Although cancer cells are frequently faced with nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anti-cancer strategy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) was downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low glucose condition. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo UTP synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.
Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang
X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift towards mono-unsaturated fatty acids relieved toxicity. In human ALD fibroblasts chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts whereas mono-unsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated towards mono-unsaturated VLCFA, and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to mono-unsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.
Quentin Raas, Malu-Clair van de Beek, Sonja Forss-Petter, Inge M.E. Dijkstra, Abigail DeSchiffart, Briana C. Freshner, Tamara J. Stevenson, Yorrick R.J. Jaspers, Liselotte M. Nagtzaam, Ronald J.A. Wanders, Michel van Weeghel, Joo-Yeon Engelen-Lee, Marc Engelen, Florian Eichler, Johannes Berger, Joshua L. Bonkowsky, Stephan Kemp
A(H3N2) Influenza vaccine effectiveness (VE) were low during 2016-2019 seasons and varied by age. We analyzed neutralizing antibody responses to egg- and cell-propagated vaccine and circulating viruses following vaccination in 375 individuals (aged 7 months to 82 years) across all vaccine eligible age groups in 3 influenza seasons. Antibody responses to cell- compared to egg-propagated vaccine viruses were significantly reduced due to egg-adapted changes T160K, D225G, and L194P in the vaccine hemagglutinins. Vaccine egg-adaptation had differential impact on antibody responses across different age groups. Immunologically naive children immunized with egg-adapted vaccines mostly mounted antibodies targeting egg-adapted epitopes, whereas those previously primed with infection produced broader responses even when vaccinated with egg-based vaccines. In elderly, repeated boost of vaccine egg-adapted epitopes significantly reduced antibody responses to the wild type cell-grown viruses. Analysis with reverse genetics viruses suggested that the response to each egg-adapted substitution varied by age. Antibody responses did not differ in male versus female vaccinees. Here, the combination of age-specific responses to vaccine egg-adapted substitutions, diverse host immune priming histories and virus antigenic drift impacted antibody responses following vaccination and may have led to the low and variable VE against A(H3N2) viruses across different age groups.
Feng Liu, F. Liaini Gross, Stacie N. Jefferson, Crystal Holiday, Yaohui Bai, Li Wang, Bin Zhou, Min Z. Levine
BACKGROUND. Rejection is the primary barrier to broader implementation of vascularized composite allografts (VCA), including face and limb transplants. The immunologic pathways activated in face transplant rejection have not been fully characterized. METHODS. Utilizing skin biopsies prospectively collected over nine years from seven face transplant patients, we studied rejection by gene expression profiling, histology, immunostaining and T cell receptor sequencing. RESULTS. Grade 1 rejection did not differ significantly from non-rejection, suggesting that it does not represent a pathologic state and that watchful waiting is warranted. In Grade 2, there was a balanced upregulation of both pro-inflammatory T cell activation pathways and anti-inflammatory checkpoint and immunomodulatory pathways, with a net result of no tissue injury. In Grade 3, IFNγ-driven inflammation, antigen presenting cell activation and infiltration of the skin by proliferative T cells bearing markers of antigen specific activation and cytotoxic effector molecules tipped the balance towards tissue injury. Rejection of VCA and solid organ transplants had both distinct and common features. VCA rejection was uniquely associated with upregulation of immunoregulatory genes, including SOCS1, induction of lipid antigen-presenting CD1 proteins, and infiltration by T cells predicted to recognize CD1b and CD1c. CONCLUSIONS. Our findings suggest that the distinct features of VCA rejection reflect the unique immunobiology of skin and that enhancing cutaneous immunoregulatory networks may be a useful strategy in combatting rejection.
