistochemical analysis in cancer cells derived from surgically resected tissues of human scirrhous gastric cancer. Reduction of CDCP1 expression by siRNA suppressed the migration, invasion and anchorage-independence, without affecting the proliferative potential, of highly invasive scirrhous gastric cancer cells. On the other hand, overexpression of CDCP1 promoted migration of gastric cancer cells with a low potential of invasion. These findings indicate that CDCP1 may be a novel target for treating cancer-specific processes such as metastasis and invasion.

Cortactin was frequently shown to be phosphorylated in cancer cells by various tyrosine kinases. We analyzed the effect of suppression of cortactin expression using RNAi in gastric and breast cancer cell lines. The results revealed that loss of cortactin blocked cell migration in cancer cells in the presence of a low level of phosphorylation of cortactin. On the other hand, loss of cortactin enhanced cell migration and tyrosine phosphorylation of p130Cas in the presence of high levels of phosphorylation cortactin. Consistent results were obtained when exogenous Fyn kinase was stably expressed in the breast cancer cell line MCF7. This study suggested that hyperphosphorylated cortactin inhibits tyrosine phosphorylation of p130Cas, resulting in suppression of cell migration.

A novel molecule, ADI1, which regulates cancer invasion through association with metalloproteinases was also analyzed ⁽⁶²⁾.

The RET proto-oncogene codes for receptor-type tyrosine kinase and is the causative gene of multiple endocrine neoplasia type 2. The effect of suppression of Ret on the growth, migration and survival of medullary thyroid carcinoma cells and neuroblastoma cells was analyzed using the RNAi method. The role of RET protein expression in GDNF-dependent enhancement of proliferation of carcinoma cells has been demonstrated.

The Shc family docking proteins possess two different phosphotyrosines-binding motifs and transmit signals of various receptor tyrosine kinases, such as ALK, TRK and RET. We have shown that ShcC, a neuro-specific member of the Shc family proteins, is hyperphosphorylated in some of the neuroblastoma cell lines that show activation of the ALK protein and plays essential roles in the proliferation of these cell lines. Many other neuronlastoma cell lines show overexpression of moderately phosphorylated ShcC protein. Suppression of the expression of ShcC by specific siRNA induced neurite outgrowth of neuroblastoma cells, such as TNB-1, which overexpress ShcC. At the same time, appearance of several neuronal markers and sustained activation of ERK was observed, suggesting at suppression of ShcC induced differentiation of neuroblastoma cells. Suppression of the ShcC protein in TNB-1 cells significantly inhibited tumor formation in the nude mice. By overexpression of the wild type and mutant ShcC proteins, it was revealed that ShcC negatively regulates the ShcA pathway in a phosphorylation-independent manner.