bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒09‒06
ninety-one papers selected by
Kıvanç Görgülü
Technical University of Munich


  1. Cell Metab. 2020 Aug 31. pii: S1550-4131(20)30420-4. [Epub ahead of print]
    Batista TM, Jayavelu AK, Wewer Albrechtsen NJ, Iovino S, Lebastchi J, Pan H, Dreyfuss JM, Krook A, Zierath JR, Mann M, Kahn CR.
      Skeletal muscle insulin resistance is the earliest defect in type 2 diabetes (T2D), preceding and predicting disease development. To what extent this reflects a primary defect or is secondary to tissue cross talk due to changes in hormones or circulating metabolites is unknown. To address this question, we have developed an in vitro disease-in-a-dish model using iPS cells from T2D patients differentiated into myoblasts (iMyos). We find that T2D iMyos in culture exhibit multiple defects mirroring human disease, including an altered insulin signaling, decreased insulin-stimulated glucose uptake, and reduced mitochondrial oxidation. More strikingly, global phosphoproteomic analysis reveals a multidimensional network of signaling defects in T2D iMyos going beyond the canonical insulin-signaling cascade, including proteins involved in regulation of Rho GTPases, mRNA splicing and/or processing, vesicular trafficking, gene transcription, and chromatin remodeling. These cell-autonomous defects and the dysregulated network of protein phosphorylation reveal a new dimension in the cellular mechanisms underlying the fundamental defects in T2D.
    Keywords:  chromatin remodeling; glucose transport; iPSC; insulin resistance; mRNA splicing; mitochondrial oxidation; phosphoproteomics; skeletal muscle; type 2 diabetes; vesicle trafficking
    DOI:  https://doi.org/10.1016/j.cmet.2020.08.007
  2. Nat Genet. 2020 Aug 31.
    Gao S, Chen S, Han D, Wang Z, Li M, Han W, Besschetnova A, Liu M, Zhou F, Barrett D, Luong MP, Owiredu J, Liang Y, Ahmed M, Petricca J, Patalano S, Macoska JA, Corey E, Chen S, Balk SP, He HH, Cai C.
      FOXA1 functions as a pioneer transcription factor by facilitating the access to chromatin for steroid hormone receptors, such as androgen receptor and estrogen receptor1-4, but mechanisms regulating its binding to chromatin remain elusive. LSD1 (KDM1A) acts as a transcriptional repressor by demethylating mono/dimethylated histone H3 lysine 4 (H3K4me1/2)5,6, but also acts as a steroid hormone receptor coactivator through mechanisms that are unclear. Here we show, in prostate cancer cells, that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 inhibition globally disrupts FOXA1 chromatin binding. Mechanistically, we demonstrate that LSD1 positively regulates FOXA1 binding by demethylating lysine 270, adjacent to the wing2 region of the FOXA1 DNA-binding domain. Acting through FOXA1, LSD1 inhibition broadly disrupted androgen-receptor binding and its transcriptional output, and dramatically decreased prostate cancer growth alone and in synergy with androgen-receptor antagonist treatment in vivo. These mechanistic insights suggest new therapeutic strategies in steroid-driven cancers.
    DOI:  https://doi.org/10.1038/s41588-020-0681-7
  3. Nat Cell Biol. 2020 Sep;22(9): 1042-1048
    Riegman M, Sagie L, Galed C, Levin T, Steinberg N, Dixon SJ, Wiesner U, Bradbury MS, Niethammer P, Zaritsky A, Overholtzer M.
      Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.
    DOI:  https://doi.org/10.1038/s41556-020-0565-1
  4. Autophagy. 2020 Sep 02.
    González-Rodríguez P, Cheray M, Füllgrabe J, Salli M, Engskog-Vlachos P, Keane L, Cunha V, Lupa A, Li W, Ma Q, Dreij K, Rosenfeld MG, Joseph B.
      Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.
    Keywords:  Autophagy; DNA methylation; MAP1LC3; epigenetics; transcription
    DOI:  https://doi.org/10.1080/15548627.2020.1816664
  5. Cancers (Basel). 2020 Sep 01. pii: E2477. [Epub ahead of print]12(9):
    Lee JS, Oh SJ, Choi HJ, Kang JH, Lee SH, Ha JS, Woo SM, Jang H, Lee H, Kim SY.
      Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.
    Keywords:  ATP production; KC mouse; PDAC; fatty acid oxidation; glycolysis
    DOI:  https://doi.org/10.3390/cancers12092477
  6. Nature. 2020 Sep 02.
    Watkins TBK, Lim EL, Petkovic M, Elizalde S, Birkbak NJ, Wilson GA, Moore DA, Grönroos E, Rowan A, Dewhurst SM, Demeulemeester J, Dentro SC, Horswell S, Au L, Haase K, Escudero M, Rosenthal R, Bakir MA, Xu H, Litchfield K, Lu WT, Mourikis TP, Dietzen M, Spain L, Cresswell GD, Biswas D, Lamy P, Nordentoft I, Harbst K, Castro-Giner F, Yates LR, Caramia F, Jaulin F, Vicier C, Tomlinson IPM, Brastianos PK, Cho RJ, Bastian BC, Dyrskjøt L, Jönsson GB, Savas P, Loi S, Campbell PJ, Andre F, Luscombe NM, Steeghs N, Tjan-Heijnen VCG, Szallasi Z, Turajlic S, Jamal-Hanjani M, Van Loo P, Bakhoum SF, Schwarz RF, McGranahan N, Swanton C.
      Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.
    DOI:  https://doi.org/10.1038/s41586-020-2698-6
  7. Cell Mol Gastroenterol Hepatol. 2020 Aug 31. pii: S2352-345X(20)30135-1. [Epub ahead of print]
    Hoffman MT, Kemp SB, Salas-Escabillas DJ, Zhang Y, Steele NG, The S, Long D, Benitz S, Yan W, Margolskee RF, Bednar F, Pasca di Magliano M, Wen HJ, Crawford HC.
      BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) initiation and progression is accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression.METHODS: Gnat3-null (Gnat3-/-) mice were crossbred with animals harboring a Cre-inducible KrasLSL-G12D/+ allele with either Ptf1aCre/+ (KC) or tamoxifen-inducible Ptf1aCreERT/+ (KCERT) mice to drive oncogenic KRAS expression in the pancreas. Ex vivo organoid conditioned media generated from KC and Gnat3-/-;KC acinar cells was analyzed for cytokine secretion. Experimental pancreatitis was induced in KCERT and Gnat3-/-;KCERT mice to accelerate tumorigenesis followed by analysis using mass cytometry and single-cell RNA sequencing. To study PDA progression, KC and Gnat3-/-;KC mice were aged to morbidity or 52-weeks.
    RESULTS: Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3-/-;KCERT pancreata found altered expression of immunomodulatory genes in Cxcr2 expressing myeloid-derived suppressor cells (MDSC) and an increased number of granulocytic MDSCs (gMDSC), a subset of tumor promoting MDSCs. Importantly, expression levels of CXCL1 and CXCL2, known ligands for CXCR2, were also elevated in Gnat3-/-;KCERT pancreata. Consistent with the tumor-promoting role of MDSCs, aged Gnat3-/-;KC mice progressed more rapidly to metastatic carcinoma compared to KC controls.
    CONCLUSIONS: Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2 - CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA.
    Keywords:  CXCL1; CXCL2; MDSC; Tuft cell
    DOI:  https://doi.org/10.1016/j.jcmgh.2020.08.011
  8. Nat Cell Biol. 2020 Sep;22(9): 1091-1102
    Miceli C, Roccio F, Penalva-Mousset L, Burtin M, Leroy C, Nemazanyy I, Kuperwasser N, Pontoglio M, Friedlander G, Morel E, Terzi F, Codogno P, Dupont N.
      Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through β-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.
    DOI:  https://doi.org/10.1038/s41556-020-0566-0
  9. Proc Natl Acad Sci U S A. 2020 Sep 01. pii: 202001562. [Epub ahead of print]
    Heckmann D, Campeau A, Lloyd CJ, Phaneuf PV, Hefner Y, Carrillo-Terrazas M, Feist AM, Gonzalez DJ, Palsson BO.
