ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

Mutant p53 regulates Survivin to foster lung metastasis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.

Long non-coding RNA: a versatile regulator of the nuclear factor-κB signalling circuit.

The nuclear factor-κB (NF-κB) family of transcription factors play an essential role for the regulation of inflammatory responses, immune function and malignant transformation. Aberrant activity of this signalling pathway may lead to inflammation, autoimmune diseases and oncogenesis. Over the last two decades great progress has been made in the understanding of NF-κB activation and how the response is counteracted for maintaining tissue homeostasis. Therapeutic targeting of this pathway has largely remained ineffective due to the widespread role of this vital pathway and the lack of specificity of the therapies currently available. Besides regulatory proteins and microRNAs, long non-coding RNA (lncRNA) is emerging as another critical layer of the intricate modulatory architecture for the control of the NF-κB signalling circuit. In this paper we focus on recent progress concerning lncRNA-mediated modulation of the NF-κB pathway, and evaluate the potential therapeutic uses and challenges of using lncRNAs that regulate NF-κB activity.

Apoptosis, autophagy, necroptosis, and cancer metastasis.

Metastasis is a crucial hallmark of cancer progression, which involves numerous factors including the degradation of the extracellular matrix (ECM), the epithelial-to-mesenchymal transition (EMT), tumor angiogenesis, the development of an inflammatory tumor microenvironment, and defects in programmed cell death. Programmed cell death, such as apoptosis, autophagy, and necroptosis, plays crucial roles in metastatic processes. Malignant tumor cells must overcome these various forms of cell death to metastasize. This review summarizes the recent advances in the understanding of the mechanisms by which key regulators of apoptosis, autophagy, and necroptosis participate in cancer metastasis and discusses the crosstalk between apoptosis, autophagy, and necroptosis involved in the regulation of cancer metastasis.

The calcineurin B subunit (CnB) is a new ligand of integrin αM that mediates CnB-induced Apo2L/TRAIL expression in macrophages.

We showed previously that the calcineurin B subunit (CnB) plays an important role in activation of peritoneal macrophage, but the underlying mechanism remained unknown. To examine whether there is a CnB receptor on peritoneal macrophages, we performed the radioligand binding assay of receptors. The receptor saturation binding curve demonstrated high-affinity and specific binding; the maximum binding was 1090 fmol/10(5) cells, and the K(d) was 70.59 pM. Then, we used a CnB affinity resin to trap potential receptors from highly purified peritoneal macrophage membranes. Mass spectrometry analysis showed that the binding protein was mouse integrin αM. We next performed a competition binding experiment to confirm the binding of CnB to integrin αM. This showed that FITC-CnB bound specifically to peritoneal macrophages and that binding was blocked by the addition of integrin αM Ab. We observed that CnB could induce TRAIL gene expression in peritoneal macrophages in vitro and in vivo. Integrin αM Ab blocking, RNA interference, and ligand competition experiments demonstrated that CnB-induced TRAIL expression is dependent on integrin αM. Furthermore, the tumoricidal activity of CnB-activated peritoneal macrophages is partially dependent on TRAIL. In addition, CnB treatment significantly prolongs the survival of mice bearing H22 ascites tumors, which has a positive correlation with the induction level of TRAIL. These results reveal a novel function of the CnB in innate immunity and cancer surveillance. They also point to a new signaling pathway leading to induction of TRAIL and suggest a possible application of CnB in cancer therapy.

Understanding the complexity of p53 in a new era of tumor suppression.

p53 was discovered 45 years ago as an SV40 large T antigen binding protein, coded by the most frequently mutated TP53 gene in human cancers. As a transcription factor, p53 is tightly regulated by a rich network of post-translational modifications to execute its diverse functions in tumor suppression. Although early studies established p53-mediated cell-cycle arrest, apoptosis, and senescence as the classic barriers in cancer development, a growing number of new functions of p53 have been discovered and the scope of p53-mediated anti-tumor activity is largely expanded. Here, we review the complexity of different layers of p53 regulation, and the recent advance of the p53 pathway in metabolism, ferroptosis, immunity, and others that contribute to tumor suppression. We also discuss the challenge regarding how to activate p53 function specifically effective in inhibiting tumor growth without harming normal homeostasis for cancer therapy.

An unexpected role for the ketogenic diet in triggering tumor metastasis by modulating BACH1-mediated transcription.

