Liu Ching-Ann, Wang Mei-Jung, Chi Chin-Wen, Wu Chew-Wun, Chen Jeou-Yuan
Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan, ROC.
Oncogene. 2004 Nov 18;23(54):8731-42. doi: 10.1038/sj.onc.1208106.
Rhotekin (RTKN), the gene coding for the Rho effector, RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN is expressed at a low level in normal cells and is overexpressed in many cancer-derived cell lines. The function of RTKN as an effector protein in Rho GTPase-mediated pathways regulating apoptosis was investigated. By transfection and expression of RTKN in cells that expressed endogenous RTKN at a low basal level, we showed that RTKN overexpression conferred cell resistance to apoptosis induced by serum deprivation or treatment with sodium butyrate, and the increased resistance correlated to the level of RTKN. Conversely, reducing RTKN expression by small interfering RNAs greatly sensitized cells to apoptosis. The RTKN-mediated antiapoptotic effect was blocked by the nuclear factor-kappaB (NF-kappaB) inhibitors, curcumin or parthenolide, but not by the phosphatidylinositol 3'-OH-kinase inhibitor, LY294002, or the MAP kinase inhibitor, PD98059. Reporter gene assays and electrophoretic mobility shift assay confirmed that RTKN overexpression led to constitutive activation of NF-kappaB through the phosphorylation of IkappaB by IKKbeta. By using the RTKN truncation mutants, we showed that RTKN mediated Rho activity eliciting signaling pathway to activate NF-kappaB, with a concomitant induction of expression of the NF-kappaB antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. Consistent with these data, RTKN-expressing cells showed increased chemoresistance to 5-fluorouracil and paclitaxol, and the resistance was greatly attenuated by NF-kappaB inhibitor. In conclusion, overactivated Rho/RTKN/NF-kappaB signaling pathway through overexpression of RTKN may play a key role in gastric tumorigenesis by conferring cells resistance to apoptosis, and this signaling pathway may serve as an important target for novel therapeutic approaches to the treatment of human GC.
Rhotekin（RTKN）是编码Rho效应蛋白RTKN的基因，已证实在人胃癌（GC）中过表达。在本研究中，我们进一步表明RTKN在正常细胞中低表达，而在许多癌症衍生细胞系中过表达。我们研究了RTKN作为Rho GTP酶介导的调节凋亡途径中的效应蛋白的功能。通过在基础水平低表达内源性RTKN的细胞中转染并表达RTKN，我们发现RTKN过表达赋予细胞对血清剥夺或丁酸钠处理诱导的凋亡的抗性，且增加的抗性与RTKN水平相关。相反，用小干扰RNA降低RTKN表达可使细胞对凋亡高度敏感。RTKN介导的抗凋亡作用被核因子-κB（NF-κB）抑制剂姜黄素或小白菊内酯阻断，但未被磷脂酰肌醇3'-OH-激酶抑制剂LY294002或丝裂原活化蛋白激酶抑制剂PD98059阻断。报告基因检测和电泳迁移率变动分析证实RTKN过表达通过IKKβ使IκB磷酸化导致NF-κB的组成型活化。通过使用RTKN截短突变体，我们表明RTKN介导Rho活性引发信号通路以激活NF-κB，同时诱导NF-κB抗凋亡基因cIAP-2、Bcl-xL、A1和A20的表达。与这些数据一致，表达RTKN的细胞对5-氟尿嘧啶和紫杉醇表现出增加的化疗抗性，且NF-κB抑制剂可大大减弱这种抗性。总之，通过RTKN过表达过度激活的Rho/RTKN/NF-κB信号通路可能通过赋予细胞抗凋亡能力在胃癌发生中起关键作用，并且该信号通路可能成为治疗人类GC新治疗方法的重要靶点。