      Enzyme turnover numbers (k cats) are essential for a quantitative understanding of cells. Because k cats are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo k cats using metabolic specialist Escherichia coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo k cats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo k cats predict unseen proteomics data with much higher precision than in vitro k cats. The results demonstrate that in vivo k cats can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.
    Keywords:  gene knockout; in vivo; kcat; proteomics; turnover number
    DOI:  https://doi.org/10.1073/pnas.2001562117
  10. Oncogene. 2020 Sep 03.
    Lian EY, Hyndman BD, Moodley S, Maritan SM, Mulligan LM.
      Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.
    DOI:  https://doi.org/10.1038/s41388-020-01448-z
  11. J Exp Med. 2020 Dec 07. pii: e20191473. [Epub ahead of print]217(12):
    Macleod BL, Elsaesser HJ, Snell LM, Dickson RJ, Guo M, Hezaveh K, Xu W, Kothari A, McGaha TL, Guidos CJ, Brooks DG.
      Many pathogens subvert intestinal immunity to persist within the gastrointestinal tract (GIT); yet, the underlying mechanisms that enable sanctuary specifically in this reservoir are unclear. Using mass cytometry and network analysis, we demonstrate that chronic LCMV infection of the GIT leads to dysregulated microbial composition, a cascade of metabolic alterations, increased susceptibility to GI disease, and a system-wide recalibration of immune composition that defines viral persistence. Chronic infection led to outgrowth of activated Tbet-expressing T reg cell populations unique to the GIT and the rapid erosion of pathogen-specific CD8 tissue-resident memory T cells. Mechanistically, T reg cells and coinhibitory receptors maintained long-term viral sanctuary within the GIT, and their targeting reactivated T cells and eliminated this viral reservoir. Thus, our data provide a high-dimensional definition of the mechanisms of immune regulation that chronic viruses implement to exploit the unique microenvironment of the GIT and identify T reg cells as key modulators of viral persistence in the intestinal tract.
    DOI:  https://doi.org/10.1084/jem.20191473
  12. Nat Rev Mol Cell Biol. 2020 Sep 01.
    Lafontaine DLJ, Riback JA, Bascetin R, Brangwynne CP.
      The nucleolus is the most prominent nuclear body and serves a fundamentally important biological role as a site of ribonucleoprotein particle assembly, primarily dedicated to ribosome biogenesis. Despite being one of the first intracellular structures visualized historically, the biophysical rules governing its assembly and function are only starting to become clear. Recent studies have provided increasing support for the concept that the nucleolus represents a multilayered biomolecular condensate, whose formation by liquid-liquid phase separation (LLPS) facilitates the initial steps of ribosome biogenesis and other functions. Here, we review these biophysical insights in the context of the molecular and cell biology of the nucleolus. We discuss how nucleolar function is linked to its organization as a multiphase condensate and how dysregulation of this organization could provide insights into still poorly understood aspects of nucleolus-associated diseases, including cancer, ribosomopathies and neurodegeneration as well as ageing. We suggest that the LLPS model provides the starting point for a unifying quantitative framework for the assembly, structural maintenance and function of the nucleolus, with implications for gene regulation and ribonucleoprotein particle assembly throughout the nucleus. The LLPS concept is also likely useful in designing new therapeutic strategies to target nucleolar dysfunction.
    DOI:  https://doi.org/10.1038/s41580-020-0272-6
  13. Cell. 2020 Aug 21. pii: S0092-8674(20)30994-6. [Epub ahead of print]
    Drokhlyansky E, Smillie CS, Van Wittenberghe N, Ericsson M, Griffin GK, Eraslan G, Dionne D, Cuoco MS, Goder-Reiser MN, Sharova T, Kuksenko O, Aguirre AJ, Boland GM, Graham D, Rozenblatt-Rosen O, Xavier RJ, Regev A.
      The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.
    Keywords:  ENS; GWAS; aging; circadian; colon; enteric nervous system; enteric neuron; ileum; neuro-immune; single cell
    DOI:  https://doi.org/10.1016/j.cell.2020.08.003
  14. J Cell Biol. 2020 Nov 02. pii: e201908212. [Epub ahead of print]219(11):
    Valente LJ, Tarangelo A, Li AM, Naciri M, Raj N, Boutelle AM, Li Y, Mello SS, Bieging-Rolett K, DeBerardinis RJ, Ye J, Dixon SJ, Attardi LD.
      The mechanisms by which TP53, the most frequently mutated gene in human cancer, suppresses tumorigenesis remain unclear. p53 modulates various cellular processes, such as apoptosis and proliferation, which has led to distinct cellular mechanisms being proposed for p53-mediated tumor suppression in different contexts. Here, we asked whether during tumor suppression p53 might instead regulate a wide range of cellular processes. Analysis of mouse and human oncogene-expressing wild-type and p53-deficient cells in physiological oxygen conditions revealed that p53 loss concurrently impacts numerous distinct cellular processes, including apoptosis, genome stabilization, DNA repair, metabolism, migration, and invasion. Notably, some phenotypes were uncovered only in physiological oxygen. Transcriptomic analysis in this setting highlighted underappreciated functions modulated by p53, including actin dynamics. Collectively, these results suggest that p53 simultaneously governs diverse cellular processes during transformation suppression, an aspect of p53 function that would provide a clear rationale for its frequent inactivation in human cancer.
    DOI:  https://doi.org/10.1083/jcb.201908212
  15. EMBO Mol Med. 2020 Sep 04. e11210
    Menga A, Serra M, Todisco S, Riera-Domingo C, Ammarah U, Ehling M, Palmieri EM, Di Noia MA, Gissi R, Favia M, Pierri CL, Porporato PE, Castegna A, Mazzone M.
      Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.
    Keywords:  glufosinate; glutamine synthetase; immunometabolism; macrophages; metastasis
    DOI:  https://doi.org/10.15252/emmm.201911210
  16. Nature. 2020 Sep 02.
    Fiore VF, Krajnc M, Quiroz FG, Levorse J, Pasolli HA, Shvartsman SY, Fuchs E.
      Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.
    DOI:  https://doi.org/10.1038/s41586-020-2695-9
  17. Nat Methods. 2020 Aug 31.
    Qiu Q, Hu P, Qiu X, Govek KW, Cámara PG, Wu H.
      Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.
    DOI:  https://doi.org/10.1038/s41592-020-0935-4
  18. Cancers (Basel). 2020 Aug 27. pii: E2437. [Epub ahead of print]12(9):
    Chen R, Jäättelä M, Liu B.
      Cancer cells generate large quantities of cytoplasmic protons as byproducts of aberrantly activated aerobic glycolysis and lactate fermentation. To avoid potentially detrimental acidification of the intracellular milieu, cancer cells activate multiple acid-removal pathways that promote cytosolic alkalization and extracellular acidification. Accumulating evidence suggests that in addition to the well-characterized ion pumps and exchangers in the plasma membrane, cancer cell lysosomes are also reprogrammed for this purpose. On the one hand, the increased expression and activity of the vacuolar-type H+-ATPase (V-ATPase) on the lysosomal limiting membrane combined with the larger volume of the lysosomal compartment increases the lysosomal proton storage capacity substantially. On the other hand, enhanced lysosome exocytosis enables the efficient release of lysosomal protons to the extracellular space. Together, these two steps dynamically drive proton flow from the cytosol to extracellular space. In this perspective, we provide mechanistic insight into how lysosomes contribute to the rewiring of pH homeostasis in cancer cells.
    Keywords:  V-ATPase; lysosomal exocytosis; lysosome; pH regulation
    DOI:  https://doi.org/10.3390/cancers12092437
  19. Ann Oncol. 2020 Aug 28. pii: S0923-7534(20)42170-2. [Epub ahead of print]
    Marinelli D, Mazzotta M, Scalera S, Terrenato I, Sperati F, D'Ambrosio L, Pallocca M, Corleone G, Krasniqi E, Pizzuti L, Barba M, Carpano S, Vici P, Filetti M, Giusti R, Vecchione A, Occhipinti M, Gelibter A, Botticelli A, De Nicola F, Ciuffreda L, Goeman F, Gallo E, Visca P, Pescarmona E, Fanciulli M, De Maria R, Marchetti P, Ciliberto G, Maugeri-Saccà M.
