Metabolic stress induces a double-positive feedback loop between AMPK and SQSTM1/p62 conferring dual activation of AMPK and NFE2L2/NRF2 to synergize antioxidant defense.

Co-occurring mutations in KEAP1 in STK11/LKB1-mutant NSCLC activate NFE2L2/NRF2 to compensate for the loss of STK11-AMPK activity during metabolic adaptation. Characterizing the regulatory crosstalk between the STK11-AMPK and KEAP1-NFE2L2 pathways during metabolic stress is crucial for understanding the implications of co-occurring mutations. Here, we found that metabolic stress increased the expression and phosphorylation of SQSTM1/p62, which is essential for the activation of NFE2L2 and AMPK, synergizing antioxidant defense and tumor growth. The SQSTM1-driven dual activation of NFE2L2 and AMPK was achieved by inducing macroautophagic/autophagic degradation of KEAP1 and facilitating the AXIN-STK11-AMPK complex formation on the lysosomal membrane, respectively. In contrast, the STK11-AMPK activity was also required for metabolic stress-induced expression and phosphorylation of SQSTM1, suggesting a double-positive feedback loop between AMPK and SQSTM1. Mechanistically, SQSTM1 expression was increased by the PPP2/PP2A-dependent dephosphorylation of TFEB and TFE3, which was induced by the lysosomal deacidification caused by low glucose metabolism and AMPK-dependent proton reduction. Furthermore, SQSTM1 phosphorylation was increased by MAP3K7/TAK1, which was activated by ROS and pH-dependent secretion of lysosomal Ca2+. Importantly, phosphorylation of SQSTM1 at S24 and S226 was critical for the activation of AMPK and NFE2L2. Notably, the effects caused by metabolic stress were abrogated by the protons provided by lactic acid. Collectively, our data reveal a novel double-positive feedback loop between AMPK and SQSTM1 leading to the dual activation of AMPK and NFE2L2, potentially explaining why co-occurring mutations in STK11 and KEAP1 happen and providing promising therapeutic strategies for lung cancer.Abbreviations: AMPK: AMP-activated protein kinase; BAF1: bafilomycin A1; ConA: concanamycin A; DOX: doxycycline; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; LN: low nutrient; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NSCLC: non-small cell lung cancer; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PPP2/PP2A: protein phosphatase 2; ROS: reactive oxygen species; PPP3/calcineurin: protein phosphatase 3; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TCL: total cell lysate; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; V-ATPase: vacuolar-type H+-translocating ATPase.

Design and Synthesis of a Novel 4-aryl-N-(2-alkoxythieno [2,3-b]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide DGG200064 Showed Therapeutic Effect on Colon Cancer through G2/M Arrest.

Cancer cells are characterized by an abnormal cell cycle. Therefore, the cell cycle has been a potential target for cancer therapeutic agents. We developed a new lead compound, DGG200064 (7c) with a 2-alkoxythieno [2,3-b]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide core skeleton. To evaluate its properties, compound DGG200064 was tested in vivo through a xenograft mouse model of colorectal cancer using HCT116 cells. The in vivo results showed high cell growth inhibition efficacy. Our results confirmed that the newly synthesized DGG200064 inhibits the growth of colorectal cancer cells by inducing G2/M arrest. Unlike the known cell cycle inhibitors, DGG200064 (GI50 = 12 nM in an HCT116 cell-based assay) induced G2/M arrest by selectively inhibiting the interaction of FBXW7 and c-Jun proteins. Additionally, the physicochemical properties of the lead compounds were analyzed. Based on the results of the study, we suggested further development of DGG200064 as a novel oral anti-colorectal cancer drug.

Reply to Krupenko et al. Comment on "Lee et al. The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer Cancers 2020, 12, 1382".

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...].

Oxoglutarate Carrier Inhibition Reduced Melanoma Growth and Invasion by Reducing ATP Production.

Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.

Phosphorylation of OCT4 Serine 236 Inhibits Germ Cell Tumor Growth by Inducing Differentiation.

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

ATP Production Relies on Fatty Acid Oxidation Rather than Glycolysis in Pancreatic Ductal Adenocarcinoma.

Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.

