Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo. Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.

Ketogenic Diet Alters the Epigenetic and Immune Landscape of Prostate Cancer to Overcome Resistance to Immune Checkpoint Blockade Therapy.

Resistance to immune checkpoint blockade (ICB) therapy represents a formidable clinical challenge limiting the efficacy of immunotherapy. In particular, prostate cancer poses a challenge for ICB therapy due to its immunosuppressive features. A ketogenic diet (KD) has been reported to enhance response to ICB therapy in some other cancer models. However, adverse effects associated with continuous KD were also observed, demanding better mechanistic understanding and optimized regimens for using KD as an immunotherapy sensitizer. In this study, we established a series of ICB-resistant prostate cancer cell lines and developed a highly effective strategy of combining anti-PD1 and anti-CTLA4 antibodies with histone deacetylase inhibitor (HDACi) vorinostat, a cyclic KD (CKD), or dietary supplementation of the ketone body ß-hydroxybutyrate (BHB), which is an endogenous HDACi. CKD and BHB supplementation each delayed prostate cancer tumor growth as monotherapy, and both BHB and adaptive immunity were required for the antitumor activity of CKD. Single-cell transcriptomic and proteomic profiling revealed that HDACi and ketogenesis enhanced ICB efficacy through both cancer cell-intrinsic mechanisms, including upregulation of MHC class I molecules, and -extrinsic mechanisms, such as CD8+ T-cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen-presenting cells, and diminished neutrophil infiltration. Overall, these findings illuminate a potential clinical path of using HDACi and optimized KD regimens to enhance ICB therapy for prostate cancer. SIGNIFICANCE: Optimized cyclic ketogenic diet and 1,3-butanediol supplementation regimens enhance the efficacy of immune checkpoint blockade in prostate cancer through epigenetic and immune modulations, providing dietary interventions to sensitize tumors to immunotherapy.

Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.

Targeting programmed cell death protein 1 (PD-1) is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment. Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1-resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on the secretion of ITA but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest.

Ferroptosis is a non-apoptotic form of cell death that can be triggered by inhibiting the system xc- cystine/glutamate antiporter or the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). We have investigated how cell cycle arrest caused by stabilization of p53 or inhibition of cyclin-dependent kinase 4/6 (CDK4/6) impacts ferroptosis sensitivity. Here, we show that cell cycle arrest can enhance sensitivity to ferroptosis induced by covalent GPX4 inhibitors (GPX4i) but not system xc- inhibitors. Greater sensitivity to GPX4i is associated with increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids (PUFA-PLs). Higher PUFA-PL abundance upon cell cycle arrest involves reduced expression of membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) and epithelial membrane protein 2 (EMP2). A candidate orally bioavailable GPX4 inhibitor increases lipid peroxidation and shrinks tumor volumes when combined with a CDK4/6 inhibitor. Thus, cell cycle arrest may make certain cancer cells more susceptible to ferroptosis in vivo.

Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides.

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1 resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naïve CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

A Cell Cycle-Dependent Ferroptosis Sensitivity Switch Governed by EMP2.

Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. Ferroptosis can be induced by system xc- cystine/glutamate antiporter inhibition or by direct inhibition of the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). The regulation of ferroptosis in response to system xc- inhibition versus direct GPX4 inhibition may be distinct. Here, we show that cell cycle arrest enhances sensitivity to ferroptosis triggered by GPX4 inhibition but not system xc- inhibition. Arrested cells have increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids, which drives sensitivity to GPX4 inhibition. Epithelial membrane protein 2 (EMP2) expression is reduced upon cell cycle arrest and is sufficient to enhance ferroptosis in response to direct GPX4 inhibition. An orally bioavailable GPX4 inhibitor increased markers of ferroptotic lipid peroxidation in vivo in combination with a cell cycle arresting agent. Thus, responses to different ferroptosis-inducing stimuli can be regulated by cell cycle state.

Regulators mount up: the metabolic roles of apoptotic proteins.

The induction of apoptosis, a programmed cell death pathway governed by activation of caspases, can result in fundamental changes in metabolism that either facilitate or restrict the execution of cell death. In addition, metabolic adaptations can significantly impact whether cells in fact initiate the apoptotic cascade. In this mini-review, we will highlight and discuss the interconnectedness of apoptotic regulation and metabolic alterations, two biological outcomes whose regulators are intertwined.

A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells.

