背景:对癌细胞的体内研究具有发现癌症进展机制和临床前候选药物的强大工具。在这些体内实验模型中,使用异种移植物建立高度恶性细胞系已被频繁使用。然而,先前的研究很少针对蛋白质水平翻译改变的恶性肿瘤相关基因。因此,本研究旨在鉴定在体内选择的癌细胞系中导致癌症进展并在蛋白质水平发生改变的恶性肿瘤相关基因.
方法:我们通过原位异种移植建立了高恶性乳腺癌细胞系(LM05)作为体内选择方法。探索通过翻译或翻译后调节改变的基因,我们通过蛋白质印迹分析了高度恶性乳腺癌细胞系中蛋白质的产生。通过体外和体内实验对改变的基因进行功能分析。揭示蛋白质水平调控的分子机制,我们通过免疫沉淀评估翻译后修饰。此外,我们通过基于点击反应的新生蛋白纯化来评估翻译生产。
结果:因此,NF-κB诱导激酶(NIK)在蛋白水平上增加,并促进高恶性乳腺癌细胞系中NF-κB2(p52)和RelB的核定位。功能分析表明,NIK上调通过癌症相关成纤维细胞(CAF)的吸引力和部分抗凋亡活性导致肿瘤恶性肿瘤。此外,免疫沉淀实验显示,LM05细胞中NIK的泛素化降低。NIK泛素化的下降归因于cIAP1的翻译下调。
结论:我们的研究通过抑制NIK后修饰和cIAP1翻译确定了NIK产生的失调机制。异常的NIK积累促进了高度恶性乳腺癌细胞系中的肿瘤生长。
BACKGROUND: In vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines.
METHODS: We established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein.
RESULTS: As a result, NF-κB inducing kinase (
NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of
NIK decreased in LM05 cells. The decline in
NIK ubiquitination was attributed to the translational downregulation of cIAP1.
CONCLUSIONS: Our study identified a dysregulated mechanism of NIK production by the suppression of
NIK post-modification and cIAP1 translation. The aberrant
NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.