      BACKGROUND: Immune checkpoint inhibitors (ICIs) have demonstrated significant overall survival (OS) benefit in lung adenocarcinoma (LUAD). Nevertheless, a remarkable interpatient heterogeneity characterizes immunotherapy efficacy, regardless of programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). KEAP1 mutations are associated with shorter survival in LUAD patients receiving chemotherapy. We hypothesized that the pattern of KEAP1 co-mutations and mutual exclusivity may identify LUAD patients unresponsive to immunotherapy.PATIENTS AND METHODS: KEAP1 mutational co-occurrences and somatic interactions were studied in the whole MSKCC LUAD dataset. The impact of coexisting alterations on survival outcomes in ICI-treated LUAD patients was verified in the randomized phase II/III POPLAR/OAK trials (blood-based sequencing, bNGS cohort, N=253). Three tissue-based sequencing studies (Rome, MSKCC and DFCI) were used for independent validation (tNGS cohort, N=289). Immunogenomic features were analyzed using the TCGA LUAD study.
    RESULTS: On the basis of KEAP1 mutational co-occurrences, we identified four genes potentially associated with reduced efficacy of immunotherapy (KEAP1, PBRM1, SMARCA4 and STK11). Independent of the nature of co-occurring alterations, tumors with coexisting mutations (CoMut) had inferior survival as compared to single-mutant (SM) and wild-type (WT) tumors (bNGS cohort: CoMut vs SM log-rank P=0.048, CoMut vs WT log-rank P<0.001; tNGS cohort: CoMut vs SM log-rank P=0.037, CoMut vs WT log-rank P=0.006). The CoMut subset harbored higher TMB than the WT disease, and the adverse significance of coexisting alterations was maintained in LUAD with high TMB. Significant immunogenomic differences were observed between the CoMut and WT groups in terms of core immune signatures, T cell receptor repertoire, T helper cell signatures and immunomodulatory genes.
    CONCLUSIONS: This study indicates that coexisting alterations in a limited set of genes characterize a subset of LUAD unresponsive to immunotherapy and with high TMB. An immune-cold microenvironment may account for the clinical course of the disease.
    Keywords:  KEAP1 co-mutations; immunogenomic; immunotherapy; lung adenocarcinoma; tumor mutational burden
    DOI:  https://doi.org/10.1016/j.annonc.2020.08.2105
  20. Cells. 2020 Sep 01. pii: E2013. [Epub ahead of print]9(9):
    Lee JS, Lee H, Jang H, Woo SM, Park JB, Lee SH, Kang JH, Kim HY, Song J, Kim SY.
      The greatest challenge in cancer therapy is posed by drug-resistant recurrence following treatment. Anticancer chemotherapy is largely focused on targeting the rapid proliferation and biosynthesis of cancer cells. This strategy has the potential to trigger autophagy, enabling cancer cell survival through the recycling of molecules and energy essential for biosynthesis, leading to drug resistance. Autophagy recycling contributes amino acids and ATP to restore mTOR complex 1 (mTORC1) activity, which leads to cell survival. However, autophagy with mTORC1 activation can be stalled by reducing the ATP level. We have previously shown that cytosolic NADH production supported by aldehyde dehydrogenase (ALDH) is critical for supplying ATP through oxidative phosphorylation (OxPhos) in cancer cell mitochondria. Inhibitors of the mitochondrial complex I of the OxPhos electron transfer chain and ALDH significantly reduce the ATP level selectively in cancer cells, terminating autophagy triggered by anticancer drug treatment. With the aim of overcoming drug resistance, we investigated combining the inhibition of mitochondrial complex I, using phenformin, and ALDH, using gossypol, with anticancer drug treatment. Here, we show that OxPhos targeting combined with anticancer drugs acts synergistically to enhance the anticancer effect in mouse xenograft models of various cancers, which suggests a potential therapeutic approach for drug-resistant cancer.
    Keywords:  ATP production; aldehyde dehydrogenase; cancer metabolism; energy metabolism; oxidative phosphorylation (OxPhos)
    DOI:  https://doi.org/10.3390/cells9092013
  21. Gut. 2020 Sep 01. pii: gutjnl-2019-319970. [Epub ahead of print]
    Gout J, Perkhofer L, Morawe M, Arnold F, Ihle M, Biber S, Lange S, Roger E, Kraus JM, Stifter K, Hahn SA, Zamperone A, Engleitner T, Müller M, Walter K, Rodriguez-Aznar E, Sainz B, Hermann PC, Hessmann E, Müller S, Azoitei N, Lechel A, Liebau S, Wagner M, Simeone DM, Kestler HA, Seufferlein T, Wiesmüller L, Rad R, Frappart PO, Kleger A.
      OBJECTIVE: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC).DESIGN: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy.
    RESULTS: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance.
    CONCLUSION: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.
    Keywords:  DNA damage; drug resistance; pancreas; pancreatic cancer; pancreatic tumours
    DOI:  https://doi.org/10.1136/gutjnl-2019-319970
  22. Nature. 2020 Sep 02.
    Bian Y, Li W, Kremer DM, Sajjakulnukit P, Li S, Crespo J, Nwosu ZC, Zhang L, Czerwonka A, Pawłowska A, Xia H, Li J, Liao P, Yu J, Vatan L, Szeliga W, Wei S, Grove S, Liu JR, McLean K, Cieslik M, Chinnaiyan AM, Zgodziński W, Wallner G, Wertel I, Okła K, Kryczek I, Lyssiotis CA, Zou W.
      Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours1-4, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.
    DOI:  https://doi.org/10.1038/s41586-020-2682-1
  23. Nature. 2020 Sep 02.
    Allegretti M, Zimmerli CE, Rantos V, Wilfling F, Ronchi P, Fung HKH, Lee CW, Hagen W, Turoňová B, Karius K, Börmel M, Zhang X, Müller CW, Schwab Y, Mahamid J, Pfander B, Kosinski J, Beck M.
      Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.
    DOI:  https://doi.org/10.1038/s41586-020-2670-5
  24. Mol Cancer. 2020 Sep 04. 19(1): 136
    Wang Y, Luo J, Alu A, Han X, Wei Y, Wei X.
      The activation of the cGAS-STING pathway has tremendous potential to improve anti-tumor immunity by generating type I interferons. In recent decades, we have witnessed that producing dsDNA upon various stimuli is an initiative factor, triggering the cGAS-SING pathway for a defensive host. The understanding of both intracellular cascade reaction and the changes of molecular components gains insight into type I IFNs and adaptive immunity. Based on the immunological study, the STING-cGAS pathway is coupled to cancer biotherapy. The most challenging problem is the limited therapeutic effect. Therefore, people view 5, 6-dimethylxanthenone-4-acetic acid, cyclic dinucleotides and various derivative as cGAS-STING pathway agonists. Even so, these agonists have flaws in decreasing biotherapeutic efficacy. Subsequently, we exploited agonist delivery systems (nanocarriers, microparticles and hydrogels). The article will discuss the activation of the cGAS-STING pathway and underlying mechanisms, with an introduction of cGAS-STING agonists, related clinical trials and agonist delivery systems.
    Keywords:  Agonist; Cancer biotherapy; Clinical trials; Cyclic dinucleotide; Delivery system; Interferon; cGAS-STING pathway
    DOI:  https://doi.org/10.1186/s12943-020-01247-w
  25. Nat Immunol. 2020 Aug 31.
    Kumagai S, Togashi Y, Kamada T, Sugiyama E, Nishinakamura H, Takeuchi Y, Vitaly K, Itahashi K, Maeda Y, Matsui S, Shibahara T, Yamashita Y, Irie T, Tsuge A, Fukuoka S, Kawazoe A, Udagawa H, Kirita K, Aokage K, Ishii G, Kuwata T, Nakama K, Kawazu M, Ueno T, Yamazaki N, Goto K, Tsuboi M, Mano H, Doi T, Shitara K, Nishikawa H.
      Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.
    DOI:  https://doi.org/10.1038/s41590-020-0769-3
  26. Autophagy. 2020 Aug 31.
    Qiang L, Yang S, Cui YH, He YY.