We have found that the ketogenic (Keto) diet is able to, unexpectedly, promote the metastatic potential of cancer cells in complementary mouse models. Notably, the Keto diet-induced tumor metastasis is dependent on BTB domain and CNC homolog 1 (BACH1) and its up-regulation of pro-metastatic targets, including cell migration-inducing hyaluronidase 1, in response to the Keto diet. By contrast, upon genetic knockout or pharmacological inhibition of endogenous BACH1, the Keto diet-mediated activation of those targets is largely diminished, and the effects on tumor metastasis are completely abolished. Mechanistically, upon administration of the Keto diet, the levels of activating transcription factor 4 (ATF4) are markedly induced. Through direct interaction with BACH1, ATF4 is recruited to those pro-metastatic target promoters and enhances BACH1-mediated transcriptional activation. Together, these data implicate a distinct transcription regulatory program of BACH1 for tumor metastasis induced by the Keto diet. Our study also raises a potential health risk of the Keto diet in human patients with cancer.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

The calcineurin B subunit induces TNF-related apoptosis-inducing ligand (TRAIL) expression via CD11b-NF-κB pathway in RAW264.7 macrophages.

We showed previously that calcineurin B subunit (CnB) could inhibit S180 solid tumor growth in mice and prolong the survival of mice bearing H22 ascites tumors, but the underlying antitumor mechanism remained unclear. Here, we report that the calcineurin B subunit binds to CD11b on RAW264.7 macrophages and induces TRAIL expression and NF-κB activation in a dose and time dependent manner, and that CnB-induced TRAIL expression and NF-κB activation are both dependent on this CD11b. Furthermore, CnB-induced TRAIL expression is mediated by NF-κB. These findings reveal a novel signaling pathway (CnB-CD11b-NF-κB-TRAIL) regulating TRAIL expression and may help to understand the roles of the calcineurin B subunit in the regulation of innate immunity.

Calcineurin subunit B activates dendritic cells and acts as a cancer vaccine adjuvant.

Safe and potent adjuvants are required to establish effective vaccines. In the present work, we show that calcineurin subunit B promotes the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-12p70 (IL-12 p70), IL-6 and the chemokine IL-8. It also up-regulates transcript levels of chemokines in bone marrow-derived dendritic cells. In an animal model, C57BL/6 mice were divided into four groups, immunized with ovalbumin (OVA), OVA mixed with calcineurin subunit B (CnB), CnB and PBS, respectively. The splenocytes from mice immunized with OVA in combination with CnB produced higher levels of IFN-γ and CTL when in vitro stimulated with OVA protein. Subcutaneous (s.c.) immunization of C57BL/6 mice with OVA plus CnB conferred greater protection against tumor-forming E.G7-OVA cells than did injection of OVA alone, and the survival rate of mice immunized intraperitoneally was higher than that of mice immunized s.c. Thus, CnB exerts potent adjuvant effects that polarize responses toward T(h)1 and elicit anti-tumor and anti-infection immunity. CnB could be of use as a prophylactic adjuvant as it is a human-derived safe agent and has immune-modulating effects that promote the control of cancer and infectious diseases.

Epigenome Programming by H3.3K27M Mutation Creates a Dependence of Pediatric Glioma on SMARCA4.

Patients with diffuse midline gliomas that are H3K27 altered (DMG) display a dismal prognosis. However, the molecular mechanisms underlying DMG tumorigenesis remain poorly defined. Here we show that SMARCA4, the catalytic subunit of the mammalian SWI/SNF chromatin remodeling complex, is essential for the proliferation, migration, and invasion of DMG cells and tumor growth in patient-derived DMG xenograft models. SMARCA4 colocalizes with SOX10 at gene regulatory elements to control the expression of genes involved in cell growth and the extracellular matrix (ECM). Moreover, SMARCA4 chromatin binding is reduced upon depletion of SOX10 or H3.3K27M, a mutation occurring in about 60% DMG tumors. Furthermore, the SMARCA4 occupancy at enhancers marked by both SOX10 and H3K27 acetylation is reduced the most upon depleting the H3.3K27M mutation. Taken together, our results support a model in which epigenome reprogramming by H3.3K27M creates a dependence on SMARCA4-mediated chromatin remodeling to drive gene expression and the pathogenesis of H3.3K27M DMG. SIGNIFICANCE: DMG is a deadly pediatric glioma currently without effective treatments. We discovered that the chromatin remodeler SMARCA4 is essential for the proliferation of DMG with H3K27M mutation in vitro and in vivo, identifying a potentially novel therapeutic approach to this disease. See related commentary by Beytagh and Weiss, p. 2730. See related article by Panditharatna et al., p. 2880. This article is highlighted in the In This Issue feature, p. 2711.

Mutation of calcineurin subunit B M118 influences the activities of NF-AT and p53, but not calcineurin expression level.

