L-2-hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule L-2-hdroxyglutarate (L-2HG) is elevated in the most common histology of renal cancer. Similar to other oncometabolites, L-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that L-2HG remodels amino acid metabolism in renal cancer cells through the combined effects on histone methylation and RNA N6-methyladenosine (m6A). The combined effects of L-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high L-2HG kidney cancers demonstrates reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high L-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Transcriptome-wide quantitative profiling of PUS7-dependent pseudouridylation by nanopore direct long read RNA sequencing.

Pseudouridylation is a prevalent post-transcriptional RNA modification that impacts many aspects of RNA biology and function. The conversion of uridine to pseudouridine (Ψ) is catalyzed by the family of pseudouridine synthases (PUSs). Development of robust methods to determine PUS-dependent regulation of Ψ location and stoichiometry in low abundant mRNA is essential for biological and functional understanding of pseudouridylation. Here, we present a framework, NanoPsiPy, for identifying Ψ sites and quantify their levels in poly-A RNA at single-nucleotide resolution using direct RNA long-read Nanopore sequencing, based on the observation that Ψ can cause characteristic U-to-C basecalling errors in Nanopore direct RNA sequencing data. Our method was able to detect low and high stoichiometric Ψ sites in human mRNA. We validated our method by transcriptome-wide quantitative profiling of PUS7-dependent Ψ sites in poly-A RNA from a MYCN -amplified neuroblastoma cell line. We identified 8,625 PUS7-dependent Ψ sites in 1,246 mRNAs that encode proteins involved primarily in ribosome biogenesis, translation, and mitochondrial energy metabolism. Our work provides the first example of using direct RNA long-read Nanopore sequencing for transcriptome-wide quantitative profiling of mRNA pseudouridylation regulated by a PUS. We envision that our method will facilitate functional interrogation of PUSs in biological and pathological processes.

Caffeine Supplementation and FOXM1 Inhibition Enhance the Antitumor Effect of Statins in Neuroblastoma.

High-risk neuroblastoma exhibits transcriptional activation of the mevalonate pathway that produces cholesterol and nonsterol isoprenoids. A better understanding of how this metabolic reprogramming contributes to neuroblastoma development could help identify potential prevention and treatment strategies. Here, we report that both the cholesterol and nonsterol geranylgeranyl-pyrophosphate branches of the mevalonate pathway are critical to sustain neuroblastoma cell growth. Blocking the mevalonate pathway by simvastatin, a cholesterol-lowering drug, impeded neuroblastoma growth in neuroblastoma cell line xenograft, patient-derived xenograft (PDX), and TH-MYCN transgenic mouse models. Transcriptional profiling revealed that the mevalonate pathway was required to maintain the FOXM1-mediated transcriptional program that drives mitosis. High FOXM1 expression contributed to statin resistance and led to a therapeutic vulnerability to the combination of simvastatin and FOXM1 inhibition. Furthermore, caffeine synergized with simvastatin to inhibit the growth of neuroblastoma cells and PDX tumors by blocking statin-induced feedback activation of the mevalonate pathway. This function of caffeine depended on its activity as an adenosine receptor antagonist, and the A2A adenosine receptor antagonist istradefylline, an add-on drug for Parkinson's disease, could recapitulate the synergistic effect of caffeine with simvastatin. This study reveals that the FOXM1-mediated mitotic program is a molecular statin target in cancer and identifies classes of agents for maximizing the therapeutic efficacy of statins, with implications for treatment of high-risk neuroblastoma. SIGNIFICANCE: Caffeine treatment and FOXM1 inhibition can both enhance the antitumor effect of statins by blocking the molecular and metabolic processes that confer statin resistance, indicating potential combination therapeutic strategies for neuroblastoma. See related commentary by Stouth et al., p. 2091.

Tubular cell senescence promotes maladaptive kidney repair and chronic kidney disease after cisplatin nephrotoxicity.