      Macroautophagy/autophagy is a cellular catabolic process that is implicated in several physiological and pathological processes. However, the role of epidermal autophagy in wound healing remains unknown. Here, using mice with genetic ablation of the essential Atg5 (autophagy related 5) or Atg7 in their epidermis to inhibit autophagy, we show that keratinocyte autophagy regulates wound healing in mice. Wounding induces the expression of autophagy genes in mouse skin. Epidermis-specific autophagy deficiency inhibits wound closure, re-epithelialization, keratinocyte proliferation and differentiation, dermal granulation tissue formation, and infiltration of immune cells including macrophages, neutrophils, and mast cells, while it does not affect angiogenesis. Using cytokine array screening, we found that autophagy deficiency inhibits the transcription and production of the cytokine CCL2/MCP-1 by TNF. At the molecular level, TNF induces autophagic flux and the expression of autophagy genes through NFKB in epidermal keratinocytes. TNF promotes CCL2 transcription through the autophagy-AMPK-BRAF-MAPK/ERK-activator protein 1 (AP-1) pathway. Indeed, treating mice with recombinant CCL2 can reverse the effect of autophagy deficiency in keratinocytes. At the cellular level, we found that CCL2 induction via autophagy in keratinocytes is required not only for keratinocyte migration and proliferation but also for dermal fibroblast activation. Our findings demonstrate a critical role of epidermal autophagy in wound healing in vivo and elucidate a critical molecular machinery coordinating keratinocyte-fibroblast interaction in skin repair.
    Keywords:  CCL2/MCP-1; TNF; autophagy; differentiation; fibroblast; inflammation; keratinocyte; migration; proliferation; wound healing
    DOI:  https://doi.org/10.1080/15548627.2020.1816342
  27. Cell Metab. 2020 Sep 01. pii: S1550-4131(20)30417-4. [Epub ahead of print]32(3): 447-456.e6
    Asadi Shahmirzadi A, Edgar D, Liao CY, Hsu YM, Lucanic M, Asadi Shahmirzadi A, Wiley CD, Gan G, Kim DE, Kasler HG, Kuehnemann C, Kaplowitz B, Bhaumik D, Riley RR, Kennedy BK, Lithgow GJ.
      Metabolism and aging are tightly connected. Alpha-ketoglutarate is a key metabolite in the tricarboxylic acid (TCA) cycle, and its levels change upon fasting, exercise, and aging. Here, we investigate the effect of alpha-ketoglutarate (delivered in the form of a calcium salt, CaAKG) on healthspan and lifespan in C57BL/6 mice. To probe the relationship between healthspan and lifespan extension in mammals, we performed a series of longitudinal, clinically relevant measurements. We find that CaAKG promotes a longer, healthier life associated with a decrease in levels of systemic inflammatory cytokines. We propose that induction of IL-10 by dietary AKG suppresses chronic inflammation, leading to health benefits. By simultaneously reducing frailty and enhancing longevity, AKG, at least in the murine model, results in a compression of morbidity.
    Keywords:  IL-10; SASP; alpha-ketoglutarate; frailty; healthspan; inflammation; lifespan; longevity
    DOI:  https://doi.org/10.1016/j.cmet.2020.08.004
  28. EMBO Mol Med. 2020 Aug 16. e12423
    Villa-Bellosta R.
      Aging is associated with redox imbalance according to the redox theory of aging. Consistently, a mouse model of premature aging (LmnaG609G/+) showed an increased level of mitochondrial reactive oxygen species (ROS) and a reduced basal antioxidant capacity, including loss of the NADPH-coupled glutathione redox system. LmnaG609G/+ mice also exhibited reduced mitochondrial ATP synthesis secondary to ROS-induced mitochondrial dysfunction. Treatment of LmnaG609G/+ vascular smooth muscle cells with magnesium-enriched medium improved the intracellular ATP level, enhanced the antioxidant capacity, and thereby reduced mitochondrial ROS production. Moreover, treatment of LmnaG609G/+ mice with dietary magnesium improved the proton pumps (complexes I, III, and IV), stimulated extramitochondrial NADH oxidation and enhanced the coupled mitochondrial membrane potential, and thereby increased H+-coupled mitochondrial NADPH and ATP synthesis, which is necessary for cellular energy supply and survival. Consistently, magnesium treatment reduced calcification of vascular smooth muscle cells in vitro and in vivo, and improved the longevity of mice. This antioxidant property of magnesium may be beneficial in children with HGPS.
    Keywords:  HGPS; aging; magnesium; progeria; vascular calcification
    DOI:  https://doi.org/10.15252/emmm.202012423
  29. Clin Cancer Res. 2020 Sep 01. pii: clincanres.1359.2020. [Epub ahead of print]
    Batra SK, Cannon A, Thompson CM, Maurer C, Atri P, Bhatia R, West S, Ghersi D, Olive KP, Kumar S.
      PURPOSE: The cytokine milieu in pancreatic ductal adenocarcinoma (PDAC) promotes tumor progression and immune suppression, contributing to the dismal prognosis of patients with PDAC. The roles of many of these cytokines, however, have not been thoroughly investigated in PDAC.EXPERIMENTAL DESIGN: PDAC microarray and TCGA datasets were analyzed to identify cytokines and cognate receptors overexpressed in PDAC and associated with survival. Pathway and CIBERSORT analyses were used to elucidate potential mechanisms of altered patient survival. Comparative analysis of cytokine expression in KPC (K-rasG12D; TP53R172H; Pdx-1cre) and KC (K-rasG12D; Pdx-1cre) PDAC models and multicolor immunofluorescence (IF) staining of human PDAC-resected samples were used to validate these findings.
    RESULTS: CXCL9 and CXCL10 were among the most highly overexpressed cytokines by bioinformatics analyses while their receptor, CXCR3, was significantly overexpressed by bioinformatics and IHC analysis. Higher CXCR3-ligand expression was associated with shorter overall survival, while high CXCR3 expression was associated with better survival. The CXCR3-ligands, CXCL4, 9, and 10 were overexpressed in KPC compared to KC mice. Pathway analysis of CXCR3- and CXCR3-ligand-associated genes showed that CXCR3 is a marker of anti-tumor immunity, while its ligands may promote immunosuppression. CIBERSORT and IF studies of PDAC tissues demonstrated that high CXCR3 expression was associated with increased CD8+ T-cell and naïve B-cell signatures and loss of plasma cell signatures. CXCR3-ligand expression was associated with increased CD8+ T-cell signatures and loss of NK-cell signatures.
    CONCLUSION: CXCR3-ligands are overexpressed in PDAC and are associated with poor survival likely related to alterations in tumor immune infiltrate/activity.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-1359
  30. Elife. 2020 Sep 03. pii: e61103. [Epub ahead of print]9
    Mulier M, Van Ranst N, Corthout N, Munck S, Vanden Berghe P, Vriens J, Voets T, Moilanen L.
      Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates inflammatory heat hyperalgesia, but the underlying mechanisms are unknown. We induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, and augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. In particular, inflammation provoked a pronounced increase in nociceptors with functional co-expression of TRPM3, TRPV1 and TRPA1. Finally, pharmacological inhibition of TRPM3 dampened TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw, but not in those innervating healthy tissue. These insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.
    Keywords:  TRP channels; inflammatory pain; mouse; neuroscience; sensory neurons
    DOI:  https://doi.org/10.7554/eLife.61103
  31. Biochem J. 2020 Sep 04. pii: BCJ20200551. [Epub ahead of print]
    Voisin P, Bernard M, Bergès T, Regnacq M.
      Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast.  It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.
    Keywords:  Autophagy; Saccharomyces cerevisiae; catabolite repression; lipid droplets; mitochondrial dysfunction
    DOI:  https://doi.org/10.1042/BCJ20200551
  32. Cell Metab. 2020 Aug 26. pii: S1550-4131(20)30414-9. [Epub ahead of print]
    Lyu K, Zhang Y, Zhang D, Kahn M, Ter Horst KW, Rodrigues MRS, Gaspar RC, Hirabara SM, Luukkonen PK, Lee S, Bhanot S, Rinehart J, Blume N, Rasch MG, Serlie MJ, Bogan JS, Cline GW, Samuel VT, Shulman GI.
      Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.
    Keywords:  ceramides; dicylglycerols; hepatic glucose production; hepatic glycogen synthesis; hepatic insulin resistance; insulin receptor phosphorylation; liquid chromatography-tandem mass spectrometry; nonalcoholic fatty liver disease; protein kinase C-epsilon; type 2 diabetes
    DOI:  https://doi.org/10.1016/j.cmet.2020.08.001
  33. Eur J Clin Nutr. 2020 Aug;74(Suppl 1): 27-32
    Frassetto LA, Sebastian A, DuBose TD.
