Targeted therapies disrupt oncogenic signaling while inducing adaptive metabolic rewiring for cancer cell survival. However, the roles of acute metabolic shifts remain poorly understood. Here, we showed that inhibiting EGFR, KRAS, or BRAF drove resistance to cystine deprivation-induced ferroptosis in cancer cells harboring each driver mutation. Resistance to cystine deprivation emerged within 24 h of drug treatment and persisted during prolonged 9-day exposure. However, acquiring drug resistance during the 2-month drug exposure abolished the resistance to cystine deprivation, coupled with reactivation of MAPK signaling. Mechanistically, GPX4, which was induced by drug treatment, was crucial to prevent ferroptosis despite a reduction in intracellular cysteine and glutathione levels during cystine deprivation. Additionally, the oncogenic inhibitors altered ALDH isozyme expression profiles, potentially inducing resistance to 4-hydroxynonenal, which is a lipid aldehyde associated with ferroptosis. Our findings identify a novel cellular adaptation mechanism to oncogenic signaling inhibition, providing mechanistic insights into how cancer cells adjust their lipid redox balance during therapy.

Oncogenic signaling and stress response pathways interact to drive tumorigenesis and therapy resistance. However, little is known about such interactions for HER3, a member of the HER/ErbB receptor family that is aberrantly expressed in many tumors, including breast cancer. Here, we show that HER3 cooperates with HER2 to enhance induction of ATF4, a central transcription factor of the integrated stress response and the unfolded protein response, during endoplasmic reticulum (ER) stress. ATF4 induction was enhanced by ligand-activated HER3 and conversely reduced by genetic knockdown or pharmacological inhibition of HER2/HER3-mediated signaling in both HER2-overexpressing SKBR3 and non-overexpressing MCF7 breast cancer cells. HER3 knockdown in SKBR3 cells also increased cell death during ER stress. Notably, depletion of HER3, likely occurring through ER stress-associated downregulation mechanisms, was accompanied by attenuation of ATF4 induction during sustained stress. These findings suggest that the HER3-ATF4 axis functions as a dynamically regulated mechanism for tuning the cellular stress response.

Austocystin D is a natural compound that induces cytochrome P450 (CYP) monooxygenase-dependent DNA damage and growth inhibition in certain cancer cell lines. Cancer cells exhibiting higher sensitivity to austocystin D often display elevated CYP2J2 expression. However, the essentiality and the role of CYP2J2 for the cytotoxicity of this compound remain unclear. In this study, we demonstrate that CYP2J2 depletion alleviates austocystin D sensitivity and DNA damage induction, while CYP2J2 overexpression enhances them. Moreover, the investigation into genes involved in austocystin D cytotoxicity identified POR and PGRMC1, positive regulators for CYP activity, and KAT7, a histone acetyltransferase. Through genetic manipulation and analysis of multiomics data, we elucidated a role for KAT7 in CYP2J2 transcriptional regulation. These findings strongly suggest that CYP2J2 is crucial for austocystin D metabolism and its subsequent cytotoxic effects. The potential use of austocystin D as a therapeutic prodrug is underscored, particularly in cancers where elevated CYP2J2 expression serves as a biomarker.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.

Ferroptosis, one of the programmed cell deaths, is induced by accumulation of lipid peroxidation. To protect from ferroptosis, cells possess a wide range of reduction systems including glutathione peroxidase 4 (GPX4), which reduces lipid peroxides to non-toxic lipid alcohols. Indeed, inhibiting GPX4 leads to ferroptosis in various cancer cells, but cellular sensitivity to GPX4 inhibition often varies depending on cell conditions. Some previous studies reported that an increase in cell density induces resistance to GPX4 inhibition, but the mechanisms of cell density-dependent resistance are not fully understood. In this study, we have also found that several melanoma cell lines become resistant to ferroptosis induced by GPX4 inhibition in a density-dependent manner. For example, a selective GPX4 inhibitor, RSL3, immediately induced cell death at low cell density of A375 within 6-8 hours, but did not at high density even after 24 hours. The acute cell death at low density was rescued by ferroptosis inhibitors (lipid peroxidation inhibitors or iron chelator), but not those of apoptosis and necrosis, supporting that this type of cell death is ferroptosis. To elucidate the mechanisms of ferroptosis resistance at high density, we first conducted transcriptome and metabolome analyses using the difference between low and high densities of A375. The gene ontology analysis on upregulated genes at high density indicated an increase in fatty acid metabolism. In contrast, nutritional stress was not implied from the transcriptional analysis, resulting from frequent changes of culture medium to avoid nutrition starvation. The enrichment analysis based on upregulated metabolites at high density similarly suggested increased de novo lipogenesis. Particularly, gene and protein levels of stearoyl-CoA desaturase (SCD) were induced in a density-dependent manner. Similar induction of SCD was observed in several melanoma cell lines. We further examined the role of SCD in density-dependent ferroptosis resistance. With this aim, pharmacological inhibition or genetic knockout of SCD was conducted on A375 at different densities (low and high) treated with RSL3. The result showed that SCD inhibition reduced RSL3 resistance at high density while having no effect on RSL3 sensitivity at low density. In summary, we found that SCD is upregulated in melanoma cells at high density and protects cells from ferroptosis induced by GPX4 inhibition. It is known that metastasizing melanomas in the blood are sensitive to ferroptosis, resembling cells at low density. This report might provide an explanation for the ferroptosis sensitivity of metastasizing melanomas and a strategy for ferroptosis-based therapy. Citation Format: Hitomi Shirahama, Yuri Tani, Satomi Tsukahara, Yuka Okamoto, Akiko Hasebe, Shingo Dan, Akihiro Tomida. Stearoyl-CoA desaturase confers cell density-dependent resistance to ferroptosis induced by inhibition of glutathione peroxidase 4 in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1384.

Supplementary Table S1 from IL12RB2 and ABCA1 Genes Are Associated with Susceptibility to Radiation Dermatitis

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

Supplementary Methods, Figures 1-4, Tables 1-7 from Chemical Genomics Identifies the Unfolded Protein Response as a Target for Selective Cancer Cell Killing during Glucose Deprivation

PDF file - 292K, hyperactivation of 4E-BP1 in UPR inhibition