Cisplatin is a widely used chemotherapy drug; however, it induces both acute and chronic kidney diseases (CKD) in patients with cancer. The pathogenesis of cisplatin-induced CKD is unclear, and effective renoprotective approaches are not available. Here, we report that repeated low-dose cisplatin (RLDC) treatment of C57BL/6 mice induced chronic cellular senescence in kidney tubules, accompanied with tubular degeneration and profibrotic phenotype transformation that culminated in maladaptive repair and renal fibrosis. Suppression of tubular senescence by senolytic drugs ABT-263 and Fisetin attenuated renal fibrosis and improved tubular repair, as indicated by restoration of tubular regeneration and renal function. In vitro, RLDC also induced senescence in mouse proximal tubular (BUMPT) cells. ABT-263 eliminated senescent BUMPT cells following RLDC treatment, reversed the profibrotic phenotype of the cells, and increased their clonogenic activity. Moreover, ABT-263 alleviated the paracrine effect of RLDC-treated BUMPT cells on fibroblasts for fibrosis. Consistently, knockdown of p16 suppressed post-RLDC senescence and fibrotic changes in BUMPT cells and alleviated their paracrine effects on renal fibroblast proliferation. These results indicate that persistent induction of tubular senescence plays an important role in promoting cisplatin-induced CKD. Targeting senescent tubular cells may be efficient for improvement of kidney repair and for the prevention and treatment of cisplatin-induced CKD.

MYCN and Metabolic Reprogramming in Neuroblastoma.

Neuroblastoma is a pediatric cancer responsible for approximately 15% of all childhood cancer deaths. Aberrant MYCN activation, as a result of genomic MYCN amplification, is a major driver of high-risk neuroblastoma, which has an overall survival rate of less than 50%, despite the best treatments currently available. Metabolic reprogramming is an integral part of the growth-promoting program driven by MYCN, which fuels cell growth and proliferation by increasing the uptake and catabolism of nutrients, biosynthesis of macromolecules, and production of energy. This reprogramming process also generates metabolic vulnerabilities that can be exploited for therapy. In this review, we present our current understanding of metabolic reprogramming in neuroblastoma, focusing on transcriptional regulation as a key mechanism in driving the reprogramming process. We also highlight some important areas that need to be explored for the successful development of metabolism-based therapy against high-risk neuroblastoma.

DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

A Negative Regulatory Role for RKIP in Breast Cancer Immune Response.

Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathways, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes.

BACKGROUND: Granzyme B is a key effector of cytotoxic T lymphocytes (CTLs), and its expression level positively correlates with the response of patients with mesothelioma to immune checkpoint inhibitor immunotherapy. Whether metabolic pathways regulate Gzmb expression in CTLs is incompletely understood. METHODS: A tumor-specific CTL and tumor coculture model and a tumor-bearing mouse model were used to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in CTL function and tumor immune evasion. A link between granzyme B expression and patient survival was analyzed in human patients with epithelioid mesothelioma. RESULTS: Mesothelioma cells alone are sufficient to activate tumor-specific CTLs and to enhance aerobic glycolysis to induce a PD-1hi Gzmblo CTL phenotype. However, inhibition of lactate dehydrogenase A, the key enzyme of the aerobic glycolysis pathway, has no significant effect on tumor-induced CTL activation. Tumor cells induce H3K9me3 deposition at the promoter of G6pd, the gene that encodes the rate-limiting enzyme G6PD in the pentose phosphate pathway, to downregulate G6pd expression in tumor-specific CTLs. G6PD activation increases acetyl-coenzyme A (CoA) production to increase H3K9ac deposition at the Gzmb promoter and to increase Gzmb expression in tumor-specific CTLs converting them from a Gzmblo to a Gzmbhi phenotype, thus increasing CTL tumor lytic activity. Activation of G6PD increases Gzmb+ tumor-specific CTLs and suppresses tumor growth in tumor-bearing mice. Consistent with these findings, GZMB expression level was found to correlate with increased survival in patients with epithelioid mesothelioma. CONCLUSION: G6PD is a metabolic checkpoint in tumor-activated CTLs. The H3K9me3/G6PD/acetyl-CoA/H3K9ac/Gzmb pathway is particularly important in CTL activation and immune evasion in epithelioid mesothelioma.