      Consuming a lower acid (and particularly lower phosphate) diet and/or supplementing the diet with base precursors, such as bicarbonate, might have a number of mitigating effects on the aging process. These include: (1) slowing progression of fibrosis by reduction of high endogenous acid production to preserve net acid excretion and minimize the degree of systemic acidosis; (2) avoiding the downregulation of klotho, a membrane and soluble factor associated with aging. Klotho declines when constant high dietary phosphate intake leads to an increase in FGF23 production; and (3) increasing activity of the enzyme telomerase, an important factor in maintaining telomere length, another factor associated with longer lifespan. Current evidence is based on studies in invertebrate and small animal models. These results, and extrapolations of associated human studies, suggest that low acid-producing diets, or neutralization of the low grade metabolic acidosis seen in humans with age-related renal dysfunction could potentially lead to a longer, healthier lifespan.
    DOI:  https://doi.org/10.1038/s41430-020-0693-5
  34. Proc Natl Acad Sci U S A. 2020 Aug 31. pii: 202005136. [Epub ahead of print]
    Gahan JM, Rentzsch F, Schnitzler CE.
      Polycomb group proteins are essential regulators of developmental processes across animals. Despite their importance, studies on Polycomb are often restricted to classical model systems and, as such, little is known about the evolution of these important chromatin regulators. Here we focus on Polycomb Repressive Complex 1 (PRC1) and trace the evolution of core components of canonical and non-canonical PRC1 complexes in animals. Previous work suggested that a major expansion in the number of PRC1 complexes occurred in the vertebrate lineage. We show that the expansion of the Polycomb Group RING Finger (PCGF) protein family, an essential step for the establishment of the large diversity of PRC1 complexes found in vertebrates, predates the bilaterian-cnidarian ancestor. This means that the genetic repertoire necessary to form all major vertebrate PRC1 complexes emerged early in animal evolution, over 550 million years ago. We further show that PCGF5, a gene conserved in cnidarians and vertebrates but lost in all other studied groups, is expressed in the nervous system in the sea anemone Nematostella vectensis, similar to its mammalian counterpart. Together this work provides a framework for understanding the evolution of PRC1 complex diversity and it establishes Nematostella as a promising model system in which the functional ramifications of this diversification can be further explored.
    Keywords:  Nematostella; PCGF; PRC1; Polycomb; cnidarian
    DOI:  https://doi.org/10.1073/pnas.2005136117
  35. Cancer Immunol Res. 2020 Sep 02. pii: canimm.0828.2019. [Epub ahead of print]
    Son J, Cho JW, Park HJ, Moon J, Park S, Lee H, Lee J, Kim G, Park SM, Lira SA, McKenzie A, Kim HY, Choi CY, Lim YT, Park SY, Kim HR, Park SH, Shin EC, Lee I, Ha SJ.
      Regulatory T cells (Tregs) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity. However, the molecular mechanism underlying the accumulation of Tregs in the TME are poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in TME and had significantly higher expression immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared to tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA sequencing and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an interleukin-33 (IL33) receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-19-0828
  36. J Proteomics. 2020 Aug 31. pii: S1874-3919(20)30317-1. [Epub ahead of print] 103949
    Sulkshane P, Duek I, Ram J, Thakur A, Reis N, Ziv T, Glickman MH.
      Strict quality control for mitochondrial proteins is necessary to ensure cell homeostasis. Two cellular pathways-Ubiquitin Proteasome System (UPS) and autophagy-contribute to mitochondrial homeostasis under stressful conditions. Here, we investigate changes to the mitochondria proteome and to the ubiquitin landscape at mitochondria in response to proteasome inhibition. Treatment of HeLa cells devoid of Parkin, the primary E3 ligase responsible for mitophagy, with proteasome inhibitor MG132 for a few hours caused mitochondrial oxidative stress and fragmentation, reduced energy output, and increased mitochondrial ubiquitination without inducing mitophagy. Overexpression of Parkin did not show any induction of mitophagy in response to MG132 treatment. Analysis of ubiquitin chains on isolated mitochondria revealed predominance of K48, K29 and K63-linked polyubiquitin. Interestingly, of all ubiquitinated mitochondrial proteins detected in response to MG132 treatment, a majority (≥90%) were intramitochondrial irrespective of Parkin expression. However, overall levels of these ubiquitinated mitochondrial proteins did not change significantly upon proteasome inhibition when evaluated by quantitative proteomics (LFQ and SILAC), suggesting that only a small portion are ubiquitinated under basal conditions. Another aspect of proteasome inhibition is significant enrichment of UPS, lysosomal and phagosomal components, and other heat shock proteins associated with isolated mitochondria. Taken together, our study highlights a critical role of UPS for ubiquitinating and removing imported proteins as part of a basal mitochondrial quality control system independent of Parkin. SIGNIFICANCE: As centers of cellular bioenergetics, numerous metabolic pathways and signaling cascades, the health of mitochondria is of utmost importance for ensuring cell survival. Due to their unique physiology, mitochondria are constantly subjected to damaging oxidative radicals (ROS) and protein import-related stress due to buildup of unfolded aggregate-prone proteins. Thus, for quality control purposes, mitochondria are constantly under surveillance by Autophagy and the Ubiquitin Proteasome System (UPS), both of which share ubiquitin as a common signal. The ubiquitin landscape of mitochondria has been studied in detail under stressful conditions, however, little is known about basal mitochondrial ubiquitination. Our study reveals that the extent of ubiquitination at mitochondria greatly increases upon proteasome inhibition, pointing to a large number of potential substrates for proteasomal degradation. Interestingly, most of the ubiquitination occurs on intramitochondrial proteins, components of the electron transport chain (ETC) and matrix-resident metabolic enzymes in particular. Moreover, numerous cytosolic UPS components, chaperones and autophagy-lysosomal proteins were recruited to mitochondria upon proteasome inhibition. Taken together, this suggests that the levels and functions of mitochondrial proteins are constantly regulated through ubiquitin-dependent proteasomal degradation even under basal conditions. Unclogging mitochondrial import channels may provide a mechanism to alleviate stress associated with mitochondrial protein import or to adapt cells according to their metabolic needs. Therefore, targeting the mitochondrial ubiquitination/deubiquitination machinery, such as improving the therapeutic potency of proteasome inhibitors, may provide an additional therapeutic arsenal against tumors.
    Keywords:  Mitochondria; Mitostasis; Proteasome; Quantitative proteomics; Ubiquitin
    DOI:  https://doi.org/10.1016/j.jprot.2020.103949
  37. Science. 2020 Sep 04. pii: eaaz7714. [Epub ahead of print]369(6508):
    Sawa-Makarska J, Baumann V, Coudevylle N, von Bülow S, Nogellova V, Abert C, Schuschnig M, Graef M, Hummer G, Martens S.
      Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.
    DOI:  https://doi.org/10.1126/science.aaz7714
  38. Clin Cancer Res. 2020 Sep 01. pii: clincanres.1497.2020. [Epub ahead of print]
    Bassaganyas L, Pinyol R, Esteban-Fabró R, Torrens L, Torrecilla S, Willoughby CE, Franch-Expósito S, Vila-Casadesús M, Salaverria I, Montal R, Mazzaferro V, Camps J, Sia D, Llovet JM.
      BACKGROUND AND AIMS: Chromosomal instability is a hallmark of cancer that results in broad and focal copy number alterations (CNAs), two events associated with distinct molecular, immunological and clinical features. In hepatocellular carcinoma (HCC) the CNAs role has not been thoroughly assessed. Thus, we dissected the impact of CNA burdens on HCC molecular and immune features.METHODS: We analyzed SNP-array data from 452 paired tumor/adjacent resected HCCs and 25 dysplastic nodules. For each sample, broad and focal CNA burdens were quantified using CNApp, and the resulting broad scores (BS) and focal scores (FS) were correlated with transcriptomic, mutational and methylation profiles, tumor immune composition, and with clinico-pathological data.
