Transcriptional and metabolic remodeling in clear cell renal cell carcinoma caused by ATF4 activation and the integrated stress response (ISR)

Abstract

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.

Further delineation of the phenotypic and metabolomic profile of ALDH1L2‐related neurodevelopmental disorder

“…2 In line with these findings, another report showed increased glutathione in clear cell renal cell carcinoma associated with the ATF4-dependent activation of ALDH1L2 expression. 39 The function of ALDH1L2 in supporting the redox status of the cell was also demonstrated in our Aldh1l2 knockout (Aldh1l2 À/À ) mouse model. 5 Aldh1l2 À/À mice compared to Aldh1l2 +/+ mice had significantly decreased levels of NADPH and experienced increased oxidative stress as judged based on the overall metabotype analysis.…”

“…In agreement with this mechanism, interference with this pathway in cultured cells resulted in decreased cellular NADPH/NADP + and reduced/oxidized glutathione ratios, and increased cell sensitivity to oxidative stress 2 . In line with these findings, another report showed increased glutathione in clear cell renal cell carcinoma associated with the ATF4‐dependent activation of ALDH1L2 expression 39 …”

Metabolic plasticity of serine metabolism is crucial for cGAS/STING-signalling and innate immune response to viral infections in the gut

“…Although we did not intensively study the mechanistic details how the metabolic reprogramming is regulated in IECs, several recent publications suggest that a PERK-ATF4-dependent activation of the integrated stress response (ISR) is important for the observed metabolic adaptation upon ER-stress (73)(74)(75). CHIPseq data already demonstrated that PHGDH, PSAT1, SHMT2, MTHFD2 and ALDH1L2 are direct ATF4 targets (76).…”

“…Although the mechanistic basis for the metabolic reprogramming in IECs deserves to be resolved in more detail, recent studies suggest a critical role for the PERK-ATF4-pathway and the integrated stress response (ISR) (72)(73)(74). CHIPseq data already demonstrated that PHGDH, PSAT1, SHMT2, MTHFD2 and ALDH1L2 are direct ATF4 targets (75).…”

Identification and validation of m6A‐associated ferroptosis genes in renal clear cell carcinoma

“…Further studies found that the expression of TRIB3 increased with the development of clinical stage, pathological grade, and primary tumor size, indicating that TIRB3 promoted the ccRCC progression and increased the risk of invasion and deterioration of ccRCC (Wu et al, 2022). In addition, normoxic HIF1α signaling in the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early‐stage ccRCC, increase TRIB3 messenger RNA levels, and increase levels of lipids and GSH (van der Mijn et al, 2022). Elevated TRIB3 expression is a prognostic marker in patients with ccRCC, suggesting that TRIB3 has a novel molecular mechanism in ccRCC.…”