Cancer-induced Muscle Wasting is IKKβ-dependent and NF-kappaB-independent. Mus musculus

Existing data suggest that NF-kappaB signaling is a key regulator of cancer-induced skeletal muscle wasting. However, identification of the components of this signaling pathway and of the NF-κB transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of d.n. IKKβ blocked muscle wasting by 69%, the IκBα-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKKα or d.n. NIK did not block C26-induced wasting. Surprisingly, overexpression of d.n. p65 or d.n. c-Rel did not significantly block muscle wasting. Genome-wide mRNA expression arrays showed upregulation of many genes previously implicated in muscle atrophy. To test if these upregulated genes were direct targets of NF-κB transcription factors, we compared genome-wide p65 or p50 binding to DNA in control and cachectic muscle using ChIP-sequencing. Bioinformatic analysis of ChIP-seq data from control and C26 muscles showed increased p65 and p50 binding to a few regulatory and structural genes but only two of these genes were upregulated with atrophy. The p65 and p50 ChIP-seq data are consistent with our finding of no significant change in protein binding to an NF-κB oligo in a gel shift assay. Taken together, these data support the idea that although inhibition of IκBα, and particularly IKKβ, blocks cancer-induced wasting, the alternative NF-κB signaling pathway is not required. In addition, the downstream NF-κB transcription factors do not regulate the transcriptional changes. These data are consistent with the growing body of literature showing that there are NF-κB-independent substrates of IKKβ and IκBα that regulate physiological processes. Overall design: To compare gene expression changes in atrophied muscles from C26 tumor bearing mice, gastrocnemius/plantaris muscles were harvested from 4 C26 tumor-bearing mice, and 3 control non tumor-bearing mice. Total RNA were isolated and pooled (2-3 muslces in the same group per RNA sample ) to make equal amount of total RNA per sample. Three pooled total RNA samples from healthy control muscles and 3 pooled total RNA from muscles of C26 tumor bearing mice were labelled and hybridized on 6 Mouse Affyemtrix Gene 1.0 ST arrays. Two-side t-tests and multiple test corrections were performed to identify differentially expressed genes due to C26 tumor bearing induced cachexia.

现有研究数据表明，核因子κB（NF-κB）信号通路是癌症诱导骨骼肌萎缩的关键调控因子。然而，目前对该信号通路的组成成分以及调控萎缩的NF-κB转录因子的鉴定仍远未完成。在C26荷瘤小鼠的骨骼肌中，显性负性（dominant negative, d.n.）IKKβ的过表达可阻断69%的肌肉萎缩，IκBα超级阻遏蛋白的过表达可阻断41%的肌肉萎缩。与之相反，显性负性IKKα或显性负性NIK的过表达无法阻断C26诱导的肌肉萎缩。令人意外的是，显性负性p65或显性负性c-Rel的过表达并未显著阻断肌肉萎缩。全基因组mRNA表达芯片分析显示，诸多此前被证实与肌肉萎缩相关的基因均出现上调。为验证这些上调基因是否为NF-κB转录因子的直接靶标，我们采用染色质免疫共沉淀测序（ChIP-seq）技术，对比了正常对照与恶病质骨骼肌中p65或p50与DNA的结合情况。对正常对照与C26荷瘤小鼠骨骼肌的ChIP-seq数据进行生物信息学分析后发现，p65与p50在少数调控基因及结构基因上的结合出现增强，但其中仅2个基因在肌肉萎缩时出现上调。p65与p50的ChIP-seq数据与我们在凝胶迁移实验中观察到的"蛋白质与NF-κB寡核苷酸结合无显著变化"的结果一致。综合来看，这些数据支持以下观点：尽管抑制IκBα（尤其是IKKβ）可阻断癌症诱导的肌肉萎缩，但非经典NF-κB信号通路并非必需。此外，下游的NF-κB转录因子并未调控上述转录组变化。这些数据与日益增多的研究文献相符，后者证实IKKβ与IκBα存在不依赖NF-κB的底物，可调控多种生理过程。实验整体设计：为对比C26荷瘤小鼠萎缩骨骼肌的基因表达变化，我们分别采集了4只C26荷瘤小鼠与3只正常非荷瘤小鼠的腓肠肌/跖肌。提取总RNA并进行混合（每组2~3块肌肉对应1份RNA样本），以确保每份样本的总RNA量一致。将来自健康对照骨骼肌的3份混合总RNA样本，与来自C26荷瘤小鼠骨骼肌的3份混合总RNA样本分别标记后，在6张小鼠Affymetrix基因1.0 ST芯片阵列上进行杂交。通过双侧t检验与多重检验校正，筛选出因C26荷瘤诱导恶病质而产生差异表达的基因。