    RESULTS: HCCs with low broad CNA burdens (defined as BS≤4; 17%) presented high inflammation, active infiltrate signalling, high cytolytic activity, and enrichment of the 'HCC-Immune-Class' and gene signatures related to antigen presentation. Conversely, tumors with chromosomal instability (high broad CNA loads, BS≥11; 40%), displayed immune excluded traits and were linked to proliferation, TP53 dysfunction, and DNA repair. Candidate determinants of the low cytotoxicity and immune exclusion features of high-BS tumors included alterations in antigen presenting machinery (i.e. HLA), widespread hypomethylation and decreased rates of observed/expected neoantigenic mutations. High focal scores were independent of tumor immune features, but were related to proliferation, TP53 dysfunction and progenitor cell traits.
    CONCLUSIONS: HCCs with high chromosomal instability exhibit features of immune exclusion, whereas tumors displaying low burdens of broad CNAs present an immune active profile. These CNA scores can be tested to predict response to immunotherapies.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-1497
  39. Nat Metab. 2020 Aug 31.
    Carvalho-Santos Z, Cardoso-Figueiredo R, Elias AP, Tastekin I, Baltazar C, Ribeiro C.
      Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. Here, we show that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. We provide functional evidence that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. Our findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes.
    DOI:  https://doi.org/10.1038/s42255-020-0266-x
  40. Nat Biotechnol. 2020 Aug 31.
    Hartmann FJ, Mrdjen D, McCaffrey E, Glass DR, Greenwald NF, Bharadwaj A, Khair Z, Verberk SGS, Baranski A, Baskar R, Graf W, Van Valen D, Van den Bossche J, Angelo M, Bendall SC.
      Cellular metabolism regulates immune cell activation, differentiation and effector functions, but current metabolic approaches lack single-cell resolution and simultaneous characterization of cellular phenotype. In this study, we developed an approach to characterize the metabolic regulome of single cells together with their phenotypic identity. The method, termed single-cell metabolic regulome profiling (scMEP), quantifies proteins that regulate metabolic pathway activity using high-dimensional antibody-based technologies. We employed mass cytometry (cytometry by time of flight, CyTOF) to benchmark scMEP against bulk metabolic assays by reconstructing the metabolic remodeling of in vitro-activated naive and memory CD8+ T cells. We applied the approach to clinical samples and identified tissue-restricted, metabolically repressed cytotoxic T cells in human colorectal carcinoma. Combining our method with multiplexed ion beam imaging by time of flight (MIBI-TOF), we uncovered the spatial organization of metabolic programs in human tissues, which indicated exclusion of metabolically repressed immune cells from the tumor-immune boundary. Overall, our approach enables robust approximation of metabolic and functional states in individual cells.
    DOI:  https://doi.org/10.1038/s41587-020-0651-8
  41. Cancer Res. 2020 Aug 31. pii: canres.0505.2020. [Epub ahead of print]
    Li G, Kanagasabai T, Lu W, Zou MR, Zhang SM, Celada SI, Izban MG, Liu Q, Lu T, Ballard BR, Zhou X, Adunyah SE, Matusik RJ, Yan Q, Chen Z.
      KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer (PCa). KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to PCa tumorigenesis remains unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and PCa progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of PCa in Pten-null mice, while Kdm5b loss alone caused no morphological abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared to that of Pten mice. Pathological analysis revealed Pten/Kdm5b mice displayed mild morphological changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high grade-prostatic intraepithelial neoplasia (HG-PIN) and PCa. Mechanistically, KDM5B governed phosphatidylinositol 3-kinase (PI3K)/AKT signaling in PCa in vitro and in vivo. KDM5B directly bound the PIK3CA promoter and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human PCa cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against PCa.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0505
  42. Open Biol. 2020 Sep;10(9): 200148
    Zhou L, Jilderda LJ, Foijer F.
      Aneuploidy, an irregular number of chromosomes in cells, is a hallmark feature of cancer. Aneuploidy results from chromosomal instability (CIN) and occurs in almost 90% of all tumours. While many cancers display an ongoing CIN phenotype, cells can also be aneuploid without displaying CIN. CIN drives tumour evolution as ongoing chromosomal missegregation will yield a progeny of cells with variable aneuploid karyotypes. The resulting aneuploidy is initially toxic to cells because it leads to proteotoxic and metabolic stress, cell cycle arrest, cell death, immune cell activation and further genomic instability. In order to overcome these aneuploidy-imposed stresses and adopt a malignant fate, aneuploid cancer cells must develop aneuploidy-tolerating mechanisms to cope with CIN. Aneuploidy-coping mechanisms can thus be considered as promising therapeutic targets. However, before such therapies can make it into the clinic, we first need to better understand the molecular mechanisms that are activated upon aneuploidization and the coping mechanisms that are selected for in aneuploid cancer cells. In this review, we discuss the key biological responses to aneuploidization, some of the recently uncovered aneuploidy-coping mechanisms and some strategies to exploit these in cancer therapy.
    Keywords:  aneuploidy; aneuploidy tolerance; cancer; chromosomal instability; intervention
    DOI:  https://doi.org/10.1098/rsob.200148
  43. J Cell Sci. 2020 Sep 02. pii: jcs.248492. [Epub ahead of print]
    Rawat S, Ghosh S, Mondal D, Anusha V, Raychaudhuri S.
      Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard mitochondrial proteome and function despite increased aggregation during proteasome-inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supra-organizations of respiratory complexes (RCs) in proteasome-inhibited cells simultaneous to pelletable aggregation of RC-subunits inside mitochondria. Complex-II (CII) and CV-subunits are increasingly incorporated into oligomers. CI, CIII and CIV-subunits are engaged into supercomplex formation. We unravel unique quinary-states of supercomplexes at early-stress that exhibit plasticity and inequivalence of constituent RCs. Core stoichiometry of CI and CIII is preserved whereas CIV-composition varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC-subunits into holocomplex and supercomplexes re-establish steady-state stoichiometry. Overall, the mechanism of increased supra-organization of RCs mimics the cooperative unfolding and folding pathways for protein-folding, restricted to RCs only and not observed for any other mitochondrial protein complexes.
    Keywords:  Increased supercomplex; Multistep proteome remodelling; Proteostasis; Quinary supercomplex; Respiratory complex biogenesis; Two-dimensional complexome profiling
    DOI:  https://doi.org/10.1242/jcs.248492
  44. JCI Insight. 2020 Sep 03. pii: 136215. [Epub ahead of print]5(17):
    Li Y, Su X, Rohatgi N, Zhang Y, Brestoff JR, Shoghi KI, Xu Y, Semenkovich CF, Harris CA, Peterson LL, Weibaecher KN, Teitelbaum SL, Zou W.
      Obesity predisposes to cancer and a virtual universality of nonalcoholic fatty liver disease (NAFLD). However, the impact of hepatic steatosis on liver metastasis is enigmatic. We find that while control mice were relatively resistant to hepatic metastasis, those which were lipodystrophic or obese, with NAFLD, had a dramatic increase in breast cancer and melanoma liver metastases. NAFLD promotes liver metastasis by reciprocal activation initiated by tumor-induced triglyceride lipolysis in juxtaposed hepatocytes. The lipolytic products are transferred to cancer cells via fatty acid transporter protein 1, where they are metabolized by mitochondrial oxidation to promote tumor growth. The histology of human liver metastasis indicated the same occurs in humans. Furthermore, comparison of isolates of normal and fatty liver established that steatotic lipids had enhanced tumor-stimulating capacity. Normalization of glucose metabolism by metformin did not reduce steatosis-induced metastasis, establishing the process is not mediated by the metabolic syndrome. Alternatively, eradication of NAFLD in lipodystrophic mice by adipose tissue transplantation reduced breast cancer metastasis to that of control mice, indicating the steatosis-induced predisposition is reversible.
    Keywords:  Cancer; Hepatology; Obesity; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.136215
  45. Epigenomics. 2020 Sep 01.
    Huang BZ, Binder AM, Sugar CA, Chao CR, Setiawan VW, Zhang ZF.