In normal tissue homeostasis, bidirectional communication between different cell types can shape numerous biological outcomes. Many studies have documented instances of reciprocal communication between fibroblasts and cancer cells that functionally change cancer cell behavior. However, less is known about how these heterotypic interactions shape epithelial cell function in the absence of oncogenic transformation. Furthermore, fibroblasts are prone to undergo senescence, which is typified by an irreversible cell cycle arrest. Senescent fibroblasts are also known to secrete various cytokines into the extracellular space; a phenomenon that is termed the senescence-associated secretory phenotype (SASP). While the role of fibroblast-derived SASP factors on cancer cells has been well studied, the impact of these factors on normal epithelial cells remains poorly understood. We discovered that treatment of normal mammary epithelial cells with conditioned media from senescent fibroblasts (SASP CM) results in a caspase-dependent cell death. This capacity of SASP CM to cause cell death is maintained across multiple senescence-inducing stimuli. However, the activation of oncogenic signaling in mammary epithelial cells mitigates the ability of SASP CM to induce cell death. Despite the reliance of this cell death on caspase activation, we discovered that SASP CM does not cause cell death by the extrinsic or intrinsic apoptotic pathway. Instead, these cells die by an NLRP3, caspase-1, and gasdermin D-dependent induction of pyroptosis. Taken together, our findings reveal that senescent fibroblasts can cause pyroptosis in neighboring mammary epithelial cells, which has implications for therapeutic strategies that perturb the behavior of senescent cells.

Assessment of Metabolic Pathways and Parameters in Extracellular Matrix-Detached Cells.

Changes in metabolism can alter a variety of distinct cellular parameters in a number of physiological and pathological contexts. Relatedly, the loss of integrin-mediated attachment to extracellular matrix (ECM) is now appreciated to alter metabolism in a variety of distinct fashions. As such, assays to quantify and assess metabolism during ECM detachment are critical to better understanding the cellular and molecular changes that impact the behavior and survival of ECM-detached cells. Here, we discuss assays and approaches commonly used to study metabolism during ECM detachment.

Reprogramming of iron metabolism confers ferroptosis resistance in ECM-detached cells.

Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. Although alterations in membrane lipid content are observed during ECM detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM detachment because of changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of ferritin sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy toward ECM-detached cells.

A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells.

In normal tissue homeostasis, bidirectional communication between different cell types can shape numerous biological outcomes. Many studies have documented instances of reciprocal communication between fibroblasts and cancer cells that functionally change cancer cell behavior. However, less is known about how these heterotypic interactions shape epithelial cell function in the absence of oncogenic transformation. Furthermore, fibroblasts are prone to undergo senescence, which is typified by an irreversible cell cycle arrest. Senescent fibroblasts are also known to secrete various cytokines into the extracellular space; a phenomenon that is termed the senescence-associated secretory phenotype (SASP). While the role of fibroblast derived SASP factors on cancer cells has been well studied, the impact of these factors on normal epithelial cells remains poorly understood. We discovered that treatment of normal mammary epithelial cells with conditioned media (CM) from senescent fibroblasts (SASP CM) results in a caspase-dependent cell death. This capacity of SASP CM to cause cell death is maintained across multiple senescence-inducing stimuli. However, the activation of oncogenic signaling in mammary epithelial cells mitigates the ability of SASP CM to induce cell death. Despite the reliance of this cell death on caspase activation, we discovered that SASP CM does not cause cell death by the extrinsic or intrinsic apoptotic pathway. Instead, these cells die by an NLRP3, caspase-1, and gasdermin D (GSDMD)-dependent induction of pyroptosis. Taken together, our findings reveal that senescent fibroblasts can cause pyroptosis in neighboring mammary epithelial cells, which has implications for therapeutic strategies that perturb the behavior of senescent cells.

A paradox promoted by microglia cannibalism shortens the lifespan of developmental microglia.