Targeting Oxidative Phosphorylation Reverses Drug Resistance in Cancer Cells by Blocking Autophagy Recycling.

The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid proliferation and biosynthesis of cancer cells. This strategy has the potential to trigger autophagy, enabling cancer cell survival through the recycling of molecules and energy essential for biosynthesis, leading to drug resistance. Autophagy recycling contributes amino acids and ATP to restore mTOR complex 1 (mTORC1) activity, which leads to cell survival. However, autophagy with mTORC1 activation can be stalled by reducing the ATP level. We have previously shown that cytosolic NADH production supported by aldehyde dehydrogenase (ALDH) is critical for supplying ATP through oxidative phosphorylation (OxPhos) in cancer cell mitochondria. Inhibitors of the mitochondrial complex I of the OxPhos electron transfer chain and ALDH significantly reduce the ATP level selectively in cancer cells, terminating autophagy triggered by anticancer drug treatment. With the aim of overcoming drug resistance, we investigated combining the inhibition of mitochondrial complex I, using phenformin, and ALDH, using gossypol, with anticancer drug treatment. Here, we show that OxPhos targeting combined with anticancer drugs acts synergistically to enhance the anticancer effect in mouse xenograft models of various cancers, which suggests a potential therapeutic approach for drug-resistant cancer.

Transglutaminase 2-Mediated p53 Depletion Promotes Angiogenesis by Increasing HIF-1α-p300 Binding in Renal Cell Carcinoma.

Angiogenesis and the expression of vascular endothelial growth factor (VEGF) are increased in renal cell carcinoma (RCC). Transglutaminase 2 (TGase 2), which promotes angiogenesis in endothelial cells during wound healing, is upregulated in RCC. Tumor angiogenesis involves three domains: cancer cells, the extracellular matrix, and endothelial cells. TGase 2 stabilizes VEGF in the extracellular matrix and promotes VEGFR-2 nuclear translocation in endothelial cells. However, the role of TGase 2 in angiogenesis in the cancer cell domain remains unclear. Hypoxia-inducible factor (HIF)-1α-mediated VEGF production underlies the induction of angiogenesis in cancer cells. In this study, we show that p53 downregulated HIF-1α in RCC, and p53 overexpression decreased VEGF production. Increased TGase 2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. The interaction of HIF-1α and p53 with the cofactor p300 is required for stable transcriptional activation. We found that TGase 2-mediated p53 depletion increased the availability of p300 for HIF-1α-p300 binding. A preclinical xenograft model suggested that TGase 2 inhibition can reverse angiogenesis in RCC.

The Combination of Loss of ALDH1L1 Function and Phenformin Treatment Decreases Tumor Growth in KRAS-Driven Lung Cancer.

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1-/- mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1-/-; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

Inhibition of Transglutaminase 2 but Not of MDM2 Has a Significant Therapeutic Effect on Renal Cell Carcinoma.

More than 50% of human cancers harbor TP53 mutations and increased expression of Mouse double minute 2 homolog(MDM2), which contribute to cancer progression and drug resistance. Renal cell carcinoma (RCC) has an unusually high incidence of wild-type p53, with a mutation rate of less than 4%. MDM2 is master regulator of apoptosis in cancer cells, which is triggered through proteasomal degradation of wild-type p53. Recently, we found that p53 protein levels in RCC are regulated by autophagic degradation. Transglutaminase 2 (TGase 2) was responsible for p53 degradation through this pathway. Knocking down TGase 2 increased p53-mediated apoptosis in RCC. Therefore, we asked whether depleting p53 from RCC cells occurs via MDM2-mediated proteasomal degradation or via TGase 2-mediated autophagic degradation. In vitro gene knockdown experiments revealed that stability of p53 in RCC was inversely related to levels of both MDM2 and TGase 2 protein. Therefore, we examined the therapeutic efficacy of inhibitors of TGase 2 and MDM2 in an in vivo model of RCC. The results showed that inhibiting TGase 2 but not MDM2 had efficient anticancer effects.

Snail augments fatty acid oxidation by suppression of mitochondrial ACC2 during cancer progression.

