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How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a G protein, that promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes non-homologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard of care genotoxic treatments. The GTP-Rac1-PP5-Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in non-malignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment.

Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron–sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC.Significance:Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin (“ferritinophagy”) as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC.See related commentary by Jain and Amaravadi, p. 2023.See related article by Ravichandran et al., p. 2198.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Figure from NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron–Sulfur Cluster Proteins

Supplementary Table from NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron–Sulfur Cluster Proteins

Supplementary Data from NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron–Sulfur Cluster Proteins

Intestinal iron absorption is activated during increased systemic demand for iron. The best-studied example is iron deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor 2α (HIF2α). However, pathways for selective targeting of intestine-mediated iron overload remain unknown. Nuclear receptor coactivator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin and the subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated into systemic iron demand via HIF2α. To demonstrate the importance of the intestinal HIF2α/ferritinophagy axis in systemic iron homeostasis, whole-body and intestine-specific NCOA4−/− mouse lines were generated and assessed. The analyses revealed that the intestinal and systemic response to iron deficiency was not altered after disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.

Percentage of APC, CTNNB1 and ZNF148 mutations in CRC (TCGA)

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

Supplemental Methods, Figure Legends and References for Supplemental Methods

Immunohistochemistry for ZBP-89 and Ki67 in WT and VC:Zfp148F/F mice

ZBP-89 Probe Sets:BFCOL1 and BERF1

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Angiogenic programming in the vascular endothelium is a tightly regulated process to maintain tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Herein, we report that hypoxic upregulation of NO synthesis in endothelial cells reprograms the transsulfuration pathway and increases H2S biogenesis. Furthermore, H2S oxidation by mitochondrial sulfide quinone oxidoreductase (SQOR) rather than downstream persulfides, synergizes with hypoxia to induce a reductive shift, limiting endothelial cell proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body WBCreSQORfl/flknockout mice exhibit lower mass and reduced angiogenesis compared to SQORfl/flcontrols. WBCreSQORfl/flmice also exhibit reduced muscle angiogenesis following femoral artery ligation, compared to controls. Collectively, our data reveal the molecular intersections between H2S, O2and NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.HighlightsHypoxic induction of •NO in endothelial cells inhibits CBS and switches CTH reaction specificityHypoxic interruption of the canonical transsulfuration pathway promotes H2S synthesisSynergizing with hypoxia, SQOR deficiency induces a reductive shift in the ETC and restricts proliferationSQOR KO mice exhibit lower neovascularization in tumor xenograft and hind limb ischemia models

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and used unbiased multi-omic analyses to reveal a robust accumulation of tumoral ammonia. The high ammonia levels induce T cell metabolic reprogramming, increase exhaustion, and decrease proliferation. CRC patients have increased serum ammonia, and the ammonia-related gene signature correlates with altered T cell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates T cells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. These findings indicate that enhancing ammonia detoxification can reactivate T cells, highlighting a new approach to enhance the efficacy of immunotherapies.

