ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1

α-Enolase 1 (ENO1) is a critical glycolytic enzyme whose aberrant expression drives the pathogenesis of various cancers. ENO1 has been indicated to have additional roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we show that ENO1 suppresses iron regulatory protein 1 (IRP1) expression to regulate iron homeostasis and survival of hepatocellular carcinoma (HCC) cells. Mechanistically, we unprecedentedly uncover that ENO1, as an RNA-binding protein, recruits CNOT6 to accelerate the mRNA decay of IRP1 in cancer cells, leading to inhibition of mtioferin-1 (Mfrn1) expression and subsequent repression of mitochondrial iron-induced ferroptosis. Moreover, through in vitro and in vivo experiments and clinical sample analysis, we identified IRP1 and Mfrn1 as tumor suppressors by inducing ferroptosis in HCC cells. Taken together, this study establishes a novel role for the ENO1/IRP1/Mfrn1 pathway in the pathogenesis of HCC and reveals a previously unknown connection between the ENO1/IRP1/Mfrn1 pathway and ferroptosis, suggesting a potential innovative cancer therapy.

Distinct TP53 Mutation Types Exhibit Increased Sensitivity to Ferroptosis Independently of Changes in Iron Regulatory Protein Activity

The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. In addition to loss of tumor suppressor functions, mutations in TP53 promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the coordination of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron-mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status affects the cellular response to ferroptosis induction. Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type (WT) TP53 gene, or a representative mutated TP53 gene from six exemplary “hotspot” mutations in the DNA binding domain (R273H, R248Q, R282W, R175H, G245S, and R249S). TP53 mutants (R273H, R248Q, R175H, G245S, and R249S) exhibited increased sensitivity ferroptosis compared to cells expressing WT TP53. As iron-mediated lipid peroxidation is critical for ferroptosis induction, we hypothesized that iron acquisition pathways would be upregulated in mutant TP53-expressing cells. However, only cells expressing the R248Q, R175H, and G245S TP53 mutation types exhibited statistically significant increases in spontaneous iron regulatory protein (IRP) RNA binding activity following ferroptosis activation. Moreover, changes in the expression of downstream IRP targets were inconsistent with the observed differences in sensitivity to ferroptosis. These findings reveal that canonical iron regulatory pathways are bypassed during ferroptotic cell death. These results also indicate that induction of ferroptosis may be an effective therapeutic approach for tumor cells expressing distinct TP53 mutation types.

FeS-cluster coordination of vertebrate thioredoxins regulates suppression of hypoxia-induced factor 2α through iron regulatory protein 1

Thioredoxins (Trxs) provide electrons to essential cellular processes such as DNA synthesis. Here, we characterize human and murine Trx1 as new iron-sulfur proteins. The [2Fe-2S] cluster is complexed using cysteinyl side chains 32 and 73 in a dimeric holocomplex. Formation of the holo-dimer depends on small structural changes of the loop connecting helices three and four and is stabilized by the formation of a direct electrostatic interaction between Lys72 and Asp60 of two monomers. The not strictly conserved Cys73 in vertebrates co-evolved with the regulation of cellular iron homeostasis through the iron-regulatory proteins (IRP). Active apo-Trx1 is required for the reduction of cysteinyl residues in IRP1 and its binding to the iron-responsive elements in the mRNA encoding hypoxiainducible factor (HIF) 2α. Depletion of Trx1 increased the mRNA levels of HIF2α, an important target of IRP1. Hence, translation of the HIF2α mRNA requires either sufficient iron-supply or the lack of reducing power of the Trx system under iron-limiting conditions. Only then, HIF2α protein may accumulate under hypoxic conditions to transcriptionally regulate processes like erythropoiesis.Significance StatementThioredoxins are, in general, cofactor-less key proteins in redox regulation and provide electrons to many essential cellular processes such as DNA synthesis. 55 years after its discovery, we show that mammalian thioredoxin 1 coordinates an iron-sulfur cluster using one of its active site cysteinyl residues and a non-conserved additional cysteinyl residue located outside the active site. This particular residue co-evolved with the vertebratespecific iron regulatory system. Our study demonstrates that this system is regulated by thioredoxin 1 at the level of the iron-regulatory protein 1, thus linking redox and iron homeostases.

TP53 Mutation Status Influences Iron Regulatory Protein RNA Binding Activity and Sensitivity to Ferroptotic Cell Death

Objectives The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer, but mutations in TP53 do not just result in loss of tumor suppressor function, they can also promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the mediation of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status effects iron-mediated cell death in response to ferroptosis induction. We also measured TP53 dependent differences in iron regulatory protein (IRP) RNA binding activity to begin to clarify the mechanisms by which TP53 mutation status may influence sensitivity to ferroptosis. Methods Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type TP53 gene, or a representative mutated TP53 gene from exemplary “hotspot” mutations in the DNA binding domain (R248, R273, R282, G245, R249 and R175). These six mutation types were selected because they represent 25% of all TP53 mutations in human cancer. To determine the influence of TP53 mutation status on sensitivity to ferroptotic cell death, we treated cells with erastin, a potent inducer of ferroptosis and measured differences in cell viability between these cell lines using PrestoBlue cell viability reagent. To assess mutant TP53-depenent differences in IRP RNA binding activity during ferroptosis we measured differences in IRP RNA binding activity via Electrophoretic Mobility-Shift Assay. Results We found that TP53 mutants (R273, R248, R175, G245, and R249) were significantly less viable (P < 0.05) after initiation of ferroptosis compared to cells expressing WT TP53. Following ferroptosis induction, we observed a significant (P < 0.05) increase in IRP RNA binding in G245, R248, and R175 mutants. Conclusions Our preliminary analyses indicate that TP53 mutants may be more sensitive to ferroptosis, but IRPs do not seem to be solely responsible for the increase in iron during ferroptotic cell death. Furthermore, ferroptosis may be a potential therapeutic target for cancers with these TP53 mutations but further investigation is warranted. Funding Sources Internal funding at Oklahoma State University.