Calcineurin (CN) is a Ca(2+)/calmodulin-dependent phosphatase, which consists of a catalytic A-subunit (CnA) and a regulatory B-subunit (CnB). Endogenous CnA and CnB have a strong corelationship in cancer cell lines. Through the introduction of CnB and its mutants in cells, we show that CnB does not increase the expression of CnA but protects it from degradation. CnB M118 is necessary for tight binding to CnA. Point mutations of CnB M118 also do not increase the expression of CnA but protect it from degradation. Furthermore, CnB M118K fails to enhance the activities of NF-AT and p53 induced by CnA in HeLa-s cells. Mutations in CnB M118 may prove to be a valuable marker in the diagnostics of some important illnesses such as Alzheimer's disease.

A new function for the calcineurin b subunit: antiplatelet aggregation and anticoagulation.

Calcineurin is the only Ca(2+) /calmodulin-dependent serine/threonine protein phosphatase. The roles of the cytosolic calcineurin have been well researched; however, the roles of the serum calcineurin remain unknown. Here, we report that the recombinant human calcineurin B subunit (CnB) can bind to rabbit platelets and show an antiplatelet aggregation activity. Furthermore, CnB exerts an anticoagulant effect by prolonging the activated partial thromboplastin time and thrombin time and reducing the plasma fibrinogen concentration in a dose-dependent manner. We further reveal that the functional domain associated with the anticoagulant activity of CnB is located in the C-terminus. Hemolysis test and intravenous stimulation study show that the recombinant CnB does not cause obvious hemolysis and is safe for intravenous injection. These results reveal a new function of calcineurin B subunit. They also give an explanation for the roles of calcineurin B subunit in serum and point to a possible implication in antithrombotic therapy.

ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer.

ABIN-1, also called TNIP1, is an ubiquitin-binding protein that serves an important role in suppressing RIPK1-independent apoptosis, necroptosis, and NF-κB activation. However, the involvement of ABIN-1 in the regulation of RIPK1-dependent apoptosis (RDA) is unknown. In this study, we found that poly(I:C) + TAK1 inhibitor 5Z-7-oxozeaenol (P5) concurrently induces RDA and necroptosis in Abin-1-/-, but not in Abin-1+/+ mouse embryonic fibroblasts (MEFs). Upon P5 stimulation, cells initially die by necroptosis and subsequently by RDA. Furthermore, we explored the therapeutic effect of ABIN-1 deficiency in necroptosis-based cancer therapy in colorectal cancer (CRC). We found that poly(I:C) + 5Z-7-oxozeaenol + IDN-6556 (P5I) yields a robust pro-necroptosis response, and ABIN-1 deficiency additionally enhances this P5I-induced necroptosis. Moreover, phase I/II cIAP inhibitor birinapant with clinical caspase inhibitor IDN-6556 (BI) alone and 5-fluorouracil with IDN-6556 (FI) alone are sufficient to induce necroptotic cell death in CRC cells by promoting auto-secretion of tumor necrosis factor (TNF); ABIN-1 deficiency amplifies the BI- or FI-induced necroptosis. Two independent xenograft experiments using HT-29 or COLO205 cells show that both BI and P5I remarkably inhibit tumor growth via necroptosis activation. For poly(I:C)-induced cell death, the sensitizing effect of ABIN-1 deficiency on cell death may be attributed to increased expression of TLR3. In TNF-induced necroptosis, ABIN-1 deficiency increases TNF-induced RIPK1 polyubiquitination by reducing the recruitment of ubiquitin-editing enzyme A20 to the TNFR1 signaling complex and induces more TNF secretion in CRC cells upon pro-necroptosis stimulation. With this combined data, ABIN-1 deficiency promotes greater sensitization of CRC cells to necroptosis.

Mutant p53 on the Path to Metastasis.

Metastasis contributes to the vast majority of cancer-related mortality. Regulatory mechanisms of the multistep invasion-metastasis cascade are being unraveled. TP53 is the most frequently mutated gene across human cancers. Accumulating evidence has shown that mutations of TP53 not only lead to loss of function or dominant negative effects, but also promotes a gain of function. Specifically, gain of function mutant p53 promotes cancer cell motility, invasion, and metastasis. Here, we summarize the mechanisms and functions of mutant p53 that foster metastasis in different types of cancers. We also discuss the prognostic value of mutant p53 and current status of therapeutic strategies targeting mutant p53. Future studies will shed light on discovering novel mechanisms of mutant p53-driven cancer metastasis and developing innovative therapeutics to improve clinical outcomes in patients harboring p53 mutations.