Drug‐induced hepatotoxicity limits development of new effective medications. Drugs and numerous endogenous/exogenous agents are metabolized/detoxified by hepatocytes, during which reactive oxygen species (ROS) are generated as a by‐product. ROS has broad adverse effects on liver function and integrity, including damaging hepatocyte proteins, lipids, and DNA and promoting liver inflammation and fibrosis. ROS in concert with iron overload drives ferroptosis. Hepatic nuclear factor kappa B (NF‐κB)‐inducing kinase (NIK) is aberrantly activated in a broad spectrum of liver disease. NIK phosphorylates and activates inhibitor of NF‐κB kinase subunit alpha (IKKα), and the hepatic NIK/IKKα cascade suppresses liver regeneration. However, the NIK/IKKα pathway has not been explored in drug‐induced liver injury. Here, we identify hepatic NIK as a previously unrecognized mediator for acetaminophen (APAP)‐induced acute liver failure. APAP treatment increased both NIK transcription and NIK protein stability in primary hepatocytes as well as in liver in mice. Hepatocyte‐specific overexpression of NIK augmented APAP‐induced liver oxidative stress in mice and increased hepatocyte death and mortality in a ROS‐dependent manner. Conversely, hepatocyte‐specific ablation of NIK or IKKα mitigated APAP‐elicited hepatotoxicity and mortality. NIK increased lipid peroxidation and cell death in APAP‐stimulated primary hepatocytes. Pretreatment with antioxidants or ferroptosis inhibitors blocked NIK/APAP‐induced hepatocyte death. Conclusion: We unravel a previously unrecognized NIK/IKKα/ROS/ferroptosis axis engaged in liver disease progression., Hepatic NIK is upregulated in response to hepatic toxicants. Ablation of hepatic NIK attenuates, whereas hepatocyte‐specific overexpression of NIK aggravates, APAP‐induced liver injury. NIK promotes hepatic oxidative stress and ferroptosis.

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. We show that PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth following nutrient stress. However, the mechanism by which PINK1 maintains colon cancer growth remains equivocal. Inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited colon cancer cell proliferation, whereas disruption of other mitophagy receptors did not similarly impact cellular proliferation. Mechanistically, we observed a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione following PINK1 KD. Mitochondria are important hubs for storing iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. An increase iron storage protein ferritin and a decrease labile iron pool was observed in PINK1 KD cells. However, neither total cellular iron nor markers of iron starvation/overload were affected. Cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). Overexpressing nuclear receptor coactivator 4 (NCOA4), a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. We demonstrate that PINK1 regulates intracellular iron availability by integrating mitophagy to ferritinophagy. In conclusion, these results indicate that PINK1 is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.

Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron–sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC. Significance: Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin (“ferritinophagy”) as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC. See related commentary by Jain and Amaravadi, p. 2023. See related article by Ravichandran et al., p. 2198. This article is highlighted in the In This Issue feature, p. 2007

Hypoxia-inducible factors (HIFs) are transcription factors critical for the adaptive response to hypoxia. There is also an essential link between hypoxia and inflammation, and HIFs have been implicated in the dysregulated immune response to various insults. Despite the prevalence of hypoxia in tissue trauma, especially involving the lungs, there remains a dearth of studies investigating the role of HIFs in clinically relevant injury models. Here, we summarize the effects of HIF-1α on the vasculature, metabolism, inflammation, and apoptosis in the lungs and review the role of HIFs in direct lung injuries, including lung contusion, acid aspiration, pneumonia, and COVID-19. We present data that implicates HIF-1α in the context of arguments both in favor and against its role as adaptive or injurious in the propagation of the acute inflammatory response in lung injuries. Finally, we discuss the potential for pharmacological modulation of HIFs as a new class of therapeutics in the modern intensive care unit.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling and metabolically leverages iron are poorly understood. We demonstrate that the liver-derived, endocrine regulator of systemic iron balance, hepcidin, is activated ectopically in CRC. Hepcidin binds to the only known mammalian iron exporter ferroportin, resulting in degradation of ferroportin and intracellular iron trapping. Mice deficient for the hepcidin gene specifically in colon tumor epithelium exhibited significant decreases in tumor number, burden, and size compared to wild-type littermates in a sporadic model of CRC, whereas ferroportin deletion exacerbated these tumor parameters. To further understand the biochemical and metabolic utilization of iron in CRC, we subjected a three-dimensional patient-derived CRC tumor enteroid model to metabolomics and found that iron is prioritized in CRC for the production of nucleotides. These metabolomics findings were recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that a decrease in mitochondrial function alters nucleotide synthesis following iron chelation. Restoration of nucleotide metabolism with exogenous supplementation of nucleosides led to a partial rescue of growth in patient-derived tumor enteroids and CRC cell lines in the presence of an iron chelator. Moreover, aspartate, a critical metabolite which links mitochondrial respiration and nucleotide synthesis, also partially rescued growth of iron deficient CRC cells. Collectively, these data suggest that ectopic hepcidin in the tumor epithelium establishes an axis to degrade ferroportin and sequester iron in colorectal tumors in order to maintain the nucleotide pool and sustain proliferation.

