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Autophagy is an attractive therapeutic target in cancer. Successful autophagy-focused clinical intervention will require a detailed understanding of when and where autophagy is important during tumorigenesis. In this issue of Cancer Research, Khayati and colleagues use state-of-the-art genetically engineered mouse models to demonstrate that transient systemic inhibition of autophagy can irreversibly impair the growth of established lung tumors with a good tolerability in normal tissues, suggesting a therapeutic strategy for cancer treatment.See related article by Khayati et al., p. 4429

Ferroptosis is an iron-dependent form of regulated cell death that is triggered by the toxic build-up of lipid peroxides on cellular membranes. In recent years, ferroptosis has garnered enormous interest in cancer research communities, partly because it is a unique cell death modality that is mechanistically and morphologically different from other forms of cell death, such as apoptosis, and therefore holds great potential for cancer therapy. In this Review, we summarize the current understanding of ferroptosis-inducing and ferroptosis defence mechanisms, dissect the roles and mechanisms of ferroptosis in tumour suppression and tumour immunity, conceptualize the diverse vulnerabilities of cancer cells to ferroptosis, and explore therapeutic strategies for targeting ferroptosis in cancer.

Ferroptosis is an iron-dependent form of regulated cell death induced by the lethal overload of lipid peroxides in cellular membranes. In recent years, modulating ferroptosis has gained attention as a potential therapeutic approach for tumor suppression. In the present study, retinol saturase (RETSAT) was identified as a significant ferroptosis mediator using a publicly accessible CRISPR/Cas9 screening dataset. RETSAT depletion protected tumor cells from lipid peroxidation and subsequent cell death triggered by various ferroptosis inducers. Furthermore, exogenous supplementation with retinoids, including retinol (the substrate of RETSAT) and its derivatives retinal and retinoic acid, also suppressed ferroptosis, whereas the product of RETSAT, 13, 14-dihydroretinol, failed to do so. As effective radical-trapping antioxidant, retinoids protected the lipid membrane from autoxidation and subsequent fragmentation, thus terminating the cascade of ferroptosis. Pseudotargeted lipidomic analysis identified an association between retinoid regulation of ferroptosis and lipid metabolism. Retinoic acid, but not 13, 14-dihydroretinoic acid, interacted with its nuclear receptor and activated transcription of SCD, which introduces the first double bond into saturated fatty acid and thus catalyzes the generation of monounsaturated fatty acid, a known ferroptosis suppressor. Therefore, RETSAT promotes ferroptosis by transforming retinol to 13, 14-dihydroretinol, thereby turning a strong anti-ferroptosis regulator into a relatively weak one.

Supplementary Figure and Table Legends

Figure S1. Detection of miR-10b, miR-10a, Hoxd4, and Hoxd3. Figure S2. Body and organ weight. Figure S3. Overall survival and fertility. Figure S4. Complete blood cell count. Figure S5. Organ histology. Figure S6. Mammary tumor cell proliferation. Figure S7. PyMT antibody characterization. Figure S8. miR-10b target screen. Figure S9. TBX5 target identification. Figure S10. TBX5 inhibits breast cancer cell proliferation, migration, and invasion. Figure S11. Clinical correlation.

Legends for Supplementary Figures S1-S11

List of Oligonucleotides

Epigenetic regulation of gene transcription has been shown to coordinate with nutrient availability, yet the mechanisms underlying this coordination remain incompletely understood. Here, we show that glucose starvation suppresses histone 2A K119 monoubiquitination (H2Aub), a histone modification that correlates with gene repression. Glucose starvation suppressed H2Aub levels independently of energy stress–mediated AMP-activated protein kinase activation and possibly through NADPH depletion and subsequent inhibition of BMI1, an integral component of polycomb-repressive complex 1 (PRC1) that catalyzes H2Aub on chromatin. Integrated transcriptomic and epigenomic analyses linked glucose starvation–mediated H2Aub repression to the activation of genes involved in the endoplasmic reticulum (ER) stress response. We further showed that this epigenetic mechanism has a role in glucose starvation–induced cell death and that pharmacologic inhibition of glucose transporter 1 and PRC1 synergistically promoted ER stress and suppressed tumor growth in vivo. Together, these results reveal a hitherto unrecognized epigenetic mechanism coupling glucose availability to the ER stress response.Significance:These findings link glucose deprivation and H2A ubiquitination to regulation of the ER stress response in tumor growth and demonstrate pharmacologic susceptibility to inhibition of polycomb and glucose transporters.

