Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition

Abstract

As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy. Methods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N 6 -methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model. Results: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N 6 -methyladenosine (m 6 A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition. Conclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m 6 A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment.

Lysine Deprivation Regulates Npy Expression via GCN2 Signaling Pathway in Mandarin Fish (Siniperca chuatsi)

“…As a downstream target of eIF2α phosphorylation, activating transcription factor ATF4 can be triggered by a variety of noxious stimuli, including metabolic disturbances (such as glucose and amino acid deprivation) [ 49 ], endoplasmic reticulum stress [ 50 , 51 ], and oxidative stress [ 52 ]. Previous studies have confirmed that cells with impaired ATF4 are more susceptible to amino acid starvation [ 53 ].…”

Crosstalk among m6A RNA methylation, hypoxia and metabolic reprogramming in TME: from immunosuppressive microenvironment to clinical application

“…For instance, METTL3 silencing can be synergistically implemented with the glycolysis inhibitor 2-deoxyglucose (2-DG) to block HCC growth [ 298 ] and combined inhibition of METTL3 and autophagy increases the sensitivity of spermatocytoma to cisplatin [ 299 ]. Additionally, co-inhibition of YTHDF2, ATF4-induced autophagy, and glutamine provides a novel strategy for targeted therapy in colorectal cancer [ 300 ].…”

ATF4/CEMIP/PKCα promotes anoikis resistance by enhancing protective autophagy in prostate cancer cells

“…In addition, previous research confirmed that tumor cells could be protected from anoikis and contribute to tumor metastasis by activating the coordination program of autophagy and antioxidant response when they were stressed or separated from the ECM [ 47 ]. Another study indicated that the ATF4 pathway was activated, and the DDIT4 was upregulated to restrict mTOR and promote autophagy [ 48 ] after inhibiting glutamine decomposition in colorectal tumor cells. Therefore, ATF4 and CEMIP were activated in a time-dependent manner upon PCa cell detachment, while both the activation and peak time of ATF4 occurred earlier than CEMIP (Fig.…”