Department of Surgery, University Medical Center Utrecht, Utrecht, The Netherlands.
Gastroenterology. 2010 Jun;138(7):2357-67. doi: 10.1053/j.gastro.2010.02.046. Epub 2010 Feb 23.
BACKGROUND & AIMS: Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential.
Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach.
CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis.
Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.
肿瘤细胞表面表达的死亡受体可通过诱导细胞凋亡来阻止转移形成，但它们也可以促进迁移和侵袭。死亡受体信号输出的决定因素尚未明确。本研究旨在探讨致癌性 K-Ras 在决定死亡受体功能和转移潜能中的作用。
使用 CD95 配体和肿瘤坏死因子相关凋亡诱导配体（TRAIL）作为刺激物，对仅存在或不存在 K-Ras 癌基因的同种异体人源和鼠源结直肠癌细胞系进行凋亡和侵袭检测，以评估其功能。通过绿色荧光蛋白或荧光素酶表达肿瘤细胞的脾内注射，随后进行活体荧光显微镜或生物发光成像以及共聚焦显微镜和免疫组织化学检测，评估转移潜能。通过 RNA 干扰和抑制剂方法评估 Ras 效应器途径对 CD95 输出的控制。
CD95 配体和 TRAIL 以 K-Ras 依赖性方式刺激结直肠肿瘤细胞和肝转移的侵袭。丧失突变型 K-Ras 使 CD95 和 TRAIL 受体重新进入凋亡模式，并消除转移潜能。Raf1 对于 CD95 功能的转换、肿瘤细胞在肝脏中的存活以及 K-Ras 驱动的肝转移形成至关重要。K-Ras 和 Raf1 抑制了 Rho 激酶（ROCK）/LIM 激酶介导的肌动蛋白切割蛋白丝切蛋白的磷酸化。ROCK 或 LIM 激酶的过表达允许 CD95L 在 K-Ras 阳性细胞中诱导凋亡并阻止转移形成，而其抑制则保护 K-Ras 缺失细胞免受凋亡。
致癌性 K-Ras 及其效应因子 Raf1 通过抑制 ROCK/LIM 激酶途径将死亡受体转化为诱导侵袭的受体，这对于 K-Ras/Raf1 驱动的转移形成至关重要。