Including Supplementary Figures 1-8. Supplementary Figure 1 shows working model for different energy stress inducers. Supplementary Figures 2-7 are additional data corresponding to Figures 2-7 in the manuscript. Supplementary Figure 8 showing proposed working model from this study.

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

Additional details related to the Materials and Methods section that appears in the main text.

PDF file - 232KB, Figure S1: FoxO subcellular localization under NVP-BEZ235, BKM120, or MK-2206 treatment. Figure S2: Quantified ratios of AKT phosphorylation levels to total AKT levels in FoxO WT and KO cells after 50 nM NVP-BEZ235 treatment. Figure S3: The effect of high dosage NVP-BEZ235 on cell proliferation in FoxO WT and KO cells. Figure S4: Rictor knockdown potentiates cell proliferation suppression and cell death induction in response to NVP-BEZ235 treatment in RCC cells. Figure S5: The expression levels of p27 and Bim in FoxO deficient cells under NVPBEZ235 treatment.

PDF file - 91KB

The PI3K–AKT pathway is hyperactivated in many human cancers, and several drugs to inhibit this pathway, including the PI3K/mTOR dual inhibitor NVP-BEZ235, are currently being tested in various preclinical and clinical trials. It has been shown that pharmacologic inhibition of the PI3K–AKT pathway results in feedback activation of other oncogenic signaling pathways, which likely will limit the clinical utilization of these inhibitors in cancer treatment. However, the underlying mechanisms of such feedback regulation remain incompletely understood. The PI3K–AKT pathway is a validated therapeutic target in renal cell carcinoma (RCC). Here, we show that FoxO transcription factors serve to promote AKT phosphorylation at Ser473 in response to NVP-BEZ235 treatment in renal cancer cells. Inactivation of FoxO attenuated NVP-BEZ235–induced AKT Ser473 phosphorylation and rendered renal cancer cells more susceptible to NVP-BEZ235–mediated cell growth suppression in vitro and tumor shrinkage in vivo. Mechanistically, we showed that FoxOs upregulated the expression of Rictor, an essential component of MTOR complex 2, in response to NVP-BEZ235 treatment and revealed that Rictor is a key downstream target of FoxOs in NVP-BEZ235–mediated feedback regulation. Finally, we show that FoxOs similarly modulate the feedback response on AKT Ser473 phosphorylation and renal tumor growth by other phosphoinositide 3-kinase (PI3K) or AKT inhibitor treatment. Together, our study reveals a novel mechanism of PI3K–AKT inhibition-mediated feedback regulation and may identify FoxO as a novel biomarker to stratify patients with RCC for PI3K or AKT inhibitor treatment, or a novel therapeutic target to synergize with PI3K–AKT inhibition in RCC treatment. Cancer Res; 74(6); 1682–93. ©2014 AACR.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Glucocorticoids, both endogenous and exogenous, are known anti-inflammatory agents with associated adverse effects when administered chronically or in excess. Foxo family members, specifically Foxo1, Foxo3 and Foxo4 have demonstrated to be major regulators of metabolic complications following glucocorticoid action. We employed mouse embryonic fibroblasts as model system and using tamoxifen induced Cre+ recombinase activation, we achieved deletion of the Foxo triplets- Foxo1, Foxo3, and Foxo4. Following incubation of dexamethasone in both wild type and Foxo triplet deleted group, RNA sequencing was performed to reveal the transcriptome. Interestingly, the transcripts involved in initiating metabolic stress like PDK4 and 11 Beta HSD1 were downregulated, whereas transcripts involved in anti-viral response were upregulated following Foxo triplet deletion. The anti-inflammatory property of glucocorticoids was kept intact based on transcripts evaluated including GILZ and COX2. In summary, our study reveals a unique phenomenon where we will be able to manipulate glucocorticoid action by deleting Foxo triplets eliminating the metabolic stress components but enhancing anti-viral response and maintaining their clinical use as an anti-inflammatory agent. This work will lay foundation for a paradigm shift in clinical use of glucocorticoids according to therapeutic advantage with lesser adverse effects