The overproduction of cells and subsequent production of debris is a universal principle of neurodevelopment. Here we show an additional feature of the developing nervous system that causes neural debris - promoted by the sacrificial nature of embryonic microglia that irreversibly become phagocytic after clearing other neural debris. Described as long-lived, microglia colonize the embryonic brain and persist into adulthood. Using transgenic zebrafish to investigate the microglia debris during brain construction, we identified that unlike other neural cell-types that die in developmental stages after they have expanded, necroptotic-dependent microglial debris is prevalent when microglia are expanding in the zebrafish brain. Time-lapse imaging of microglia demonstrates that this debris is cannibalized by other microglia. To investigate features that promote microglia death and cannibalism, we used time-lapse imaging and fate-mapping strategies to track the lifespan of individual developmental microglia. These approaches revealed that instead of embryonic microglia being long-lived cells that completely digest their phagocytic debris, once most developmental microglia in zebrafish become phagocytic they eventually die, including ones that are cannibalistic. These results establish a paradox -- which we tested by increasing neural debris and manipulating phagocytosis -- that once most microglia in the embryo become phagocytic, they die, create debris and then are cannibalized by other microglia, resulting in more phagocytic microglia that are destined to die.

SGK1-regulated metabolism: key for the survival of cells detached from the extracellular matrix.

Cells that lack attachment to the extracellular matrix (ECM) experience metabolic defects that can lead to caspase-independent cell death. Recently, we discovered that serum and glucocorticoid kinase-1 (SGK1) plays a critical role in the regulation of glucose metabolism, the promotion of energy production, and ultimately the survival of ECM-detached cells. Abbreviations: ECM, extracellular matrix; SGK1, serum and glucocorticoid kinase-1; ROS, reactive oxygen species; CCCP, cyanide m-chlorophenyl hydrazine; PPP, pentose phosphate pathway; G3P, glyceraldhyde-3-phosphate; shRNA, short hairpin RNA; TCA, tricarboxylic acid.

Oncogenic signaling inhibits c-FLIPL expression and its non-apoptotic function during ECM-detachment.

Inhibition of programmed cell death pathways is frequently observed in cancer cells where it functions to facilitate tumor progression. However, some proteins involved in the regulation of cell death function dichotomously to both promote and inhibit cell death depending on the cellular context. As such, understanding how cell death proteins are regulated in a context-dependent fashion in cancer cells is of utmost importance. We have uncovered evidence that cellular FLICE-like Inhibitory Protein (c-FLIP), a well-known anti-apoptotic protein, is often downregulated in tumor tissue when compared to adjacent normal tissue. These data argue that c-FLIP may have activity distinct from its canonical role in antagonizing cell death. Interestingly, we have discovered that detachment from extracellular matrix (ECM) serves as a signal to elevate c-FLIP transcription and that oncogenic signaling blocks ECM-detachment-induced c-FLIP elevation. In addition, our data reveal that downregulation of c-FLIP promotes luminal filling in mammary acini and that c-FLIP overexpression in cancer cells inhibits colony formation in cells exposed to ECM-detachment. Taken together, our study reveals an unexpected, non-apoptotic role for c-FLIP during ECM-detachment and raises the possibility that c-FLIP may have context-dependent roles during tumorigenesis.

SGK1 signaling promotes glucose metabolism and survival in extracellular matrix detached cells.

Loss of integrin-mediated attachment to extracellular matrix (ECM) proteins can trigger a variety of cellular changes that affect cell viability. Foremost among these is the activation of anoikis, caspase-mediated cell death induced by ECM detachment. In addition, loss of ECM attachment causes profound alterations in cellular metabolism, which can lead to anoikis-independent cell death. Here, we describe a surprising role for serum and glucocorticoid kinase-1 (SGK1) in the promotion of energy production when cells are detached. Our data demonstrate that SGK1 activation is necessary and sufficient for ATP generation during ECM detachment and anchorage-independent growth. More specifically, SGK1 promotes a substantial elevation in glucose uptake because of elevated GLUT1 transcription. In addition, carbon flux into the pentose phosphate pathway (PPP) is necessary to accommodate elevated glucose uptake and PPP-mediated glyceraldehyde-3-phosphate (G3P) is necessary for ATP production. Thus, our data show SGK1 as master regulator of glucose metabolism and cell survival during ECM-detached conditions.

Mitochondrial Reactive Oxygen Species and Mitophagy: A Complex and Nuanced Relationship.

