Flotillin-1 Promotes Tumor Necrosis Factor-α Receptor Signaling and Activation of NF-κB in Esophageal Squamous Cell Carcinoma Cells
Flotillin-1 Promotes Tumor Necrosis Factor-α Receptor Signaling and Activation of NF-κB in Esophageal Squamous Cell Carcinoma Cells
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Flotillin-1 促进食管鳞状细胞癌细胞中肿瘤坏死因子-α 受体信号传导和 NF-κb 激活
DOI：
10.1053/j.gastro.2012.06.033
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发表时间：
2012-10-01
期刊：
影响因子：
29.4
通讯作者：
Li, Jun
中科院分区：
文献类型：
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作者：
Song, Libing;Gong, Hui;Li, Jun
背景和目的：flotillin 蛋白家族，包括 flotillin-1（FLOT1 或 Reggie-2），是启动受体激酶信号传导并在多种肿瘤类型中上调的脂筏蛋白。我们研究了食管鳞状细胞癌 (ESCC) 细胞中 FLOT1 信号传导和转录因子核因子 (NF)-kappa B 激活的作用。方法：我们使用免疫印迹和免疫化学分析来测定 ESCC 细胞系和 432 例患者的 ESSC 样本中脂筏相关蛋白 FLOT1 的水平；原代正常食管上皮细胞和匹配的邻近非肿瘤组织用作对照。我们使用激酶、电泳迁移率变化和荧光素酶报告基因检测确定了 FLOT1 激活 NF-kappa B 的能力。我们使用集落形成、贴壁独立生长、鸡绒毛尿囊膜、transwell 基质渗透和膜联蛋白 V 结合测定，测量了 ESCC 细胞系中 FLOT1 过表达和短发夹 RNA 敲低的影响。我们分析了裸鼠 ESCC 异种移植肿瘤的生长。结果：与对照组相比，ESCC 细胞系和患者样本中 FLOT1 水平升高；蛋白质水平与疾病阶段和生存时间相关。 Kyse30 和 Kyse510 ESCC 细胞系中 FLOT1 的过度表达增加了增殖、贴壁依赖性生长和侵袭活性，并保护它们免于凋亡。 FLOT1转导的ESCC细胞在裸鼠中形成比对照细胞（仅用载体转导）更大的肿瘤。 FLOT1 促进肿瘤坏死因子-α 受体募集至脂筏；促进信号中介肿瘤坏死因子受体相关因子 2、受体相互作用蛋白和 NEMO 的 K63 连接多聚泛素化；并持续激活 NF-kappa B。FLOT1 水平与患者 ESCC 样本中 NF-kappa B 的激活相关。结论：脂筏蛋白 FLOT1 在 ESCC 细胞系和患者样本中上调，并促进小鼠 ESCC 细胞增殖和肿瘤生长。 FLOT1 激活肿瘤坏死因子-α 受体信号传导并维持 ESCC 细胞中 NF-κ B 的激活。
BACKGROUND & AIMS: The flotillin family of proteins, including flotillin-1 (FLOT1 or Reggie-2), are lipid raft proteins that initiate receptor kinase signaling and are up-regulated in several tumor types. We investigated the role of FLOT1 signaling and activation of the transcription factor nuclear factor (NF)-kappa B in esophageal squamous cell carcinoma (ESCC) cells. METHODS: We used immunoblot and immunochemical analyses to determine levels of the lipid raft-associated protein FLOT1 in ESCC cell lines and 432 ESSC samples from patients; primary normal esophageal epithelial cells and matched adjacent nontumor tissues were used as controls. We determined the ability of FLOT1 to activate NF-kappa B using kinase, electrophoretic mobility shift, and luciferase reporter assays. We measured the effects of FLOT1 overexpression and knockdown with short hairpin RNAs in ESCC cell lines using colony formation, anchorage-independent growth, chicken chorioallantoic membrane, transwell matrix penetration, and Annexin V-binding assays. We analyzed growth of ESCC xenograft tumors in nude mice. RESULTS: Levels of FLOT1 were increased in ESCC cell lines and samples from patients, compared with controls; protein levels correlated with disease stage and survival time. Overexpression of FLOT1 in Kyse30 and Kyse510 ESCC cell lines increased proliferation, anchorage-independent growth, and invasive activity and protected them from apoptosis. FLOT1-transduced ESCC cells formed larger tumors in nude mice than control cells (transduced with only the vector). FLOT1 facilitated recruitment of the tumor necrosis factor-alpha receptor to lipid rafts; promoted K63-linked polyubiquitination of the signaling intermediaries tumor necrosis factor receptor associated factor 2, receptor interacting protein, and NEMO; and sustained the activation of NF-kappa B. Levels of FLOT1 correlated with activation of NF-kappa B in ESCC samples from patients. CONCLUSIONS: The lipid raft protein FLOT1 is up-regulated in ESCC cell lines and samples from patients and promotes ESCC cell proliferation and tumor growth in mice. FLOT1 activates tumor necrosis factor-alpha receptor signaling and sustains activation of NF-kappa B in ESCC cells.