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells ofUSP7and its closest homolog,USP47. Our proteome profiling for the first time revealed the proteome changes caused byUSP7and/orUSP47depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells withUSP7deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

Lipid peroxidation occurs under conditions where reactive oxygen species (ROS) readily react with vulnerable lipids on cell membranes. Polyunsaturated fatty acids (PUFAs) are highly susceptible to lipid peroxidation because of their unstable double bonds. Because the cell membrane is particularly rich in PUFAs, it is often the site at which many lipid peroxidation chain reactions occur. Lipid peroxidation is considered the ultimate trigger of ferroptosis, an iron-dependent form of non-apoptotic cell death. Radiotherapy is a common cancer treatment that uses high-energy ionizing radiation to kill cancer cells, and radiation-induced cell death is partially attributed to lipid peroxidation-driven ferroptosis. Here, we describe methods to assess lipid peroxidation in irradiated cells. The same techniques can be applied to a variety of lipid peroxidation measurements under different treatment conditions.

Ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation, has recently emerged as a key tumor suppression mechanism(1–4). Glutathione peroxidase 4 (GPX4)(5, 6) and ferroptosis suppressor protein 1 (FSP1)(7, 8) constitute two major ferroptosis defense systems. Here we show that treatment with GPX4 inhibitors in cancer cells results in an acute depletion of N-carbamoyl-L-aspartate (C-Asp), a pyrimidine biosynthesis intermediate, with a concomitant accumulation of uridine. Supplementation of dihydroorotate (DHO) or orotate (OA), the substrate and product of DHO dehydrogenase (DHODH), attenuates or potentiates ferroptosis induced by GPX4 inhibition, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4(low)). DHODH inactivation induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4(low) cancer cells, whereas DHODH inactivation synergizes with ferroptosis inducers to induce mitochondrial lipid peroxidation and ferroptosis in GPX4(high) cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independent of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane, through reducing ubiquinone (CoQ) to ubiquinol (CoQH(2)), a radical-trapping antioxidant with anti-ferroptosis activity. We further show that the DHODH inhibitor brequinar selectively suppresses GPX4(low) tumor growth through inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis inducing activity, synergistically induces ferroptosis and suppresses GPX4(high) tumor growth. Our results identify a DHODH-mediated ferroptosis defense mechanism in mitochondria and suggest a therapeutic strategy to target ferroptosis in cancer treatment.

Ferroptosis, a form of regulated cell death triggered by lipid peroxidation, was recently identified as an important mechanism in radiotherapy (RT)-mediated tumor suppression and radioresistance, although the exact genetic contexts in which to target ferroptosis in RT remains to be defined. p53 is the most commonly mutated gene in human cancers and a major effector to RT. Here, we identify ferroptosis as a critical mechanism to mediate p53 function in tumor radiosensitivity. Mechanistically, RT-mediated p53 activation antagonizes RT-induced SLC7A11 expression and represses glutathione synthesis, thereby promoting RT-induced lipid peroxidation and ferroptosis. p53 deficiency promotes radioresistance in cancer cells or tumors at least partly through SLC7A11-mediated ferroptosis inhibition. Ferroptosis inducers (FINs) that inhibit SLC7A11 exert significant radiosensitizing effects in tumor organoids and patient-derived xenografts with p53 mutation or deficiency. Finally, we show that RT-induced ferroptosis correlates with p53 activation and better clinical outcomes to RT in cancer patients. Together, our study uncovers a previously unappreciated role of ferroptosis in p53-mediated radiosensitization and suggest using FINs in combination with RT to treat p53-mutant cancers.

Ferroptosis, an iron-dependent form of regulated cell death driven by peroxidative damages of polyunsaturated-fatty-acid-containing phospholipids in cellular membranes, has recently been revealed to play an important role in radiotherapy-induced cell death and tumor suppression, and to mediate the synergy between radiotherapy and immunotherapy. In this review, we summarize known as well as putative mechanisms underlying the crosstalk between radiotherapy and ferroptosis, discuss the interactions between ferroptosis and other forms of regulated cell death induced by radiotherapy, and explore combination therapeutic strategies targeting ferroptosis in radiotherapy and immunotherapy. This review will provide important frameworks for future investigations of ferroptosis in cancer therapy.