ABIN-1 heterozygosity sensitizes to innate immune response in both RIPK1-dependent and RIPK1-independent manner.

ABIN-1 (encoded by the gene Tnip1) is a ubiquitin-binding protein that can interact with ubiquitin-editing enzyme A20 (encoded by the gene TNFAIP3) to restrain the activation of necroptosis and NF-κB activation. Genetic variants in the genes Tnip1 and TNFAIP3 are both strongly associated with susceptibility to autoimmune chronic inflammatory diseases such as psoriasis vulgaris and systemic lupus erythematosus (SLE) in humans. Here we investigated the mechanism by which ABIN-1 regulated innate immune responses. We show that ABIN-1 heterozygosity sensitizes cells to antiviral response by mediating NF-κB-dependent and RIPK1-independent expression of pattern recognition molecules, including TLR3, RIG-I, and MDA5, in MEFs. Furthermore, we demonstrate that increased interaction of ABIN-1 and A20 with prolonged poly(I:C) stimulation of WT cells leads to A20-dependent reduction of ABIN-1 protein. Finally, we show that ABIN-1 heterozygosity sensitizes innate immune response of Abin-1+/- mice in vivo by promoting the production of proinflammatory cytokines, which can be blocked upon inhibition of RIPK1 kinase. Inhibition of RIPK1 kinase activity in vivo partially reduces the expression of MDA5, RIG-I, and caspase-11 in Abin-1+/- mice but not in WT mice. Thus, we conclude that ABIN-1 is a suppressor of innate immune response and the interaction of ABIN-1 with A20 controls innate immunity response through the NF-κB pathway and in both RIPK1 kinase activity-independent and dependent manner.

ABIN-1 regulates RIPK1 activation by linking Met1 ubiquitylation with Lys63 deubiquitylation in TNF-RSC.

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.

Combination of calcineurin B subunit (CnB) and 5-fluorouracil reverses 5-fluorouracil-induced immunosuppressive effect and enhances the antitumor activity in hepatocellular carcinoma.

Five-fluorouracil (5-FU) is a widely used chemotherapeutic agent for digestive system tumors; however, continuous use of 5-FU may cause severe side effects, including myelosuppression and immunosuppression. Our previous study revealed that calcineurin B subunit (CnB), an innovative genetic engineering antitumor protein, possesses tumor-suppressive effects with low toxicity. CnB can bind to and activate integrin αM on macrophages, subsequently promoting the expression, and secretion of TNF-related apoptosis-inducing ligand, a specific proapoptotic cytokine. In the present study, whether the combined use of CnB and 5-FU can reverse the myelosuppression, and immunosuppressive effects of 5-FU by reactivating the immune system thus increasing antitumor efficacy, was investigated. It was demonstrated that combined treatment of 5-FU and CnB led to increased tumor-suppressive effects, as indicated by reduced tumor volume and weight when compared with 5-FU or CnB treatment alone in a hepatoma xenograph model. In addition, it was demonstrated that combined treatment inhibited the proliferation of hepatoma cells. Notably, the addition of CnB to 5-FU-based therapy completely reversed the immunosuppressive effect of 5-FU. The spleen index and total number of white blood cells in the combination group were higher compared with that of the 5-FU alone group. Furthermore, pathological examinations indicated that CnB attenuated 5-FU-induced organ damage. Based on these findings, it is proposed that CnB may serve as a novel and promising drug candidate for the improvement of 5-FU-based chemotherapy.

Fluvastatin Prevents Lung Adenocarcinoma Bone Metastasis by Triggering Autophagy.

Bone is one of the most preferred sites of metastasis in lung cancer. Currently, bisphosphonates and denosumab are major agents for controlling tumor-associated skeletal-related events (SREs). However, both bisphosphonates and denosumab significantly increase the risk for jaw osteonecrosis. Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and the most frequently prescribed cholesterol-lowering agents, have been reported to inhibit tumor progression and induce autophagy in cancer cells. However, the effects of statin and role of autophagy by statin on bone metastasis are unknown. In this study, we report that fluvastatin effectively prevented lung adenocarcinoma bone metastasis in a nude mouse model. We further reveal that fluvastatin-induced anti-bone metastatic property was largely dependent on its ability to induce autophagy in lung adenocarcinoma cells. Atg5 or Atg7 deletion, or 3-methyadenine (3-MA) or Bafilomycin A1 (Baf A1) treatment prevented the fluvastatin-induced suppression of bone metastasis. Furthermore, we reveal that fluvastatin stimulation increased the nuclear p53 expression, and fluvastatin-induced autophagy and anti-bone metastatic activity were mostly dependent on p53.