Knockdown Of ATF4 Research Articles

The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation.

The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho-eIF2alpha were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2-eIF2alpha-ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti-tumour approaches.

Abstract 1683: ATF4 down-regulation potentiates L-asparaginase activity in ovarian cancer cell lines

Abstract The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly correlated with L-ASP activity in leukemia cell lines of the NCI-60 cell line panel. Unexpectedly, however, the correlation was also apparent in ovarian cancer lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. Using siRNA to down-regulate ASNS by 5-fold produced a proportional ∼5-fold potentiation of L-ASP activity in some ovarian cancer lines (OVCAR-3 and OVCAR-4). Surprisingly, ASNS siRNAs yielded a striking >500-fold potentiation in other ovarian lines (OVCAR-8 and OVCAR-8/ADR). Taken together, those findings suggest that the L-ASP/ASNS correlation is causal but also support the hypothesis that L-ASP activity is modulated by additional molecules or pathways. To address that hypothesis and to further elucidate the L-ASP mechanism of action, we have initiated genome-wide siRNA library screening. Preliminary screens in the OVCAR-8 cell line using a 418-gene apoptosis library, a custom 392-gene metabolic library, and the MTS assay as an endpoint yielded Z′ factors >0.90, indicating a robust screen. RNAi-mediated down-regulation of apoptosis genes had little or no effect on L-ASP's anticancer activity. That was surprising because L-ASP has been reported to cause apoptosis through glutamine depletion. Knock-down of ATF4, a transcription factor involved in cell metabolism, did markedly potentiate L-ASP activity (>500-fold). Furthermore, ATF4 knock-down markedly enhanced L-ASP anticancer activity toward the highly resistant cell line, OVCAR-4. These findings suggest that ATF4 is an important mediator of L-ASP resistance and provide rationale for investigation of ATF4 as a predictive biomarker of L-ASP effect. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1683.

CHOP (C/EBP homologous protein) and ASNS (asparagine synthetase) induction in cybrid cells harboring MELAS and NARP mitochondrial DNA mutations

Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) is related to a variety of diseases including MELAS and NARP syndromes. However, little is known about the intracellular responses induced by mtDNA mutations. In order to identify genes whose expression is altered as a result of the presence of mtDNA mutations, DNA microarray analysis was performed using human 143B osteosarcoma cells harboring 3243A > G [tRNA-Leu (UUR)] and 8993T > G [ATPase6 Leu156Arg] mtDNA mutations associated with MELAS and NARP syndromes (2SD and NARP3-1 cybrid cells), respectively. We found that mRNA and protein levels of ATF4, CHOP and ASNS were upregulated in 2SD and NARP3-1 cells as compared with parental cells. Reporter assays demonstrated that transcription of CHOP and ASNS genes was upregulated through the AARE (amino acid regulatory element) and NSRE-1 (nutrient-sensing response element-1) enhancer elements to which ATF4 binds, respectively. Furthermore, knockdown of ATF4 by RNA interference reduced CHOP and ASNS transcription in 2SD and NARP3-1 cells. These results suggest that the presence of mtDNA mutations elicits upregulation of CHOP and ASNS genes through the elevation of ATF4 expression and its binding to the AARE and NSRE-1, respectively.

Proteasome Inhibitor PS-341 Induces Apoptosis in Cisplatin-resistant Squamous Cell Carcinoma Cells by Induction of Noxa

Proteasome Inhibitor PS-341 Induces Apoptosis in Cisplatin-resistant Squamous Cell Carcinoma Cells by Induction of Noxa

TRB3, a novel ER stress-inducible gene, is induced via ATF4–CHOP pathway and is involved in cell death

C/EBP homologous protein (CHOP) is a stress-inducible nuclear protein that is crucial for the development of programmed cell death and regeneration; however, the regulation of its function has not been well characterized. Slbo, a Drosophila homolog of C/EBP (CCAAT/enhancer binding protein), was shown to be unstabilized by tribbles. Here, we identified TRB3 as a tribbles ortholog in humans, which associated with CHOP to suppress the CHOP-dependent transactivation. TRB3 is induced by various forms endoplasmic reticulum (ER) stress later than CHOP. Tunicamycin treatment enhanced the TRB3 promoter activity, while dominant-negative forms of CHOP suppressed the tunicamycin-induced activation. In addition, the tunicamycin response region in the TRB3 promoter contains amino-acid response elements overlapping the CHOP-binding site, and CHOP and ATF4 cooperated to activate this promoter activity. Knockdown of endogenous ATF4 or CHOP expression dramatically repressed tunicamycin-induced TRB3 induction. Furthermore, knockdown of TRB3 expression decreased ER stress-dependent cell death. These results indicate that TRB3 is a novel target of CHOP/ATF4 and downregulates its own induction by repression of CHOP/ATF4 functions, and that it is involved in CHOP-dependent cell death during ER stress.