Significance: Mitochondria represent a major source of intracellular reactive oxygen species (ROS) generation. This is often a consequence of oxidative phosphorylation, which can produce ROS as a result of leakage from the electron transport chain. In addition, quality control mechanisms exist to protect cells from cytotoxic ROS production. One such mechanism is selective autophagic degradation of ROS-producing mitochondria, termed mitophagy, that ultimately results in elimination of mitochondria in the lysosome. Recent Advances: However, while the relationship between mitophagy and ROS production is clearly interwoven, it is yet to be fully untangled. In some circumstances, mitochondrial ROS (mtROS) are elevated as a consequence of mitophagy induction. Critical Issues: In this review, we discuss mtROS generation and their detrimental effects on cellular viability. In addition, we consider the cellular defense mechanisms that the eukaryotic cell uses to abrogate superfluous oxidative stress. In particular, we delve into the prominent mechanisms governing mitophagy induction that bear on oxidative stress. Future Directions: Finally, we examine the pathological conditions associated with defective mitophagy, where additional research may help to facilitate understanding.

Antioxidant Defenses: A Context-Specific Vulnerability of Cancer Cells.

Reactive oxygen species (ROS) are well known for their capacity to cause DNA damage, augment mutagenesis, and thereby promote oncogenic transformation. Similarly, agents that reduce ROS levels (antioxidants) are frequently thought to have anti-cancer properties given their propensity to minimize DNA damage and mutagenesis. However, numerous clinical studies focused on antioxidants suggest that this is a facile premise and that antioxidant capacity can be important for cancer cells in a similar fashion to normal cells. As a consequence of this realization, numerous laboratories have been motivated to investigate the biological underpinnings explaining how and when antioxidant activity can potentially be beneficial to cancer cells. Relatedly, it has become clear that the reliance of cancer cells on antioxidant activity in certain contexts represents a potential vulnerability that could be exploited for therapeutic gain. Here, we review some of the recent, exciting findings documenting how cancer cells utilized antioxidant activity and under what circumstances this activity could represent an opportunity for selective elimination of cancer cells.

Ras-ling with new therapeutic targets for metastasis.

Successful cancer metastasis relies on the ability of cancer cells to survive independently of attachment to the extracellular matrix (ECM) and to overcome ECM-detachment-induced death programs. This can be accomplished through activating mutations in cellular oncogenes that subsequently lead to the inhibition of anoikis and to alterations in productive metabolism. One example of such an oncogene is Ras which is found to be mutated and hyperactivated in a variety of distinct cancers. Despite numerous studies on Ras, the precise molecular mechanisms that facilitate survival during ECM-detachment remain poorly understood. Recently, we discovered that ECM-detached cells harboring oncogenic Ras mutations require signaling through the PI(3)K/SGK1 signaling axis to promote survival. Furthermore, we found that oncogenic Ras can concurrently diminish PHLPP1 phosphatase levels, which results in a decrease in p38 MAPK-mediated activation of anoikis. Thus, our data suggest that cancer cells with activating Ras mutations can survive during ECM-detachment using downstream effector molecules that modulate distinct pathways. Overall, these data suggest that new therapeutic interventions that aim to mitigate SGK1 signaling and activate the p38 MAPK activity may aid in specifically targeting and eliminating metastatic cancer cells.

RIPK1-dependent mitophagy: A novel mechanism to eliminate cells detached from the extracellular matrix.

For cancer cells to survive during extracellular matrix (ECM)-detachment, they must inhibit anoikis and rectify metabolic deficiencies that lead to the induction of non-apoptotic cell death. Here, we highlight and discuss our recent study implicating receptor-interacting protein kinase-1 (RIPK1) in the induction of mitophagy, the production of reactive oxygen species (ROS) and the consequent elimination of ECM-detached cells.

RIPK1-mediated induction of mitophagy compromises the viability of extracellular-matrix-detached cells.

For cancer cells to survive during extracellular matrix (ECM) detachment, they must inhibit anoikis and rectify metabolic deficiencies that cause non-apoptotic cell death. Previous studies in ECM-detached cells have linked non-apoptotic cell death to reactive oxygen species (ROS) generation, although the mechanistic underpinnings of this link remain poorly defined. Here, we uncover a role for receptor-interacting protein kinase 1 (RIPK1) in the modulation of ROS and cell viability during ECM detachment. We find that RIPK1 activation during ECM detachment results in mitophagy induction through a mechanism dependent on the mitochondrial phosphatase PGAM5. As a consequence of mitophagy, ECM-detached cells experience diminished NADPH production in the mitochondria, and the subsequent elevation in ROS levels leads to non-apoptotic death. Furthermore, we find that antagonizing RIPK1/PGAM5 enhances tumour formation in vivo. Thus, RIPK1-mediated induction of mitophagy may be an efficacious target for therapeutics aimed at eliminating ECM-detached cancer cells.