Thet Su Win, William J. Crisler, Beatrice Dyring-Andersen, Rachel Lopdrup, Jessica E. Teague, Qian Zhan, Victor Barrera, Shannan J. Ho Sui, Sotirios Tasigiorgos, Naoka Murakami, Anil Chandraker, Stefan G. Tullius, Bohdan Pomahac, Leonardo V. Riella, Rachael Clark
SUMOylation emerged as the inducer for the sorting of bioactive molecules into extracellular vesicles (EVs) triggering lymphangiogenesis, further driving tumor lymph node (LN) metastasis, but the precise mechanisms remain largely unclear. Herein, we identified that bladder cancer (BCa) cell-secreted EVs mediated the intercellular communication with human lymphatic endothelial cells (HLECs) through the transmission of a long noncoding RNA ELNAT1, and promoted lymphangiogenesis and LN metastasis in a SUMOylation-dependent manner in both cultured BCa cell lines and mouse models. Mechanistically, ELNAT1 induced UBC9 overexpression to catalyze the SUMOylation of hnRNPA1 at lysine-113 residue, which mediated the recognition of ELNAT1 by endosomal sorting complex required for transport (ESCRT) and facilitated their packaging into EVs. EV-mediated ELNAT1 was specifically transmitted into HLECs and epigenetically activated SOX18 transcription to induce lymphangiogenesis. Importantly, blocking the SUMOylation of tumor by downregulating UBC9 expression markedly reduced lymphatic metastasis in EV-mediated ELNAT1-treated BCa in vivo. Clinically, EV-mediated ELNAT1 was correlated with LN metastasis and poor prognosis of patients with BCa. These findings highlight a molecular mechanism that EV-mediated ELNAT1/UBC9/SOX18 regulatory axis promotes the lymphangiogenesis and LN metastasis of BCa in a SUMOylation-dependent manner, and implicate ELNAT1 as an attractive therapeutic target for LN metastatic BCa.
Changhao Chen, Hanhao Zheng, Yuming Luo, Yao Kong, Mingjie An, Yuting Li, Wang He, Bowen Gao, Yue Zhao, Hao Huang, Jian Huang, Tianxin Lin
Autophagy modulates lipid turnover, cell survival, inflammation and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34/Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescues the defective autophagy in Sr-b1-/- macrophages. Taken together, macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34/Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and novel targets for the treatment of atherosclerosis.
Huan Tao, Patricia G. Yancey, John L. Blakemore, Youmin Zhang, Lei Ding, W. Gray Jerome, Jonathan D. Brown, Kasey C. Vickers, MacRae F. Linton
The excitability of interneurons requires Nav1.1, the α subunit of voltage-gated sodium channel. Nav1.1 deficiency and mutations reduce interneuron excitability, a major pathological mechanism for epilepsy syndromes. However, the regulatory mechanisms of Nav1.1 expression remain unclear. Here we provide evidence that neddylation is critical to Nav1.1 stability. Mutant mice lacking Nae1, an obligatory component of the E1 ligase for neddylation, in parvalbumin-positive interneurons (PVINs) exhibited spontaneous epileptic seizures and premature death. Electrophysiological studies indicate that Nae1 deletion reduced in PVIN excitability and GABA release, and consequently increased the network excitability of pyramidal neurons (PyNs). Further analysis revealed a reduction in sodium current density, not a change in channel property, in mutant PVINs and decreased Nav1.1 protein level. These results suggest that insufficient neddylation in PVINs reduces Nav1.1 stability and thus the excitability of PVINs; ensuing increased PyN activity causes seizures in mice. In agreement, Nav1.1 was found reduced by proteomic analysis that revealed abnormality in synapses and metabolic pathways. Our findings, for the first time, described a role of neddylation in maintaining Nav1.1 stability for PVIN excitability and reveal a new mechanism in pathogenesis of epilepsy.
Wenbing Chen, Bin Luo, Nannan Gao, Haiwen Li, Hongsheng Wang, Lei Li, Wanpeng Cui, Lei Zhang, Dong Sun, Fang Liu, Zhaoqi Dong, Xiao Ren, Hongsheng Zhang, Huabo Su, Wen-Cheng Xiong, Lin Mei
A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multi-omic analyses using 2,872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance (155 unique variants with allele frequency < 1%; 12.9% carrier rate). We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C (FDR=2.6e-5), which is induced also in mucosal biopsies of IBD patients. DUOX2 deficient mice replicated increased IL17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in IBD patients corroborated IL17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL17C induction provided a rationale for variant stratification in case-control studies that substantiated DUOX2 as an IBD risk gene (pooled OR = 1.54 [95% CI 1.09-2.18]; P = 7.1e-4). Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.
