Tumor Necrosis Factor (TNF)-α promotes MMP-9 secretion and invasiveness of cholangiocarcinoma via TNFR2

Y Tanimura MD^a,b, T Kokuryo MD^a, K Oda MD PhD^a, Y Nimura MD PhD^a, Y Nakanuma MD PhD^c, M Hamaguchi MD PhD^b

^aDivision of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Aichi, ^bDivision of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan, ^cDepartment of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa, Japan

AIM: Inflammatory process appears to be involved in tumor promotion. High-risk factors for the prognosis of cholangiocarcinoma such as hepatolithiasis are often associated with long-standing inflammation. By cDNA array analysis, we found frequent over-expression of TNFR1and TNFR2, receptors for Tumor Necrosis Factor (TNF)-α, in tissues of cholangiocarcinoma with hepatolithiasis so that we focused on the role of pro-inflammatory cytokine, TNF-α in tumor invasion. The aim of this study is to investigate the invasiveness of the cholangiocarcinoma cells by TNF-α and intracellular signaling critical for the TNF-α dependent-invasion. METHODS: 1. Gene expression for obtained three cases of cholangiocarcinoma combined with hepatolithiasis was analyzed by cDNA array. 2. In human cholangiocarcinoma CCKS1 cells, invasiveness with TNF-α treatment was examined by a modified Boyden chamber method. TNF-α dependent Matrix Metalloproteinase (MMP) secretion was assayed by gelatin zymography. 3. TNFR1 and TNFR2 expression of human cholangiocarcinoma HuH-28, HuCCT1, TFK-1 and CCKS1 cells in protein and mRNA levels were examined by Western-blot and RT-PCR, respectively. RESUTS: We found that 1.Over-expression of TNFR1and TNFR2 gene in cholangiocarcinoma tissues. 2.TNF-α promoted invasiveness and MMP-9-secretion in CCKS1 cells. 3. Treatment of cells with Ras inhibitor (Manumycin A), MEK inhibitor (U0126) or MEK1 inhibitor (PD98059), PI3K inhibitor (LY294002) and PKC inhibitor (GF109203X) suppressed the TNF-α dependent MMP-9 secretion. 4. TNFR2, but not TNFR1, was expressed in all four human cholangiocarcinoma cell lines in protein and mRNA levels. 5. Treatment with anti-TNFR2 neutralizing antibody blocked TNF-a dependent MMP-9 secretion of CCKS1. CONCLUSIONS: In cholangiocarcinoma CCKS1 cells, TNF-α activated MMP-9-secretion and in turn augmented cancer invasion. Signaling through TNFR2, Ras-MAPK, PI3K-Akt and PKC cascades appeared to be involved in TNF-α dependent MMP-9 secretion. Taken together, our results strongly suggest inflammatory process can promote invasion and metastasis of cholangiocarcinoma. The signaling pathway that leads to the activation of MMP-9 secretion in cholangiocarcinoma can be therapeutic target.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 891 (Risk factors).