Judge Sarah M, Wu Chia-Ling, Beharry Adam W, Roberts Brandon M, Ferreira Leonardo F, Kandarian Susan C, Judge Andrew R
Department of Physical Therapy, University of Florida, 1225 Center Drive, HPNP Building 1142, Gainesville, Florida, USA.
Department of Health Sciences, Boston University, Boston, Massachusetts, USA.
BMC Cancer. 2014 Dec 24;14:997. doi: 10.1186/1471-2407-14-997.
Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype.
To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later.
Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model.
These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.
恶病质癌症患者和癌症恶病质动物模型的证据支持叉头框O（FoxO）转录因子参与驱动癌症诱导的骨骼肌萎缩。然而，在癌症恶病质期间，FoxO调控的全基因组基因网络和相关生物学过程尚不清楚。我们假设FoxO是癌症期间骨骼肌中多种基因网络的核心上游调节因子，可能协同作用以促进消瘦表型。
为了抑制内源性FoxO与DNA的结合，我们用携带显性负性（d.n.）FoxO蛋白（或绿色荧光蛋白对照）cDNA的AAV9转导小鼠的肢体和膈肌。d.n.FoxO构建体仅由与FoxO1和FoxO4高度同源的FoxO3a DNA结合域组成，它能竞争并阻断内源性FoxO与DNA的结合。随后给小鼠接种结肠26（C26）细胞，并在26天后采集肌肉。
阻断FoxO可防止C26诱导的运动肌肉和膈肌的肌纤维萎缩，并显著减轻力量缺陷。肌肉大小和功能的这种保留与2093个因C26而变化的转录本中的543个转录本的差异调节有关。对需要FoxO的上调基因转录本的生物信息学分析显示蛋白酶体、AP-1和IL-6途径富集，并且包括几个与萎缩相关的转录因子，包括Stat3、Fos和Cebpb。FoxO对于癌症诱导的几种富含细胞外基质和肌节蛋白编码基因的基因转录本的下调也是必需的。我们通过qRT-PCR在肢体肌肉和膈肌中验证了这些发现，并进一步证明FoxO1和/或FoxO3a足以增加Stat3、Fos、Cebpb以及C/EBPβ靶基因Ubr2。对Cebpb近端启动子的分析揭示了两个真正的FoxO结合元件，我们进一步证实它们对于Cebpb启动子响应IL-6（C26癌症模型中的主要细胞因子）激活是必需的。
这些发现提供了新的证据，表明FoxO依赖的转录是癌症恶病质期间控制骨骼肌中多种基因网络的核心节点，并确定了新的候选基因和网络，作为癌症诱导消瘦的致病因素有待进一步研究。