Single-Nucleus Transcriptional Profiling of Chronic Kidney Disease after Cisplatin Nephrotoxicity.

Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in patients with cancer. Presented here is the first study of single-nucleus RNA sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity. Repeated low-dose cisplatin treatment (RLDC) led to decreases in renal function and kidney weight in mice at 9 weeks. The kidneys of these mice showed tubular degeneration and dilation. snRNA-seq identified 16 cell types and 17 cell clusters in these kidneys. Cluster-by-cluster comparison demonstrated cell type-specific changes in gene expression and identified a unique proximal tubule (PT) injury/repair cluster that co-expressed the injury marker kidney injury molecule-1 (Kim1) and the proliferation marker Ki-67. Compared with control, post-RLDC kidneys had 424 differentially expressed genes in PT cells, including tubular transporters and cytochrome P450 enzymes involved in lipid metabolism. snRNA-seq also revealed transcriptional changes in potential PT injury markers (Krt222, Eda2r, Ltbp2, and Masp1) and repair marker (Bex4). RLDC induced inflammation and proinflammatory cytokines (RelB, TNF-α, Il7, Ccl2, and Cxcl2) and the expression of fibrosis markers (fibronectin, collagen I, connective tissue growth factor, vimentin, and α-smooth muscle actin). Together, these results provide new insights into RLDC-induced transcriptional changes at the single-cell level that may contribute to the development of chronic kidney problems in patients with cancer after cisplatin chemotherapy.

ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction.

The serine synthesis pathway (SSP) involving metabolic enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) drives intracellular serine biosynthesis and is indispensable for cancer cells to grow in serine-limiting environments. However, how SSP is regulated is not well understood. Here, we report that activating transcription factor 3 (ATF3) is crucial for transcriptional activation of SSP upon serine deprivation. ATF3 is rapidly induced by serine deprivation via a mechanism dependent on ATF4, which in turn binds to ATF4 and increases the stability of this master regulator of SSP. ATF3 also binds to the enhancers/promoters of PHGDH, PSAT1, and PSPH and recruits p300 to promote expression of these SSP genes. As a result, loss of ATF3 expression impairs serine biosynthesis and the growth of cancer cells in the serine-deprived medium or in mice fed with a serine/glycine-free diet. Interestingly, ATF3 expression positively correlates with PHGDH expression in a subset of TCGA cancer samples.

Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma.

Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.

p53/microRNA-214/ULK1 axis impairs renal tubular autophagy in diabetic kidney disease.

Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD.

PRMT1 promotes neuroblastoma cell survival through ATF5.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

ATF3 promotes erastin-induced ferroptosis by suppressing system Xc.

The amino acid antiporter system Xc- is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc- often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc- activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc- is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc-, depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc- and promote ferroptosis repressed by this antiporter.

Competitive ubiquitination activates the tumor suppressor p53.

Blocking p53 ubiquitination through disrupting its interaction with MDM2 or inhibiting the MDM2 catalytic activity is the central mechanism by which the tumor suppressor p53 is activated in response to genotoxic challenges. Although MDM2 is first characterized as the major E3 ubiquitin ligase for p53, it can also catalyze the conjugation of ubiquitin moieties to other proteins (e.g., activating transcription factor 3, or ATF3). Here we report that ATF3 can act as an ubiquitin "trap" and competes with p53 for MDM2-mediated ubiquitination. While ATF3-mediated p53 stabilization required ATF3 binding to the MDM2 RING domain, we demonstrated that ATF3 ubiquitination catalyzed by MDM2 was indispensable for p53 activation in response to DNA damage. Moreover, a cancer-derived ATF3 mutant (R88G) devoid of ubiquitination failed to prevent p53 from MDM2-mediated degradation and thus was unable to activate the tumor suppressor. Therefore, we have identified a previously-unknown mechanism that can activate p53 in the genotoxic response.

Glycine decarboxylase is a transcriptional target of MYCN required for neuroblastoma cell proliferation and tumorigenicity.