      Aim: Given the immunosuppressive nature of pancreatic cancer, we investigated the relationship between epigenetic modification of immune-regulatory cytokine genes and pancreatic cancer outcomes. Materials & methods: We evaluated DNA methylation of 184 pancreatic tumor samples from The Cancer Genome Atlas for 111 CpG loci in seven cytokine genes: IL10, IL6, IL8, TGFβ1, TGFβ2, TGFβ3 and TNF. We used Cox regression to evaluate the associations between methylation and overall survival, disease-specific survival and disease progression (α = 0.05). Results: Poorer survival was associated with increased methylation in fifteen CpG probes in TGFβ1, TGFβ2, TGFβ3 and TNF. We also detected improved outcomes for three loci in IL10, IL8 and IL6. Conclusion: Epigenetic regulation of cytokine-related gene expression may be associated with pancreatic cancer outcomes.
    Keywords:  cytokines; epigenetics; immune system; methylation; pancreatic cancer; progression; survival
    DOI:  https://doi.org/10.2217/epi-2019-0335
  46. ACS Cent Sci. 2020 Aug 26. 6(8): 1367-1375
    Bond MJ, Chu L, Nalawansha DA, Li K, Crews CM.
      KRAS is mutated in ∼20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered "undruggable." The discovery of potent covalent inhibitors of the KRASG12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.
    DOI:  https://doi.org/10.1021/acscentsci.0c00411
  47. Nat Rev Mol Cell Biol. 2020 Sep 01.
    Bedoui S, Herold MJ, Strasser A.
      The removal of functionally dispensable, infected or potentially neoplastic cells is driven by programmed cell death (PCD) pathways, highlighting their important roles in homeostasis, host defence against pathogens, cancer and a range of other pathologies. Several types of PCD pathways have been described, including apoptosis, necroptosis and pyroptosis; they employ distinct molecular and cellular processes and differ in their outcomes, such as the capacity to trigger inflammatory responses. Recent genetic and biochemical studies have revealed remarkable flexibility in the use of these PCD pathways and indicate a considerable degree of plasticity in their molecular regulation; for example, despite having a primary role in inducing pyroptosis, inflammatory caspases can also induce apoptosis, and conversely, apoptotic stimuli can trigger pyroptosis. Intriguingly, this flexibility is most pronounced in cellular responses to infection, while apoptosis is the dominant cell death process through which organisms prevent the development of cancer. In this Review, we summarize the mechanisms of the different types of PCD and describe the physiological and pathological processes that engage crosstalk between these pathways, focusing on infections and cancer. We discuss the intriguing notion that the different types of PCD could be seen as a single, coordinated cell death system, in which the individual pathways are highly interconnected and can flexibly compensate for one another.
    DOI:  https://doi.org/10.1038/s41580-020-0270-8
  48. J Cell Sci. 2020 Sep 01. pii: jcs242487. [Epub ahead of print]133(16):
    Riggs CL, Kedersha N, Ivanov P, Anderson P.
      Stress granules (SGs) and processing bodies (PBs) are membraneless ribonucleoprotein-based cellular compartments that assemble in response to stress. SGs and PBs form through liquid-liquid phase separation that is driven by high local concentrations of key proteins and RNAs, both of which dynamically shuttle between the granules and the cytoplasm. SGs uniquely contain certain translation initiation factors and PBs are uniquely enriched with factors related to mRNA degradation and decay, although recent analyses reveal much broader protein commonality between these granules. Despite detailed knowledge of their composition and dynamics, the function of SGs and PBs remains poorly understood. Both, however, contain mRNAs, implicating their assembly in the regulation of RNA metabolism. SGs may also serve as hubs that rewire signaling events during stress. By contrast, PBs may constitute RNA storage centers, independent of mRNA decay. The aberrant assembly or disassembly of these granules has pathological implications in cancer, viral infection and neurodegeneration. Here, we review the current concepts regarding the formation, composition, dynamics, function and involvement in disease of SGs and PBs.
    Keywords:  Cell stress; P bodies; Protein synthesis; RNA metabolism; Stress Granules
    DOI:  https://doi.org/10.1242/jcs.242487
  49. Sci Transl Med. 2020 Sep 02. pii: eaaz8048. [Epub ahead of print]12(559):
    Montgomery MK, Bayliss J, Devereux C, Bezawork-Geleta A, Roberts D, Huang C, Schittenhelm RB, Ryan A, Townley SL, Selth LA, Biden TJ, Steinberg GR, Samocha-Bonet D, Meex RCR, Watt MJ.
      Intertissue communication is a fundamental feature of metabolic regulation, and the liver is central to this process. We have identified sparc-related modular calcium-binding protein 1 (SMOC1) as a glucose-responsive hepatokine and regulator of glucose homeostasis. Acute intraperitoneal administration of SMOC1 improved glycemic control and insulin sensitivity in mice without changes in insulin secretion. SMOC1 exerted its favorable glycemic effects by inhibiting adenosine 3',5'-cyclic monophosphate (cAMP)-cAMP-dependent protein kinase (PKA)-cAMP response element-binding protein (CREB) signaling in the liver, leading to decreased gluconeogenic gene expression and suppression of hepatic glucose output. Overexpression of SMOC1 in the liver or once-weekly intraperitoneal injections of a stabilized SMOC1-FC fusion protein induced durable improvements in glucose tolerance and insulin sensitivity in db/db mice, without adverse effects on adiposity, liver histopathology, or inflammation. Furthermore, circulating SMOC1 correlated with hepatic and systemic insulin sensitivity and was decreased in obese, insulin-resistant humans. Together, these findings identify SMOC1 as a potential pharmacological target for the management of glycemic control in type 2 diabetes.
    DOI:  https://doi.org/10.1126/scitranslmed.aaz8048
  50. Cancers (Basel). 2020 Aug 31. pii: E2463. [Epub ahead of print]12(9):
    Barbeau LMO, Keulers TGH, Rouschop KMA.
      Recent advances in cancer treatment modalities reveal the limitations of the prevalent "one-size-fits-all" therapies and emphasize the necessity to develop personalized approaches. In this perspective, identification of predictive biomarkers and intrinsic vulnerabilities are an important advancement for further therapeutic strategies. Autophagy is an important lysosomal degradation and recycling pathway that provides energy and macromolecular precursors to maintain cellular homeostasis. Although all cells require autophagy, several genetic and/or cellular changes elevate the dependence of cancer cells on autophagy for their survival and indicates that autophagy inhibition in these tumors could provide a favorable addition to current therapies. In this context, we review the current literature on tumor (sub)types with elevated dependence on autophagy for their survival and highlight an exploitable vulnerability. We provide an inventory of microenvironmental factors, genetic alterations and therapies that may be exploited with autophagy-targeted approaches to improve efficacy of conventional anti-tumor therapies.
    Keywords:  autophagy-addiction; biomarkers; cancer; personalized medicine
    DOI:  https://doi.org/10.3390/cancers12092463
  51. Physiol Rep. 2020 Sep;8(17): e14547
    Grapov D, Fiehn O, Campbell C, Chandler CJ, Burnett DJ, Souza EC, Casazza GA, Keim NL, Hunter GR, Fernandez JR, Garvey WT, Hoppel CL, Harper ME, Newman JW, Adams SH.
      Very little is known about how metabolic health status, insulin resistance or metabolic challenges modulate the endocannabinoid (eCB) or polyunsaturated fatty acid (PUFA)-derived oxylipin (OxL) lipid classes. To address these questions, plasma eCB and OxL concentrations were determined at rest, 10 and 20 min during an acute exercise bout (30 min total, ~45% of preintervention V̇O2peak , ~63 W), and following 20 min recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled diet conditions. We hypothesized that increased fitness and insulin sensitivity following a ~14-week training and weight loss intervention would lead to significant changes in lipid signatures using an identical acute exercise protocol to preintervention. In the first 10 min of exercise, concentrations of a suite of OxL diols and hydroxyeicosatetraenoic acid (HETE) metabolites dropped significantly. There was no increase in 12,13-DiHOME, previously reported to increase with exercise and proposed to activate muscle fatty acid uptake and tissue metabolism. Following weight loss intervention, exercise-associated reductions were more pronounced for several linoleate and alpha-linolenate metabolites including DiHOMEs, DiHODEs, KODEs, and EpODEs, and fasting concentrations of 9,10-DiHODE, 12,13-DiHODE, and 9,10-DiHOME were reduced. These findings suggest that improved metabolic health modifies soluble epoxide hydrolase, cytochrome P450 epoxygenase (CYP), and lipoxygenase (LOX) systems. Acute exercise led to reductions for most eCB metabolites, with no evidence for concentration increases even at recovery. It is proposed that during submaximal aerobic exercise, nonoxidative fates of long-chain saturated, monounsaturated, and PUFAs are attenuated in tissues that are important contributors to the blood OxL and eCB pools.
