ケモカインレセプターCXCR4を標的とした口腔癌に対するリンパ節転移抑制療法の開発

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We have been investigating the correlation between the expression of a chemokine receptor CXCR4 and lymph node metastasis in oral squamous cell carcinoma (SCC). In these previous studies, we clarified as follows ; First, oral SCC cells, B88 and HNt, which have lymph node metastatic potentials in the orthotopic inoculation model into the masseter muscle of nude mice, express CXCR4. Second, overexpression of CXCR4 in non-metastatic oral SCC cells, IH, which does not express CXCR4, confers them with lymph node metastatic potential. These results suggest that the association between the expression of CXCR4 in oral SCC cells and stromal cell-derived factor (SDF)-1 produced by lymphatic stromal cells is critical for the lymph node metastasis of oral SCC. Thus, in the present study, I investigated the possibility of anti-CXCR4 targeting therapy against lymph node metastasis in oral SCC. In order to assess the possibility of this targeting therapy, 2 knock-down cell lines (siCXCR4-16, siCXCR4-17) were established by introduction of a vector that expressed short hairpin small interfering RNA (siRNA) against CXCR4 into B88 cells. Both cell lines exhibited a reduced expression of CXCR4 both in mRNA and in protein levels, and showed impaired migration in response to SDF-1. Moreover, when siCXCR4-17 cells and control cells were orthotopically inoculated into the masseter muscle of nude mice, lymph node metastasis in siCXCR4-17 cells were significantly inhibited in comparison with control cells. Thus, several agents against CXCR4 was experimentally tested for the inhibition of lymph node metastasis. First, I examined the effect of AMD3100, a CXCR4 antagonist, on the lymph node metastasis of B88 cells. SDF-1-induced migration of B88 cells was significantly inhibited by the treatment of AMD3100 in vitro. When cells were orthotopically inoculated into the masseter muscle of nude mice, administration of AMD3100 significantly inhibited the lymph node metastasis of B88 cells, but did not affect the growth of primary tumors. Second, I tested the effect of kinase inhibitors against phosphatidylinositol 3 kinase or mitogen-activated protein/extracellular signal-regulated kinase, which are involved both in the signal transduction of CXCR4 and in diverse growth signaling of the cells. Both inhibitors inhibited the cell migration induced by SDF-1 in vitro and lymph node metastasis of the cells in the orthotopic inoculation of nude mice ; however, severe body weight loss in the nude mice was observed. Third, I examined the trancriptional regulation of CXCR4 by conventional chemotherapeutic agents in oral SCC cells. Consequently, vesnarinone, a newly identified chemotherapeutic agent, trascriptionally downregulated CXCR4. Vesnarinone inhibited the cell migration induced by SDF-1 and lymph node metastasis of cells in the orthotopic inoculation model. By using several deletion constructs against CXCR4 promoters, the minimal vesnarinone-responsive element in the CXCR4 promoter region at -300 to -167 relative to the transcription start site was identified. Moreover, using a cDNA microarray method, I identified a transcription factor, krtippel-like factor 2 (KLF2), as a novel vesnarinone-responsive molecule which was able to bind to this region. The forced expression of KLF2 downregulated CXCR4 mRNA and impaired cell migration induced by SDF-1. When KLF2 was knocked down by siRNA against KLF2, CXCR4 mRNA was markedly upregulated. These results suggested that CXCR4 might be a possible molecular target for anti-metastatic therapy against lymph node metastases in cases of CXCR4-related oral SCC.
徳島大学の論文

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