Despite the importance of mitochondrial fatty acid oxidation (FAO) in cancer metabolism, the biological mechanisms responsible for the FAO in cancer and therapeutic intervention based on catabolic metabolism are not well defined. In this study, we observe that Snail (SNAI1), a key transcriptional repressor of epithelial-mesenchymal transition, enhances catabolic FAO, allowing pro-survival of breast cancer cells in a starved environment. Mechanistically, Snail suppresses mitochondrial ACC2 (ACACB) by binding to a series of E-boxes located in its proximal promoter, resulting in decreased malonyl-CoA level. Malonyl-CoA being a well-known endogenous inhibitor of fatty acid transporter carnitine palmitoyltransferase 1 (CPT1), the suppression of ACC2 by Snail activates CPT1-dependent FAO, generating ATP and decreasing NADPH consumption. Importantly, combinatorial pharmacologic inhibition of pentose phosphate pathway and FAO with clinically available drugs efficiently reverts Snail-mediated metabolic reprogramming and suppresses in vivo metastatic progression of breast cancer cells. Our observations provide not only a mechanistic link between epithelial-mesenchymal transition and catabolic rewiring but also a novel catabolism-based therapeutic approach for inhibition of cancer progression.

Gastric cancer depends on aldehyde dehydrogenase 3A1 for fatty acid oxidation.

The major source of ATP in cancer cells remains unclear. Here, we examined energy metabolism in gastric cancer cells and found increased fatty acid oxidation and increased expression of ALDH3A1. Metabolic analysis showed that lipid peroxidation by reactive oxygen species led to spontaneous production of 4-hydroxynonenal, which was converted to fatty acids with NADH production by ALDH3A1, resulting in further fatty acid oxidation. Inhibition of ALDH3A1 by knock down using siRNA of ALDH3A1 resulted in significantly reduced ATP production by cancer cells, leading to apoptosis. Oxidative phosphorylation by mitochondria in gastric cancer cells was driven by NADH supplied via fatty acid oxidation. Therefore, blockade of ALDH3A1 together with mitochondrial complex I using gossypol and phenformin led to significant therapeutic effects in a preclinical gastric cancer model.

Loss of SLC25A11 causes suppression of NSCLC and melanoma tumor formation.

BACKGROUND: Fast growing cancer cells require greater amounts of ATP than normal cells. Although glycolysis was suggested as a source of anabolic metabolism based on lactate production, the main source of ATP to support cancer cell metabolism remains unidentified. METHODS: We have proposed that the oxoglutarate carrier SLC25A11 is important for ATP production in cancer by NADH transportation from the cytosol to mitochondria as a malate. We have examined not only changes of ATP and NADH but also changes of metabolites after SLC25A11 knock down in cancer cells. FINDINGS: The mitochondrial electron transport chain was functionally active in cancer cells. The cytosolic to mitochondrial NADH ratio was higher in non-small cell lung cancer (NSCLC) and melanoma cells than in normal cells. This was consistent with higher levels of the oxoglutarate carrier SLC25A11. Blocking malate transport by knockdown of SLC25A11 significantly impaired ATP production and inhibited the growth of cancer cells, which was not observed in normal cells. In in vivo experiments, heterozygote of SLC25A11 knock out mice suppressed KRASLA2 lung tumor formation by cross breeding. INTERPRETATION: Cancer cells critically depended on the oxoglutarate carrier SLC25A11 for transporting NADH from cytosol to mitochondria as a malate form for the purpose of ATP production. Therefore blocking SLC25A11 may have an advantage in stopping cancer growth by reducing ATP production. FUND: The Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT to SYK (NRF-2017R1A2B2003428).

Transglutaminase 2 Promotes Autophagy by LC3 Induction through p53 Depletion in Cancer Cell.

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

Renal Cell Carcinoma Is Abrogated by p53 Stabilization through Transglutaminase 2 Inhibition.

