Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

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.

Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer.

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.

DIX domain containing 1 (DIXDC1) modulates VEGFR2 level in vasculatures to regulate embryonic and postnatal retina angiogenesis.

BACKGROUND: In sprouting angiogenesis, VEGFR2 level is regulated via a fine-tuned process involving various signaling pathways. Other than VEGF/VEGFR2 signaling pathway, Wnt/ ß-catenin signaling is also important in vascular development. However, the crosstalk between these two signaling pathways is still unknown to date. In this study, we aimed to investigate the role of DIX domain containing 1 (DIXDC1) in vasculature, facilitating the crosstalk between VEGF/VEGFR2 and Wnt/ ß-catenin signaling pathways. RESULTS: In mice, DIXDC1 deficiency delayed angiogenesis at the embryonic stage and suppressed neovascularization at the neonatal stage. DIXDC1 knockdown inhibited VEGF-induced angiogenesis in endothelial cells in vitro by downregulating VEGFR2 expression. DIXDC1 bound Dishevelled Segment Polarity Protein 2 (Dvl2) and polymerized Dvl2 stabilizing VEGFR2 protein via its direct interaction. The complex formation and stability of VEGFR2 was potentiated by Wnt signaling. Moreover, hypoxia elevated DIXDC1 expression and likely modulated both canonical Wnt/ß-catenin signaling and VEGFR2 stability in vasculatures. Pathological angiogenesis in DIXDC1 knockout mice was decreased significantly in oxygen-induced retinopathy (OIR) and in wound healing models. These results suggest that DIXDC1 is an important factor in developmental and pathological angiogenesis. CONCLUSION: We have identified DIXDC1 as an important factor in early vascular development. These results suggest that DIXDC1 represents a novel regulator of sprouting angiogenesis that links Wnt signaling and VEGFR2 stability and may have a potential role in pathological neovascularization.

Deubiquitinases: Modulators of Different Types of Regulated Cell Death.

The mechanisms and physiological implications of regulated cell death (RCD) have been extensively studied. Among the regulatory mechanisms of RCD, ubiquitination and deubiquitination enable post-translational regulation of signaling by modulating substrate degradation and signal transduction. Deubiquitinases (DUBs) are involved in diverse molecular pathways of RCD. Some DUBs modulate multiple modalities of RCD by regulating various substrates and are powerful regulators of cell fate. However, the therapeutic targeting of DUB is limited, as the physiological consequences of modulating DUBs cannot be predicted. In this review, the mechanisms of DUBs that regulate multiple types of RCD are summarized. This comprehensive summary aims to improve our understanding of the complex DUB/RCD regulatory axis comprising various molecular mechanisms for diverse physiological processes. Additionally, this review will enable the understanding of the advantages of therapeutic targeting of DUBs and developing strategies to overcome the side effects associated with the therapeutic applications of DUB modulators.

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.

Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-XL.

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-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL 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-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, 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-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

Dual loss of USP10 and p14ARF protein expression is associated with poor prognosis in patients with small intestinal adenocarcinoma.

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 < 0.001). Multivariate survival analysis revealed that the combined loss of USP10 and p14ARF expression could be an independent prognostic factor for overall survival in small intestinal adenocarcinoma. Furthermore, the aberrant hypermethylation of the USP10 and p14ARF promoter could be a key mechanism for the downregulation of USP10 and p14ARF proteins in small intestinal adenocarcinoma. These findings suggest that the dual loss of USP10 and p14ARF could be used as a prognostic indicator of small intestinal adenocarcinoma.

Camptothecin and topotecan inhibit adipocyte differentiation by inducing degradation of PPARγ.

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.

EGFR inhibits TNF-α-mediated pathway by phosphorylating TNFR1 at tyrosine 360 and 401.

Tumour necrosis factor receptor 1 (TNFR1) induces the nuclear factor kappa-B (NF-κB) signalling pathway and regulated cell death processes when TNF-α ligates with it. Although mechanisms regulating the downstream pathways of TNFR1 have been elucidated, the direct regulation of TNFR1 itself is not well known. In this study, we showed that the kinase domain of the epidermal growth factor receptor (EGFR) regulates NF-κB signalling and TNF-α-induced cell death by directly phosphorylating TNFR1 at Tyr 360 and 401 in its death domain. In contrast, EGFR inhibition by EGFR inhibitors, such as erlotinib and gefitinib, prevented their interaction. Once TNFR1 is phosphorylated, its death domain induces the suppression of the NF-κB pathways, complex II-mediated apoptosis, or necrosome-dependent necroptosis. Physiologically, in mouse models, EGF treatment mitigates TNF-α-dependent necroptotic skin inflammation induced by treatment with IAP and caspase inhibitors. Our study revealed a novel role for EGFR in directly regulating TNF-α-related pathways.

