Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography–mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.

中文翻译：

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ATF4 激活和综合应激反应 (ISR) 引起的透明细胞肾细胞癌的转录和代谢重塑

研究表明，透明细胞肾细胞癌 (ccRCC) 发生广泛的代谢重塑，并伴有谷胱甘肽 (GSH) 水平升高。我们假设激活转录因子 4 (ATF4) 和综合应激反应 (ISR) 会诱导代谢转变，包括 GSH 积累增加，而 ccRCC 中发现的维生素 A 缺乏 (VAD) 也可以激活肾脏中的 ATF4 信号传导。为了确定 ATF4 的作用，我们使用了来自癌症基因组图谱的公开 RNA 测序 (RNA-seq) 数据集。随后，我们对早期 ccRCC 的小鼠 TRansgenic Cancer of the Kidney (TRACK) 模型进行了 RNA-seq 和基于液相色谱-质谱的代谢组学分析。为了验证我们的发现，我们生成了具有 ATF4 基因编辑（ATF4 敲除 [KO]）的 RCC4 细胞系，并对这些细胞进行代谢同位素示踪。使用方差分析、双侧学生t检验和基因集富集分析 ( p  < 0.05) 来确定统计显着性。在这里，我们发现大多数人类 ccRCC 肿瘤表现出转录因子 ATF4 的激活。ATF4 的激活伴随着 ATF4 基因集的富集和 ATF4 靶基因ASNS、ALDH1L2、MTHFD2、DDIT3 ( CHOP )、DDIT4、TRIB3、EIF4EBP1、SLC7A11和PPP1R15A ( GADD34 ) 表达的升高。转录谱分析和代谢组学分析表明，TRACK ccRCC 小鼠模型中激活的缺氧诱导因子 1α (HIF1α) 信号传导也会诱导 ATF4 介导的 ISR。值得注意的是，TRACK 肾脏中的常氧 HIF1α 信号传导和野生型肾脏中的 VAD 信号传导都会降低氨基酸水平，增加ASNS、TRIB3和MTHFD2信使 RNA 水平，并增加脂质和 GSH 水平。通过人 RCC4 肾癌亲本细胞系和 ATF4 基因编辑 (ATF4-KO) 细胞系的代谢同位素示踪，我们发现 ATF4 部分通过激活线粒体一碳代谢途径来增加 GSH 积累。我们的结果首次证明，ATF4 的激活可增强 GSH 积累，增加嘌呤和嘧啶生物合成，并有助于 ccRCC 中的转录和代谢重塑。此外，仅在小鼠肾近曲小管中表达的组成型 HIF1α 会激活 ATF4，导致与 ISR 相关的代谢变化。我们的数据表明 HIF1α 可以通过 ATF4 激活促进 ccRCC。此外，肾脏缺乏维生素 A 也概括了 ISR 的各个方面。