Honma Nobuyuki, Genda Takuya, Matsuda Yasunobu, Yamagiwa Satoshi, Takamura Masaaki, Ichida Takafumi, Aoyagi Yutaka
Division of Gastroenterology and Hepatology, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Science, Niigata City, Japan.
Lab Invest. 2006 Jul;86(7):687-96. doi: 10.1038/labinvest.3700427. Epub 2006 May 8.
The human hepatocellular carcinoma (HCC)-derived cell line KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastasis of human HCC, because it often shows cell scattering in vitro and intrahepatic metastasis in vivo. We previously found that integrin-mediated extracellular signals inactivated E-cadherin in KYN-2, and caused loss of cell-cell contact with gain of cell motility, which is considered to be a critical step in the process of cancer cell invasion and metastasis. To further understand molecular mechanisms involved in biological aggressiveness of HCC, we investigated intracellular signaling involved in integrin-mediated scattering of KYN-2 cells. Cultured KYN-2 cells formed trabecular aggregates in suspension, but when adhering to integrin-stimulating substrata, they scattered according to phosphorylation of extracellular signal-regulated kinase (ERK). Upon treatment with ERK kinase (MEK) inhibitor PD98059, adhered KYN-2 cell scattering was inhibited, tight cell-to-cell contact was recovered, and both E-cadherin and actin filaments accumulated in the area of intercellular contact zone. In contrast, constitutively active MEK1-transfected KYN-2 cells showed reduced E-cadherin and actin filaments in the intercellular contact zone, showing a flattened phenotype with broad lamellipodia. Enforced signaling of MEK-ERK pathway in KYN-2 cells suppressed cadherin-mediated homotypic adhesion and increased the potential of cell motility. An antibody-based protein microarray analysis revealed that the cytoplasmic protein c-Cbl was significantly downregulated in MEK1-transfected KYN-2 cells, suggesting that c-Cbl might be a candidate downstream mediator of integrin/MEK/ERK-mediated cell scattering. In conclusion, cell scattering of the highly metastatic cell line KYN-2 is regulated through the integrin-MEK-ERK signaling cascade, suggesting that this molecular pathway may be critical in intrahepatic metastasis of human HCC.
人肝癌衍生细胞系KYN-2被认为是研究人类肝癌侵袭和转移分子基础的良好模型，因为它在体外常表现出细胞散射，在体内会发生肝内转移。我们之前发现，整合素介导的细胞外信号使KYN-2中的E-钙黏蛋白失活，并导致细胞间接触丧失，同时细胞运动性增加，这被认为是癌细胞侵袭和转移过程中的关键步骤。为了进一步了解参与肝癌生物学侵袭性的分子机制，我们研究了整合素介导的KYN-2细胞散射所涉及的细胞内信号传导。培养的KYN-2细胞在悬浮液中形成小梁状聚集体，但当黏附于整合素刺激的基质时，它们会根据细胞外信号调节激酶（ERK）的磷酸化而散射。用ERK激酶（MEK）抑制剂PD98059处理后，黏附的KYN-2细胞散射受到抑制，细胞间紧密接触得以恢复，并且E-钙黏蛋白和肌动蛋白丝在细胞间接触区域积聚。相反，组成型活性MEK1转染的KYN-2细胞在细胞间接触区域显示E-钙黏蛋白和肌动蛋白丝减少，呈现出具有宽片状伪足的扁平表型。KYN-2细胞中MEK-ERK途径的增强信号传导抑制了钙黏蛋白介导的同型黏附，并增加了细胞运动性。基于抗体的蛋白质微阵列分析显示，在MEK1转染的KYN-2细胞中，细胞质蛋白c-Cbl显著下调，这表明c-Cbl可能是整合素/MEK/ERK介导的细胞散射的候选下游介质。总之，高转移性细胞系KYN-2的细胞散射通过整合素-MEK-ERK信号级联进行调节，这表明该分子途径可能在人类肝癌的肝内转移中起关键作用。