UBX-390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer.

The androgen receptor (AR) is an attractive target for treating prostate cancer, considering its role in the development and progression of localized and metastatic prostate cancer. The high global mortality burden of prostate cancer, despite medical treatments such as androgen deprivation or AR antagonist therapy, highlights the need to explore alternative strategies. One strategy involves the use of heterobifunctional degraders, also known as proteolysis-targeting chimeras, which are novel small-molecule therapeutics that inhibit amplified or mutated targets. Here, the study reports a novel cereblon-based AR degrader, UBX-390, and demonstrates its superior activity over established AR degraders, such as ARV-110 or ARCC-4, in prostate cancer cells under short- and long-term treatment conditions. UBX-390 suppresses chromatin binding and gene expression of AR and demonstrates substantial efficacy in the degradation of AR mutants in patients with treatment-resistant prostate cancer. UBX-390 is presented as an optimized AR degrader with remarkable potential for treating castration-resistant prostate cancer.

Differential regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and apoptosis.

Makorin Ring Finger Protein 1 (MKRN1) is a transcriptional co-regulator and an E3 ligase. Here, we show that MKRN1 simultaneously functions as a differentially negative regulator of p53 and p21. In normal conditions, MKRN1 could destabilize both p53 and p21 through ubiquitination and proteasome-dependent degradation. As a result, depletion of MKRN1 induced growth arrest through activation of p53 and p21. Interestingly, MKRN1 used earlier unknown sites, K291 and K292, for p53 ubiquitination and subsequent degradation. Under severe stress conditions, however, MKRN1 primarily induced the efficient degradation of p21. This regulatory process contributed to the acceleration of DNA damage-induced apoptosis by eliminating p21. MKRN1 depletion diminished adriamycin or ultraviolet-induced cell death, whereas ectopic expression of MKRN1 facilitated apoptosis. Furthermore, MKRN1 stable cell lines that constantly produced low levels of p53 and p21 exhibited stabilization of p53, but not p21, with increased cell death on DNA damage. Our results indicate that MKRN1 exhibits dual functions of keeping cells alive by suppressing p53 under normal conditions and stimulating cell death by repressing p21 under stress conditions.

IKK-mediated TRAF6 and RIPK1 interaction stifles cell death complex assembly leading to the suppression of TNF-α-induced cell death.

Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine capable of inducing extrinsic apoptosis and necroptosis. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ligase, is a member of the TRAF family of proteins, which mediates inflammatory signals by activating nuclear factor kappa B (NFкB) and mitogen-activated protein kinase (MAPK). Although the functions of TRAF6 have been identified, its role in TNF-α-induced cell death remains poorly understood. Here, we report that TRAF6 is a negative modulator of TNF-α-induced cell death but does not affect TNF-α-induced NFκB activation. TRAF6 deficiency accelerates both TNF-α-induced apoptosis and necroptosis; however, the acceleration can be reversed by reconstituting TRAF6 or TRAF6C70A, suggesting that E3 ligase activity is not required for this activity. Mechanistically, TRAF6 directly interacts with RIPK1 during TNF-α-induced cell death signaling, which prevents RIPK1 from interacting with components of the cell death complex such as itself, FADD or RIPK3. These processes suppress the assembly of the death complex. Notably, IKK was required for TRAF6 to interact with RIPK1. In vivo, Traf6-/- embryos exhibited higher levels of cell death in the liver but could be rescued by the simultaneous knockout of Tnf. Finally, TRAF6 knockdown xenografts were highly sensitive to necroptotic stimuli. We concluded that TRAF6 suppresses TNF-α-induced cell death in coordination with IKK complexes in vivo and in vitro by suppressing the assembly of cell death complex.

MKRN1 induces degradation of West Nile virus capsid protein by functioning as an E3 ligase.

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.

Mitochondrial dysfunction induced by callyspongiolide promotes autophagy-dependent cell death.

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].

Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators.

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.

Overall survival of pancreatic ductal adenocarcinoma is doubled by Aldh7a1 deletion in the KPC mouse.

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.

Cytoplasmic MYC is an anti-necroptotic protein.

Cancer cells are often resistant to necroptosis as well as apotosis, but the underlying mechanisms are not fully understood. We recently revealed an important crosstalk between MYC, a potent oncogene, and receptor-interacting protein kinase 3 (RIPK3), a pivotal factor in inducing necroptosis. Mechanistically, cytoplasmic MYC directly binds to RIPK3, inhibiting initial necrosome complex formation.

Post-Translational Regulation of ARF: Perspective in Cancer.

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.

Author Correction: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis.

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

Beclin 1 functions as a negative modulator of MLKL oligomerisation by integrating into the necrosome complex.

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.