Helmut Grasberger, Andrew T. Magis, Elisa Sheng, Matthew P. Conomos, Min Zhang, Lea S. Garzotto, Guoqing Hou, Shrinivas Bishu, Hiroko Nagao-Kitamoto, Mohamad El-Zataari, Sho Kitamoto, Nobuhiko Kamada, Ryan Stidham, Yasutada Akiba, Jonathan Kaunitz, Yael Haberman, Subra Kugathasan, Lee A. Denson, Gilbert S. Omenn, John Y. Kao
BACKGROUND. p16 positive oropharyngeal squamous cell carcinoma (OPSCC) patients are potentially cured with definitive treatment. However, there are currently no reliable biomarkers of treatment failure in p16 positive OPSCC. Pathologist-based visual assessment of tumor cell multinucleation has been shown to be independently prognostic of disease-free survival in p16 positive OPSCC. However, its quantification is time-intensive, subjective, and at risk of interobserver variability. METHODS. We present a deep learning-based metric, the multi-nucleation index (MuNI), for prognostication in p16 positive OPSCC. This approach quantifies tumor multi-nucleation from digitally scanned hematoxylin eosin (H&E)-stained slides. Representative H&E whole slide images from 1,094 previously untreated p16 positive OPSCC patients were acquired from six institutions for optimizing and validating MuNI. RESULTS. MuNI was prognostic for disease-free (DFS), overall (OS), or distant metastasis-free (DMFS) survival in p16 positive OPSCC with HRs of 1.78(95%CI:1.37-2.30), 1.94(1.44-2.60), and 1.88(1.43-2.47), respectively, independent of age, smoking status, treatment type, and T/N-categories in multivariable analyses. It was also prognostic for DFS, OS, and DMFS in OPSCC patients at stages I and III. CONCLUSION. MuNI holds promise as a low-cost, tissue non-destructive, H&E stain based digital biomarker test for counseling, treatment, and surveillance of p16 positive OPSCC patients. These data support further confirmation of MuNI in prospective trials. FUNDING. This work was supported by the National Cancer Institute of the National Institutes of Health (under award numbers 1U24CA199374-01, R01CA202752-01A, R01CA208236-01A1, R01CA216579-01A1, R01CA220581-01A1, 1U01CA239055-01), the National Institute for Biomedical Imaging and Bioengineering (1R43EB028736-01), the National Center for Research Resources (1C06RR12463-01), the VA Merit Review Award (IBX004121A) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DoD Breast Cancer Research Program Breakthrough Level 1 Award (W81XWH-19-1-0668), the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering, and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University, the Michael E. DeBakey VA Medical Center, an institutional pilot grant (1IK2CX001953) and Dan L Duncan Comprehensive Cancer Center Support Grant (NCI-CA125123). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, the Department of Defense, or the United States Government.
Can F. Koyuncu, Cheng Lu, Kaustav Bera, Zelin Zhang, Jun Xu, Paula Andrea Toro Castaño, Germán Corredor, Deborah Chute, Pingfu Fu, Wade L. Thorstad, Farhoud Faraji, Justin A. Bishop, Mitra Mehrad, Patricia D. Castro, Andrew G. Sikora, Lester D. R. Thompson, R. D. Chernock, Krystle A. Lang Kuhs, Jingqin Luo, Vlad C. Sandulache, David J. Adelstein, Shlomo Koyfman, James S. Lewis Jr., Anant Madabhushi
Inhibitors to factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to non-inhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 mediated B-cell depletion. In naïve HA mice, prophylactic anti-BAFF therapy prior to FVIII immunization prevented inhibitors and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.
Bhavya S. Doshi, Jyoti Rana, Giancarlo Castaman, Mostafa A. Shaheen, Radoslaw Kaczmarek, John S. S. Butterfield, Shannon L. Meeks, Cindy Leissinger, Moanaro Biswas, Valder R. Arruda
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