In general, expression of transglutaminase 2 (TGase 2) is upregulated in renal cell carcinoma (RCC), resulting in p53 instability. Previous studies show that TGase 2 binds to p53 and transports it to the autophagosome. Knockdown or inhibition of TGase 2 in RCC induces p53-mediated apoptosis. Here, we screened a chemical library for TGase 2 inhibitors and identified streptonigrin as a potential therapeutic compound for RCC. Surface plasmon resonance and mass spectroscopy were used to measure streptonigrin binding to TGase 2. Mass spectrometry analysis revealed that streptonigrin binds to the N-terminus of TGase 2 (amino acids 95⁻116), which is associated with inhibition of TGase 2 activity in vitro and with p53 stabilization in RCC. The anti-cancer effects of streptonigrin on RCC cell lines were demonstrated in cell proliferation and cell death assays. In addition, a single dose of streptonigrin (0.2 mg/kg) showed marked anti-tumor effects in a preclinical RCC model by stabilizing p53. Inhibition of TGase 2 using streptonigrin increased p53 stability, which resulted in p53-mediated apoptosis of RCC. Thus, targeting TGase 2 may be a new therapeutic approach to RCC.

Targeting Mitochondrial Oxidative Phosphorylation Abrogated Irinotecan Resistance in NSCLC.

Anticancer drug resistance is a major challenge of cancer therapy. We found that irinotecan-resistant NSCLC cells showed increased mitochondrial oxidative phosphorylation compared to the drug sensitive NSCLC cells. Previously, we found that combined inhibition of aldehyde dehydrogenase using gossypol, and mitochondrial complex I using phenformin, effectively reduced oxidative phosphorylation in NSCLC. Here, we showed that targeting oxidative phosphorylation with gossypol and phenformin abrogated irinotecan resistance in NSCLC. Furthermore, irinotecan treatment by blocking oxidative phosphorylation induced synergistic anti-cancer effect in NSCLC. The pre-clinical xenograft model of human NSCLC also demonstrated a therapeutic response to the dual targeting treatment. Therefore, this combination of gossypol and phenformin increases irinotecan sensitivity as well as preventing irinotecan resistance.

Allosteric inhibition site of transglutaminase 2 is unveiled in the N terminus.

Previously we have demonstrated transglutaminase 2 (TGase 2) inhibition abrogated renal cell carcinoma (RCC) using GK921 (3-(phenylethynyl)-2-(2-(pyridin-2-yl)ethoxy)pyrido[3,2-b]pyrazine), although the mechanism of TGase 2 inhibition remains unsolved. Recently, we found that the increase of TGase 2 expression is required for p53 depletion in RCC by transporting the TGase 2 (1-139 a.a)-p53 complex to the autophagosome, through TGase 2 (472-687 a.a) binding p62. In this study, mass analysis revealed that GK921 bound to the N terminus of TGase 2 (81-116 a.a), which stabilized p53 by blocking TGase 2 binding. This suggests that RCC survival can be stopped by p53-induced cell death through blocking the p53-TGase 2 complex formation using GK921. Although GK921 does not bind to the active site of TGase 2, GK921 binding to the N terminus of TGase 2 also inactivated TGase 2 activity through acceleration of non-covalent self-polymerization of TGase 2 via conformational change. This suggests that TGase 2 has an allosteric binding site (81-116 a.a) which changes the conformation of TGase 2 enough to accelerate inactivation through self-polymer formation.

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner.

Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.

Ubiquitylation and degradation of adenomatous polyposis coli by MKRN1 enhances Wnt/ß-catenin signaling.

The adenomatous polyposis coli (APC) protein has a tumor-suppressor function by acting as a negative regulator of the Wnt signaling pathway. While its role as a tumor suppressor is well-defined, the post-translational modifications that regulate APC stability are not fully understood. Here we showed that MKRN1, an E3 ligase, could directly interact with and ubiquitylate APC, promoting its proteasomal degradation. In contrast, an E3 ligase-defective MKRN1 mutant was no longer capable of regulating APC, indicating that its E3 ligase activity is required for APC regulation by MKRN1. Strengthening these results, MKRN1 ablation resulted in reduced ß-catenin activity and decreased expression of Wnt target genes. The ability of the Wnt-dependent pathway to induce cancer cell proliferation, migration, and invasion was impaired by MKRN1 depletion, but restored by simultaneous APC knockdown. Taken together, these results demonstrate that MKRN1 functions as a novel E3 ligase of APC that positively regulates Wnt/ß-catenin-mediated biological processes.

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.