Colorectal cancer (CRC) is a devastating disease that is highly modulated by dietary nutrients. Mechanistic target of rapamycin complex 1 (mTORC1) contributes to tumor growth and limits therapy responses. Growth factor signaling is a major mechanism of mTORC1 activation. However, compensatory pathways exist to sustain mTORC1 activity after therapies that target oncogenic growth factor signaling. Amino acids potently activate mTORC1 via amino acid-sensing GTPase activity towards Rags (GATOR). The role of amino acid-sensing pathways in CRC is unclear.Human colon cancer cell lines, preclinical intestinal epithelial-specific GATOR1 and GATOR2 knockout mouse subjected to colitis-induced or sporadic colon tumor models, small interfering RNA screening targeting regulators of mTORC1, and tissues of patients with CRC were used to assess the role of amino acid sensing in CRC.We identified loss-of-function mutations of the GATOR1 complex in CRC and showed that altered expression of amino acid-sensing pathways predicted poor patient outcomes. We showed that dysregulated amino acid-sensing induced mTORC1 activation drives colon tumorigenesis in multiple mouse models. We found amino acid-sensing pathways to be essential in the cellular reprogramming of chemoresistance, and chemotherapeutic-resistant patients with colon cancer exhibited de-regulated amino acid sensing. Limiting amino acids in in vitro and in vivo models (low-protein diet) reverted drug resistance, revealing a metabolic vulnerability.Our findings suggest a critical role for amino acid-sensing pathways in driving CRC and highlight the translational implications of dietary protein intervention in CRC.

Background & Aims Fatty liver or nonalcoholic fatty liver disease (NAFLD) is the most common liver disease associated with comorbidities such as insulin resistance and cardiovascular and metabolic diseases. Chronic activation of hypoxic signaling, in particular, hypoxia-inducible factor (HIF)2α, promotes NAFLD progression by repressing genes involved in fatty acid β-oxidation through unclear mechanisms. Therefore, we assessed the precise mechanism by which HIF2α promotes fatty liver and its physiological relevance in metabolic homeostasis. Methods Primary hepatocytes from VHL (VhlΔHep) and PPARα (Ppara-null) knockout mice that were loaded with fatty acids, murine dietary protocols to induce hepatic steatosis, and fasting-refeeding dietary regimen approaches were used to test our hypothesis. Results Inhibiting autophagy using chloroquine did not decrease lipid contents in VhlΔHep primary hepatocytes. Inhibition of ERK using MEK inhibitor decreased lipid contents in primary hepatocytes from a genetic model of constitutive HIF activation and primary hepatocytes loaded with free fatty acids. Moreover, MEK-ERK inhibition potentiated ligand-dependent activation of PPARα. We also show that MEK-ERK inhibition improved diet-induced hepatic steatosis, which is associated with the induction of PPARα target genes. During fasting, fatty acid β-oxidation is induced by PPARα, and refeeding inhibits β-oxidation. Our data show that ERK is involved in the post-prandial repression of hepatic PPARα signaling. Conclusions Overall, our results demonstrate that ERK activated by hypoxia signaling plays a crucial role in fatty acid β-oxidation genes by repressing hepatocyte PPARα signaling., Graphical abstract

Aim: Sessile serrated adenomas (SSAs) are premalignant lesions driven by the BRAF(V600E) mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. Results: Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAF(V600E) mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAF(V600E) mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusion: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress and can serve as an imaging biomarker for in vivo detection of SSAs. Antioxid. Redox Signal. 36, 39–56.