Significance: RAS genes are among the most mutated proto-oncogenes in human cancer. The mechanisms supporting RAS transformation are not fully understood, particularly regarding the relative contributions of oxidant versus antioxidant pathways. Here, we report that the cystine/glutamate transporter xCT is essential for RAS-induced tumorigenicity by enhancing antioxidant glutathione synthesis. Our findings uncover that RAS controls xCT transcription by downstream activation of ETS-1 to synergize with ATF4. This has clinical relevance since xCT expression is upregulated in human cancers exhibiting an activated RAS pathway. Therefore, oncogenic RAS transformation is supported by induction of an antioxidant program, highlighting xCT as a potential vulnerability for therapeutic targeting.

Oncogenic KRAS is the key driver oncogene for several of the most aggressive human cancers. One key feature of oncogenic KRAS expression is an early increase in cellular reactive oxygen species (ROS) which promotes cellular transformation if cells manage to escape cell death, mechanisms of which remain incompletely understood. Here, we identify that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis, a recently described type of regulated necrosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation. Interestingly, KRAS-mutant cells upregulate expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS-mutant cells are responsible for mediating ferroptosis resistance and FSP1 is upregulated as a consequence of MAPK and NRF2 pathway activation downstream of KRAS. Strikingly, FSP1 activity promotes cellular transformation in soft agar and its overexpression is sufficient to promote spheroid growth in 3D in KRAS WT cells. Moreover, FSP1 expression and its activity in ferroptosis inhibition accelerates tumor onset of KRAS WT cells in the absence of oncogenic KRAS in vivo. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRAS^G12D expressing mouse model is only effective in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin and correlates with NRF2 expression in PDAC patient datasets. Based on these data, we propose that KRAS-mutant cells must navigate a ferroptosis checkpoint by upregulating FSP1 during tumor establishment. Consequently, ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers.

The RAS family of proto-oncogenes comprises HRAS, KRAS, and NRAS, which are among the most mutated genes in human cancers. The RAS family genes encode small GTPases that coordinate key signaling pathways in response to growth factors. Mutations in RAS result in a constitutively active form of the protein that supports cellular transformation and tumorigenesis. The mechanisms of oncogenic RAS-mediated transformation encompass uncontrolled proliferation and inhibition of cell death through overactivation of the RAF-MEK-ERK and the PI3K-AKT pathways, respectively. In addition, the control of redox balance by RAS has also been proposed to play a role in its oncogenic properties. However, the exact role of redox balance in mediating mutant RAS transformation is still under debate. Here, we present, on one hand, the involvement of pro-oxidant components in oncogenic RAS transformation, such as NADPH oxidases and mitochondrial reactive oxygen species, and how these promote transformation. On the other hand, we describe the contribution of antioxidant components to mutant RAS transformation, including Nrf2, glutathione biosynthesis and xCT, as well as the mechanisms by which antioxidant programs drive transformation. Finally, we aim to reconcile the seemingly opposite effects of oncogenic RAS on redox balance and discuss a model for the complementary role of both pro-oxidant and antioxidant pathways in mutant RAS-driven tumor progression.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance ofHOXATranscript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzingHOTAIRM1in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation andMGMTpromoter methylation status. Finally, the role ofHOTAIRM1in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identifiedHOTAIRM1as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation andMGMTpromoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity uponHOTAIRM1down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels uponHOTAIRM1knock-down.HOTAIRM1knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down ofTGM2mimicked the phenotype ofHOTAIRM1down-regulation in glioblastoma cells. Moreover,HOTAIRM1modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role forHOTAIRM1as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. TargetingHOTAIRM1may be a promising new therapeutic approach.

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms.