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Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein–protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis., Inflammation: Understanding cell death opens therapeutic possibilities A better understanding of the signaling pathways that drive an inflammatory mode of cell death known as necroptosis could lead to new therapies for autoimmune and neuroinflammatory conditions. Necroptosis is a regulated form of cell death often triggered by infections, tissue injuries, and chronic inflammatory conditions. It leads cells to rupture and disperse their contents, which in turn spurs an inflammatory immune response that can sometimes fuel disease. A team from South Korea led by Jaewhan Song of Yonsei University in Seoul discusses the molecular mechanisms of this process. They focus on several key mediators of the necroptosis signaling pathway and how those proteins in turn are modified to regulate their function. Several drug inhibitors of these proteins are already in clinical development for the treatment of psoriasis and other inflammatory disorders.

Callyspongiolide is a marine macrolide known to induce caspaseindependent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments. [BMB Reports 2021; 54(4): 227-232].

Rationale: The activity of aldehyde dehydrogenase 7A1 (ALDH7A1), an enzyme that catalyzes the lipid peroxidation of fatty aldehydes was found to be upregulated in pancreatic ductal adenocarcinoma (PDAC). ALDH7A1 knockdown significantly reduced tumor formation in PDAC. We raised a question how ALDH7A1 contributes to cancer progression. Methods: To answer the question, the role of ALDH7A1 in energy metabolism was investigated by knocking down and knockdown gene in mouse model, because the role of ALDH7A1 has been reported as a catabolic enzyme catalyzing fatty aldehyde from lipid peroxidation to fatty acid. Oxygen consumption rate (OCR), ATP production, mitochondrial membrane potential, proliferation assay and immunoblotting were performed. In in vivo study, two human PDAC cell lines were used for pre-clinical xenograft model as well as spontaneous PDAC model of KPC mice was also employed for anti-cancer therapeutic effect. Results: ALDH7A1 knockdown significantly reduced tumor formation with reduction of OCR and ATP production, which was inversely correlated with increase of 4-hydroxynonenal. This implies that ALDH7A1 is critical to process fatty aldehydes from lipid peroxidation. Overall survival of PDAC is doubled by cross breeding of KPC (KrasG12D; Trp53R172H; Pdx1-Cre) and Aldh7a1-/- mice. Conclusion: Inhibitions of ALDH7A1 and oxidative phosphorylation using gossypol and phenformin resulted in a regression of tumor formation in xenograft mice model and KPC mice model.

Ferroptosis is an iron-dependent regulated necrosis mediated by lipid peroxidation. Cancer cells survive under metabolic stress conditions by altering lipid metabolism, which may alter their sensitivity to ferroptosis. However, the association between lipid metabolism and ferroptosis is not completely understood. In this study, we found that the expression of elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) is up-regulated in mesenchymal-type gastric cancer cells (GCs), leading to ferroptosis sensitization. In contrast, these enzymes are silenced by DNA methylation in intestinal-type GCs, rendering cells resistant to ferroptosis. Lipid profiling and isotope tracing analyses revealed that intestinal-type GCs are unable to generate arachidonic acid (AA) and adrenic acid (AdA) from linoleic acid. AA supplementation of intestinal-type GCs restores their sensitivity to ferroptosis. Based on these data, the polyunsaturated fatty acid (PUFA) biosynthesis pathway plays an essential role in ferroptosis; thus, this pathway potentially represents a marker for predicting the efficacy of ferroptosis-mediated cancer therapy.

Significance A major yet perplexing question in the field of necroptosis is the role and involvement of necroptosis in cancer cells. Many cancer cells have protective mechanisms against necroptosis, but the underlying mechanism remains elusive. We report findings of cross-talk and a regulatory pathway that exist between MYC, a potent oncogene, and RIPK3, a pivotal factor in necroptosis. We find that MYC pathway is downregulated upon necroptotic, while MYC inhibits TNF-α–induced necroptosis. The inhibitory effect of MYC on necroptosis is unexpected because no transcriptional activity by MYC is required. Mechanistically, a direct interaction between MYC and RIPK3 takes place in the cytosol, preventing RIPK1–RIPK3 complex formation. Finally, MYC depletion enhances antitumor activity of necroptosis-inducing agents in a xenograft model., The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1–RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1–RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Necroptosis is a form of regulated cell death caused by formation of the necrosome complex. However, the factors modulating this process and the systemic pathophysiological effects of necroptosis are yet to be understood. Here, we identified that Beclin 1 functions as an anti-necroptosis factor by being recruited into the necrosome complex upon treatment with TNFα, Smac mimetic, and pan-caspase inhibitor and by repressing MLKL oligomerisation, thus preventing the disruption of the plasma membrane. Cells ablated or knocked-out for Beclin 1 become sensitised to necroptosis in an autophagy-independent manner without affecting the necrosome formation itself. Interestingly, the recruitment of Beclin 1 into the necrosome complex is dependent on the activation and phosphorylation of MLKL. Biochemically, the coiled-coil domain (CCD) of Beclin 1 binds to the CCD of MLKL, which restrains the oligomerisation of phosphorylated MLKL. Finally, Beclin 1 depletion was found to promote necroptosis in leukaemia cells and enhance regression of xenografted-tumour upon treatment with Smac mimetics and caspase inhibitors. These results suggest that Beclin 1 functions as a negative regulator in the execution of necroptosis by suppressing MLKL oligomerisation.

Summary: There are abundant peroxiredoxin (Prx) enzymes, but an increase of cellular H2O2 level always happens in apoptotic cells. Here, we show that cellular H2O2 switches different apoptosis pathways depending on which type of Prx enzyme is absent. TNF-α-induced H2O2 burst preferentially activates the DNA damage-dependent apoptosis pathway in the absence of PrxI. By contrast, the same H2O2 burst stimulates the RIPK1-dependent apoptosis pathway in the absence of PrxII by inducing the destruction of cIAP1 in caveolar membrane. Specifically, H2O2 induces the oxidation of Cys308 residue in the cIAP1-BIR3 domain, which induces the dimerization-dependent E3 ligase activation. Thus, the reduction in cIAP level by the absence of PrxII triggers cell-autonomous apoptosis in cancer cells and tumors. Such differential functions of PrxI and PrxII are mediated by interaction with H2AX and cIAP1, respectively. Collectively, this study reveals the distinct switch roles of 2-Cys Prx isoforms in apoptosis signaling. : Lee et al. show that the 2-Cys peroxiredoxin (Prx) isoforms, Prx I and Prx II, discretely regulate different apoptosis pathways. Specifically, the absence of Prx I augments apoptosis through the DNA damage response, while the absence of Prx II switches RIPK1-dependent apoptosis by causing cIAP1 depletion. Keywords: peroxiredoxin, apoptosis, H2O2, TNF-α, cIAP, DNA damage, RIPK1

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-X(L) or BCL2L1). This binding disrupted an interaction between Bcl-X(L) and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-X(L). Interestingly, the structure of the Bcl-X(L)–p73-TAD16 peptide complex revealed a novel mechanism of Bcl-X(L) recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-X(L), comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-X(L) recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

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.

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders., AMPK activation has been suggested as treatment for obesity and its complications. Here the authors show that the ubiquitin ligase MKRN1 binds to AMPK and mediates its ubiquitination and degradation. Loss of MKRN1 leads to AMPK activation, increased glucose consumption and decreased lipid accumulation.

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.

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 β-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/β-catenin-mediated biological processes.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20178-0

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Although myocardial cell death plays a significant role in myocardial infarction (MI), its underlying mechanism remains to be elucidated. To understand the progression of MI and identify potential therapeutic targets, we performed tandem mass tag (TMT)-based quantitative proteomic analysis using an MI mouse model. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) revealed that the glutathione metabolic pathway and reactive oxygen species (ROS) pathway were significantly downregulated during MI. In particular, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis (an iron-dependent programme of regulated necrosis), was downregulated in the early and middle stages of MI. RNA-seq and qRT-PCR analyses suggested that GPX4 downregulation occurred at the transcriptional level. Depletion or inhibition of GPX4 using specific siRNA or the chemical inhibitor RSL3, respectively, resulted in the accumulation of lipid peroxide, leading to cell death by ferroptosis in H9c2 cardiomyoblasts. Although neonatal rat ventricular myocytes (NRVMs) were less sensitive to GPX4 inhibition than H9c2 cells, NRVMs rapidly underwent ferroptosis in response to GPX4 inhibition under cysteine deprivation. Our study suggests that downregulation of GPX4 during MI contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress such as cysteine deprivation.

Notch, an essential factor in tissue development and homoeostasis, has been reported to play an oncogenic function in a variety of cancers. Here, we report ubiquitin-specific protease 8 (USP8) as a novel deubiquitylase of Notch1 intracellular domain (NICD). USP8 specifically stabilizes and deubiquitylates NICD through a direct interaction. The inhibition of USP8 downregulated the Notch signalling pathway via NICD destabilization, resulting in the retardation of cellular growth, wound closure, and colony forming ability of breast cancer cell lines. These phenomena were restored by the reconstitution of NICD or USP8, supporting the direct interaction between these two proteins. The expression levels of NICD and USP8 proteins were positively correlated in patients with advanced breast cancer. Taken together, our results suggest that USP8 functions as a positive regulator of Notch signalling, offering a therapeutic target for breast cancer.

The tumor suppressor function of p14ARF is regulated at a posttranslational level via mechanisms yet to be fully understood. Here, we report the identification of an unconventional p14ARF degradation pathway induced by the chaperone HSP90 in association with the E3 ubiquitin ligase C-terminus of HSP70-interacting protein (CHIP). The ternary complex of HSP90, CHIP, and p14ARF was required to induce the lysosomal degradation of p14ARF by an ubiquitination-independent but LAMP2A-dependent mechanism. Depletion of HSP90 or CHIP induced p14ARF-dependent senescence in human fibroblasts. Premature senescence observed in cells genetically deficient in CHIP was rescued in cells that were doubly deficient in CHIP and p14ARF. Notably, non–small cell lung cancer cells (NSCLC) positive for p14ARF were sensitive to treatment with the HSP90 inhibitor geldanamycin. Furthermore, overexpression of HSP90 and CHIP with a concomitant loss of p14ARF correlated with poor prognosis in patients with NSCLC. Our findings identify a relationship between p14ARF and its chaperones that suggest new therapeutic strategies in cancers that overexpress.

ARF is an alternative reading frame product of the INK4a/ARF locus, inactivated in numerous human cancers. ARF is a key regulator of cellular senescence, an irreversible cell growth arrest that suppresses tumor cell growth. It functions by sequestering MDM2 (a p53 E3 ligase) in the nucleolus, thus activating p53. Besides MDM2, ARF has numerous other interacting partners that induce either cellular senescence or apoptosis in a p53-independent manner. This further complicates the dynamics of the ARF network. Expression of ARF is frequently disrupted in human cancers, mainly due to epigenetic and transcriptional regulation. Vigorous studies on various transcription factors that either positively or negatively regulate ARF transcription have been carried out. However, recent focus on posttranslational modifications, particularly ubiquitination, indicates wider dynamic controls of ARF than previously known. In this review, we discuss the role and dynamic regulation of ARF in senescence and cancer. [BMB Reports 2016; 49(11): 598-606].

// Hye-Young Kim 1, 2 , Yunhee Cho 1, 3 , HyeokGu Kang 1, 3 , Ye-Seal Yim 1, 3 , Seok-Jun Kim 1, 3 , Jaewhan Song 2 , Kyung-Hee Chun 1, 3 1 Department of Biochemistry & Molecular Biology, Yonsei University College of Medicine, Seodaemun-gu, Seoul 03722, Korea 2 Department of Biochemistry, College of Life Science and Biotechnology, Seodaemun-gu, Seoul 03722, Korea 3 Brain Korea 21 PlusProject for Medical Science, Yonsei University, Seodaemun-gu, Seoul 03722, Korea Correspondence to: Kyung-Hee Chun, email: khchun@yuhs.ac Keywords: WEE1, AZD1775 (MK-1775), 5-FU, Paclitaxel, gastric cancer Received: September 07, 2015 Accepted: May 28, 2016 Published: June 23, 2016 ABSTRACT Wee1 is a member of the Serine/Threonine protein kinase family and is a key regulator of cell cycle progression. It has been known that WEE1 is highly expressed and has oncogenic functions in various cancers, but it is not yet studied in gastric cancers. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in gastric cancer. At first, higher expression levels of WEE1 with lower survival probability were determined in stage 4 gastric cancer patients or male patients with accompanied lymph node metastasis. To determine the function of WEE1 in gastric cancer cells, we determined that WEE1 ablation decreased the proliferation, migration, and invasion, while overexpression of WEE1 increased these effects in gastric cancer cells. We also validated the clinical application of WEE1 targeting by a small molecule, AZD1775 (MK-1775), which is a WEE1 specific inhibitor undergoing clinical trials. AZD1775 significantly inhibited cell proliferation and induced apoptosis and cell cycle arrest in gastric cancer cells, which was more effective in WEE1 high-expressing gastric cancer cells. Moreover, we performed combination treatments with AZD1775 and anti-cancer agents, 5- fluorouracil or Paclitaxel in gastric cancer cells and in gastric cancer orthotopic-transplanted mice to maximize the therapeutic effect and safety of AZD1775. The combination treatments dramatically inhibited the proliferation of gastric cancer cells and tumor burdens in stomach orthotopic-transplanted mice. Taken together, we propose that WEE1 is over-expressed and could enhance gastric cancer cell proliferation and metastasis. Therefore, we suggest that WEE1 is a potent target for gastric cancer therapy.

FLICE-like inhibitory protein (FLIP) is a critical regulator of death receptor-mediated apoptosis. Here, we found ubiquitin-specific peptidase 8 (USP8) to be a novel deubiquitylase of the long isoform of FLIP (FLIPL). USP8 directly deubiquitylates and stabilizes FLIPL, but not the short isoform. USP8 depletion induces FLIPL destabilization, promoting anti-Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha-induced extrinsic apoptosis by facilitating death-inducing signaling complex or TNFR1 complex II formation, which results in the activation of caspase-8 and caspase-3. USP8 mRNA levels are elevated in melanoma and cervical cancers, and the protein levels of USP8 and FLIPL are positively correlated in these cancer cell lines. Xenograft analyses using ME-180 cervical cancer cells showed that USP8 depletion attenuated tumor growth upon TRAIL injection. Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.

Tumorigenesis can be induced by various stresses that cause aberrant DNA mutations and unhindered cell proliferation. Under such conditions, normal cells autonomously induce defense mechanisms, thereby stimulating tumor suppressor activation. ARF, encoded by the CDKN2a locus, is one of the most frequently mutated or deleted tumor suppressors in human cancer. The safeguard roles of ARF in tumorigenesis are mainly mediated via the MDM2-p53 axis, which plays a prominent role in tumor suppression. Under normal conditions, low p53 expression is stringently regulated by its target gene, MDM2 E3 ligase, which induces p53 degradation in a ubiquitin-proteasome-dependent manner. Oncogenic signals induced by MYC, RAS, and E2Fs trap MDM2 in the inhibited state by inducing ARF expression as a safeguard measure, thereby activating the tumor-suppressive function of p53. In addition to the MDM2-p53 axis, ARF can also interact with diverse proteins and regulate various cellular functions, such as cellular senescence, apoptosis, and anoikis, in a p53-independent manner. As the evidence indicating ARF as a key tumor suppressor has been accumulated, there is growing evidence that ARF is sophisticatedly fine-tuned by the diverse factors through transcriptional and post-translational regulatory mechanisms. In this review, we mainly focused on how cancer cells employ transcriptional and post-translational regulatory mechanisms to manipulate ARF activities to circumvent the tumor-suppressive function of ARF. We further discussed the clinical implications of ARF in human cancer.

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.

C-terminus of heat shock protein 70 (HSP70)-interacting protein (CHIP) is an E3 ligase involved in a variety of protein homeostasis events implicated in diverse signaling pathways. Its involvement in varied and even opposite signaling circuits might be due to its hallmark signature of associating with molecular chaperones, including HSP90 and HSP70. Together, these proteins may be pivotal in implementing protein quality control. A curious and puzzling aspect of the function of CHIP is its capability to induce protein degradation via the proteasome- or lysosome-dependent pathways. In addition, these pathways are combined with ubiquitin-dependent or -independent pathways. This review focuses on the role of CHIP in the development or suppression of tumorigenesis. CHIP can act as a tumor suppressor by downregulating various oncogenes. CHIP also displays an oncogenic feature involving the inhibition of diverse tumor suppressors, including proteins related to intrinsic and extrinsic apoptotic pathways. The ability of CHIP to exhibit dual roles in determining the fate of cells has not been studied analytically. However, its association with various proteins involved in protein quality control might play a major role. In this review, the mechanistic roles of CHIP in tumor formation based on the regulation of diverse proteins are discussed.

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.

Oncogene-induced senescence occurs following oncogene activation in normal cells and is considered as a critical tumor-suppressing mechanism. Ubiquitin-specific protease 10 (USP10) has been reported to play a vital role in oncogene-induced senescence via the deubiquitination-dependent stabilization of p14ARF. However, knowledge of the clinical significance of USP10 and p14ARF expression in patients with small intestinal adenocarcinoma is limited. To study the clinical significance of USP10 and p14ARF expression, we performed immunohistochemistry for USP10 and p14ARF on 195 surgically resected small intestinal adenocarcinoma specimens. Furthermore, we performed methylation analysis on five small intestinal adenocarcinoma samples and matched adjacent normal intestinal tissue samples. UPS10 ( p = 0.023) and p14ARF ( p = 0.007) expression were significantly decreased in adenocarcinoma in comparison with normal tissue. The loss of USP10 was observed in 124/194 (63.9%) of small intestinal adenocarcinoma samples and was correlated with a higher pT stage ( p = 0.044), lymphatic invasion ( p = 0.033), and the absence of sporadic adenoma ( p = 0.024) and peritumoral dysplasia ( p = 0.019). p14ARF expression was downregulated in 75/195 (38.5%) of small intestinal adenocarcinoma samples and was associated with vascular ( p = 0.011) and lymphatic ( p = 0.013) invasions. The loss of USP10 expression was associated with the loss of p14ARF expression ( r = 0.342, p

Regulation of cell survival and death, including apoptosis and necroptosis, is important for normal development and tissue homeostasis, and disruption of these processes can cause cancer, inflammatory diseases, and degenerative diseases. Ubiquitination is a cellular process that induces proteasomal degradation by covalently attaching ubiquitin to the substrate protein. In addition to proteolytic ubiquitination, nonproteolytic ubiquitination, such as M1-linked and K63-linked ubiquitination, has been shown to be important in recent studies, which have demonstrated its function in cell signaling pathways that regulate inflammation and cell death pathways. In this review, we summarize the TRAIL- and TNF-induced death receptor signaling pathways along with recent advances in this field and illustrate how different types of ubiquitination control cell death and survival. In particular, we provide an overview of the different types of ubiquitination, target residues, and modifying enzymes, including E3 ligases and deubiquitinating enzymes. Given the relevance of these regulatory pathways in human disease, we hope that a better understanding of the regulatory mechanisms of cell death pathways will provide insights into and therapeutic strategies for related diseases.

ARF is a tumor suppressor protein that has a pivotal role in the prevention of cancer development through regulating cell proliferation, senescence, and apoptosis. As a factor that induces senescence, the role of ARF as a tumor suppressor is closely linked to the p53-MDM2 axis, which is a key process that restrains tumor formation. Thus, many cancer cells either lack a functional ARF or p53, which enables them to evade cell oncogenic stress-mediated cycle arrest, senescence, or apoptosis. In particular, the ARF gene is a frequent target of genetic and epigenetic alterations including promoter hyper-methylation or gene deletion. However, as many cancer cells still express ARF, pathways that negatively modulate transcriptional or post-translational regulation of ARF could be potentially important means for cancer cells to induce cellular proliferation. These recent findings of regulators affecting ARF protein stability along with its low levels in numerous human cancers indicate the significance of an ARF post-translational mechanism in cancers. Novel findings of regulators stimulating or suppressing ARF function would provide new therapeutic targets to manage cancer- and senescence-related diseases. In this review, we present the current knowledge on the regulation and alterations of ARF expression in human cancers, and indicate the importance of regulators of ARF as a prognostic marker and in potential therapeutic strategies.

Oncogene-induced senescence (OIS) is a critical tumor-suppressor mechanism, which prevents hyper-proliferation and transformation of cells. c-Myc promotes OIS through the transcriptional activation of p14ARF followed by p53 activation. Although the oncogene-mediated transcriptional regulation of p14ARF has been well addressed, the post-translational modification of p14ARF regulated by oncogenic stress has yet to be investigated. Here, we found that c-Myc increased p14ARF protein stability by inducing the transcription of ubiquitin-specific protease 10 (USP10). USP10, in turn, mediated the deubiquitination of p14ARF, preventing its proteasome-dependent degradation. USP10-null mouse embryonic fibroblasts and human primary cells depleted of USP10 bypassed c-Myc-induced senescence via the destabilization of p14ARF, and these cells displayed accelerated hyper-proliferation and transformation. Clinically the c-Myc-USP10-p14ARF axis was disrupted in non-small cell lung cancer patients, resulting in significantly worse overall survival. Our studies indicate that USP10 induced by c-Myc has a crucial role in OIS by maintaining the stability of key tumor suppressor p14ARF.

Supplemental material, Supplementary_material__Rv_(TUB-18-0160) for Dual loss of USP10 and p14ARF protein expression is associated with poor prognosis in patients with small intestinal adenocarcinoma by Joon Seon Song, Joo Mi Yi, Hanbyoul Cho, Chel Hun Choi, Yoonho Park, Eun Joo Chung, Jaewhan Song, Joon-Yong Chung and Seung-Mo Hong in Tumor Biology

Camptothecin is an anti-cancer drug extracted from Camptotheca acuminata, a tree native to mainland China. Phase III clinical trials for camptothecin have been completed, and it is now used as a chemotherapeutic reagent. We identified a novel function of camptothecin that affects adipocyte differentiation. Following treatment with camptothecin, endogenous or overexpressed PPARγ becomes destabilized; this was prevented in the presence of MG132, a proteasome inhibitor. Our findings suggest that camptothecin is able to induce proteasome-dependent degradation of PPARγ. The ubiquitylation of PPARγ increased in the presence of camptothecin. Adipogenic differentiation of 3T3-L1 cells was prevented by campothecin and topotecan, but not by irinotecan, confirming our initial findings. Our results suggest a possible role for camptothecin analogs in the regulation of PPARγ.

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.

Interactions between immune effector cells of the central nervous system appear to directly or indirectly influence the progress/regression of multiple sclerosis (MS). Here, we report that glial STAT1 and −3 are distinctively phosphorylated following the interaction of activated lymphocytes and glia, and this effect is significantly inhibited by glatiramer acetate (GA), a disease-modifying drug for MS. GA also reduces the activations of STAT1 and −3 by MS-associated stimuli such as IFNγ or LPS in primary glia, but not neurons. Experiments in IFNγ- and IFNγ receptor-deficient mice revealed that GA-induced inhibitions of STAT signaling are independent of IFNγ and its receptor. Interestingly, GA induces the expression levels of suppressor of cytokine signaling-1 and −3, representative negative regulators of STAT signaling in glia. We further found that GA attenuates the LPS-triggered enhancement of IL-2, a highly produced cytokine in patients with active MS, in CD4+ T cells co-cultured with glia, but not in CD4+ T cells alone. Collectively, these results provide that activation of glial STATs is an essential event in the interaction between glia and T cells, which is a possible underlying mechanism of GA action in MS. These findings provide an insight for the development of targeted therapies against MS.

PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis.

Oxidative stress and inflammation are common to many pathological conditions. Defense mechanisms protect cells from oxidative stress, but can become over-activated following injury and inflammation. NF-κB and Nrf2 transcription factors regulate proinflammatory and antioxidant gene expression, respectively. Studies have shown that many natural dietary compounds regulate NF-κB and Nrf2, preventing inflammation and oxidative stress. Here, we report major compounds of Prunella vulgaris var. lilacina such as rosmarinic acid, oleanolic acid, ursolic acid and caffeic acid as a potential therapeutic for oxidative stress and inflammation. The major compounds exhibited high anti-inflammatory activity, inhibiting NO, PGE2 production, NF-κB expression and activating Nrf2 expression. In addition, we examined the effect of major compounds on MafK expression. Among the compounds, oleanolic acid significantly decreased MafK expression and MafK-mediated p65 acetylation. These findings suggest that oleanolic acid as NF-κB inhibitors can potentially be used in therapeutic applications for the treatment of oxidative stressnduced diseases. [BMB Reports 2014; 47(9): 524-529]

Premature senescence induced by oncogenic stimuli or tumor suppressor activation plays opposing roles in tumorigenesis. Here, we propose that galectin-3, a β-galactoside-binding lectin, regulates premature senescence without oncogenic stress. We detected premature senescence, decreased Skp2, and increased p27(KIP1) expression in galectin-3 knockout MEFs and galectin-3-depleted gastric cancer cells. Interestingly, galectin-3 depletion did not affect other senescence inducers such as p14(ARF), p16(INK4A), and p21(WAF1/CIP1), suggesting that galectin-3-regulated senescence is p27(KIP1) dependent. We demonstrate that galectin-3 depletion decreases retinoblastoma protein (Rb) phosphorylation (Ser780, Ser807/811), cyclin D1 and CDK4 expression, and E2F1 transcriptional activation. Galectin-3 directly interacts with the cyclin D1/CDK4 complex and promotes hyperphosphorylation of Rb. It also blocks the inhibition of E2F1 transcription, thereby increasing the expression of Skp2 and reducing the stability of p27(KIP1) to promote the proliferation of gastric cancer cells. Xenograft mice with galectin-3-depleted gastric cancer cells display tumor growth retardation that is reversed by Skp2 overexpression. Increased expression of galectin-3 is also associated with the advanced TNM (tumor, lymph node, metastasis) system, clinicopathological stage, and lymph node metastases. The probability of survival was significantly decreased in gastric cancer patients with galectin-3(high) p27(KIP1-low)cells. Taken together, our results show that galectin-3 may accelerate gastric tumorigenesis by inhibiting premature senescence.