Genomic amplification of the oncogene MYCN is a major driver in the development of high-risk neuroblastoma, a pediatric cancer with poor prognosis. Given the challenge in targeting MYCN directly for therapy, we sought to identify MYCN-dependent metabolic vulnerabilities that can be targeted therapeutically. Here, we report that the gene encoding glycine decarboxylase (GLDC), which catalyzes the first and rate-limiting step in glycine breakdown with the production of the one-carbon unit 5,10-methylene-tetrahydrofolate, is a direct transcriptional target of MYCN. As a result, GLDC expression is markedly elevated in MYCN-amplified neuroblastoma tumors and cell lines. This transcriptional upregulation of GLDC expression is of functional significance, as GLDC depletion by RNA interference inhibits the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines by inducing G1 arrest. Metabolomic profiling reveals that GLDC knockdown disrupts purine and central carbon metabolism and reduces citrate production, leading to a decrease in the steady-state levels of cholesterol and fatty acids. Moreover, blocking purine or cholesterol synthesis recapitulates the growth-inhibitory effect of GLDC knockdown. These findings reveal a critical role of GLDC in sustaining the proliferation of neuroblastoma cells with high-level GLDC expression and suggest that MYCN amplification is a biomarker for GLDC-based therapeutic strategies against high-risk neuroblastoma.

Metabolic Reprogramming by MYCN Confers Dependence on the Serine-Glycine-One-Carbon Biosynthetic Pathway.

MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCN-activated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycine-one-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN-amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN-amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7-mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. SIGNIFICANCE: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy.See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818.

Bif-1 Interacts with Prohibitin-2 to Regulate Mitochondrial Inner Membrane during Cell Stress and Apoptosis.

BACKGROUND: Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics. METHODS: We used azide-induced ATP depletion to incite cell stress in mouse embryonic fibroblasts and renal proximal tubular cells, and renal ischemia-reperfusion to induce stress in mice. We also used knockout cells and mice to determine the role of Bif-1, and used multiple techniques to analyze the molecular interaction between Bif-1 and prohibitin-2. RESULTS: Upon cell stress, Bif-1 translocated to mitochondria to bind prohibitin-2, resulting in the disruption of prohibitin complex and proteolytic inactivation of the inner membrane fusion protein OPA1. Bif-1-deficiency inhibited prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis. Domain deletion analysis indicated that Bif-1 interacted with prohibitin-2 via its C-terminus. Notably, mutation of Bif-1 at its C-terminal tryptophan-344 not only prevented Bif-1/prohibitin-2 interaction but also reduced prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis, supporting a pathogenic role of Bif-1/prohibitin-2 interaction. In mice, Bif-1 bound prohibitin-2 during renal ischemia/reperfusion injury, and Bif-1-deficiency protected against OPA1 proteolysis, mitochondrial fragmentation, apoptosis and kidney injury. CONCLUSIONS: These findings suggest that during cell stress, Bif-1 regulates mitochondrial inner membrane by interacting with prohibitin-2 to disrupt prohibitin complexes and induce OPA1 proteolysis and inactivation.

BMP4 and Neuregulin regulate the direction of mouse neural crest cell differentiation.

The neural crest is a transient embryonic tissue that initially generates neural crest stem cells, which then migrate throughout the body to give rise to a variety of mature tissues. It was proposed that the fate of neural crest cells is gradually determined via environmental cues from the surrounding tissues. In the present study, neural crest cells were isolated and identified from mouse embryos. Bone morphogenetic protein 4 (BMP4) and Neuregulin (NRG) were employed to induce the differentiation of neural crest cells. Treatment with BMP4 revealed neuron-associated differentiation; cells treated with NRG exhibited differentiation into the Schwann cell lineage, a type of glia. Soft agar clonogenic and neurosphere formation assays were conducted to investigate the effects of N-Myc (MYCN) overexpression in neural crest cells; the number of colonies and neurospheres notably increased after 14 days. These findings demonstrated that the direction of cell differentiation may be affected by altering the factors present in the surrounding environment. In addition, MYCN may serve a key role in regulating neural crest cell differentiation.