    Keywords:  PUFA; lipoxygenase; oxylipid; polyunsaturated fatty acid; soluble epoxide hydrolase
    DOI:  https://doi.org/10.14814/phy2.14547
  52. Ann Surg Oncol. 2020 Sep 04.
    Parker NH, Basen-Engquist K, Rubin ML, Li Y, Prakash L, Ngo-Huang A, Gorzelitz J, Ikoma N, Lee JE, Katz MHG.
      BACKGROUND: We previously demonstrated associations between exercise during pancreatic cancer treatment and quality of life and physical fitness prior to pancreatectomy. In this study, we quantified exercise among survivors following pancreatic tumor resection and characterized concordance with established guidelines.METHODS: We quantified exercise frequency, duration, and intensity among survivors who underwent pancreatectomy for adenocarcinoma or a neuroendocrine tumor at our center from 2000 to 2017 and compared them with American College of Sports Medicine Guidelines for Cancer Survivors. Additional surveys measured motivation to exercise, barrier self-efficacy, quality of life, and fatigue. Multivariable models were constructed to evaluate associations between clinicodemographic and psychosocial variables and guideline concordance, and between guideline concordance and quality of life and fatigue.
    RESULTS: Of 504 eligible survivors, 262 (52%) returned surveys. Only 62 participants (24%) reported meeting both aerobic and strengthening guidelines; 103 (39%) reported meeting neither. Adjusted analyses demonstrated that higher autonomous motivation was associated with higher aerobic and strengthening guideline concordance (both p < 0.01). Higher barrier self-efficacy and older age were associated with higher aerobic guideline concordance (p < 0.01). We identified no significant associations between guideline concordance and tumor type, time since surgery, or recent cancer therapy (all p > 0.05). We found favorable associations between aerobic guideline concordance and both quality of life and fatigue (both p < 0.001).
    CONCLUSIONS: Less than one-quarter of participants exercised sufficiently to meet national exercise guidelines following pancreatectomy. To maximize exercise and related benefits, interventions should help survivors increase intrinsic motivation and overcome barriers to exercise.
    DOI:  https://doi.org/10.1245/s10434-020-09062-9
  53. Nat Cell Biol. 2020 Sep;22(9): 1049-1055
    Katikaneni A, Jelcic M, Gerlach GF, Ma Y, Overholtzer M, Niethammer P.
      Rapid wound detection by distant leukocytes is essential for antimicrobial defence and post-infection survival1. The reactive oxygen species hydrogen peroxide and the polyunsaturated fatty acid arachidonic acid are among the earliest known mediators of this process2-4. It is unknown whether or how these highly conserved cues collaborate to achieve wound detection over distances of several hundreds of micrometres within a few minutes. To investigate this, we locally applied arachidonic acid and skin-permeable peroxide by micropipette perfusion to unwounded zebrafish tail fins. As in wounds, arachidonic acid rapidly attracted leukocytes through dual oxidase (Duox) and 5-lipoxygenase (Alox5a). Peroxide promoted chemotaxis to arachidonic acid without being chemotactic on its own. Intravital biosensor imaging showed that wound peroxide and arachidonic acid converged on half-millimetre-long lipid peroxidation gradients that promoted leukocyte attraction. Our data suggest that lipid peroxidation functions as a spatial redox relay that enables long-range detection of early wound cues by immune cells, outlining a beneficial role for this otherwise toxic process.
    DOI:  https://doi.org/10.1038/s41556-020-0564-2
  54. Mol Biol Rep. 2020 Sep 03.
    Nicolaidou V, Papaneophytou C, Koufaris C.
      Through the process of alternative splicing, proteins with distinct biological functions and localisations are generated from a single gene. The mitochondrial folate metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been receiving attention in recent years as one of the most frequently upregulated metabolic enzymes across multiple tumour types. We hypothesized that alternative splicing of MTHFD2 could be a mechanism that generates novel isoforms of this enzyme, with potentially distinct and important biological functions. Multiple alternatively spliced MTHFD2 transcripts were first characterized in the UCSC and Ensemble genome browser. Subsequently, investigating the transcriptomic data for the Genotype-Tissue Expression (GTeX) project it was found that beyond the canonical MTHFD2 transcript, alternative transcripts lacking the second exon of MTHFD2 are also common. The presence of MTHFD2 transcripts lacking the second exon was confirmed by RT-PCR in normal and cancer cells. Translation of MTHFD2 transcripts lacking this second exon are predicted to generate a truncated protein lacking the first 102 N-terminal amino acids of the full-length protein, including the mitochondrial transport sequence. Hence, the truncated MTHFD2 protein could be an isoform with distinct localisation and functions. However, we were not able to confirm the generation of a stable truncated MTHFD2 protein in eukaryotic cells. This study characterizes for the first time alternative spliced transcripts of the enzyme MTHFD2, although further work is required to investigate their biological significance.
    Keywords:  Folate; Isoform; MTHFD2; Mitochondria; Nucleus; Splicing
    DOI:  https://doi.org/10.1007/s11033-020-05775-y
  55. Cancer Discov. 2020 Sep 04. pii: CD-19-1448. [Epub ahead of print]
    Wang G, Chow RD, Zhu L, Bai Z, Ye L, Zhang F, Renauer PA, Dong MB, Dai X, Zhang X, Du Y, Cheng Y, Niu L, Chu Z, Kim K, Liao C, Clark P, Errami Y, Chen S.
      Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMMs) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in cancer patients. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. Additionally, Kmt2d-mutant cells exhibit increased protein turnover and IFN-γ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human are characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.
    DOI:  https://doi.org/10.1158/2159-8290.CD-19-1448
  56. Nature. 2020 Sep 02.
    Cuylen-Haering S, Petrovic M, Hernandez-Armendariz A, Schneider MWG, Samwer M, Blaukopf C, Holt LJ, Gerlich DW.
      Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.
    DOI:  https://doi.org/10.1038/s41586-020-2672-3
  57. Elife. 2020 Sep 01. pii: e55794. [Epub ahead of print]9
    Willemsen D, Cui R, Reichard M, Valenzano DR.
      The evolutionary forces shaping life history divergence within species are largely unknown. Turquoise killifish display differences in lifespan among wild populations, representing an ideal natural experiment in evolution and diversification of life history. By combining genome sequencing and population genetics, we investigate the evolutionary forces shaping lifespan among wild turquoise killifish populations. We generate an improved reference genome assembly and identify genes under positive and purifying selection, as well as those evolving neutrally. Short-lived populations from the outer margin of the species range have small population size and accumulate deleterious mutations in genes significantly enriched in the WNT signaling pathway, neurodegeneration, cancer and the mTOR pathway. We propose that limited population size due to habitat fragmentation and repeated population bottlenecks, by increasing the genome-wide mutation load, exacerbates the effects of mutation accumulation and cumulatively contribute to the short adult lifespan.
    Keywords:  aging; evolution; evolutionary biology; genetics; genomics; lifespan; nothobranchius furzeri; turquoise killifish
    DOI:  https://doi.org/10.7554/eLife.55794
  58. Nat Commun. 2020 Sep 04. 11(1): 4423
    Rashkin SR, Graff RE, Kachuri L, Thai KK, Alexeeff SE, Blatchins MA, Cavazos TB, Corley DA, Emami NC, Hoffman JD, Jorgenson E, Kushi LH, Meyers TJ, Van Den Eeden SK, Ziv E, Habel LA, Hoffmann TJ, Sakoda LC, Witte JS.
      Deciphering the shared genetic basis of distinct cancers has the potential to elucidate carcinogenic mechanisms and inform broadly applicable risk assessment efforts. Here, we undertake genome-wide association studies (GWAS) and comprehensive evaluations of heritability and pleiotropy across 18 cancer types in two large, population-based cohorts: the UK Biobank (408,786 European ancestry individuals; 48,961 cancer cases) and the Kaiser Permanente Genetic Epidemiology Research on Adult Health and Aging cohorts (66,526 European ancestry individuals; 16,001 cancer cases). The GWAS detect 21 genome-wide significant associations independent of previously reported results. Investigations of pleiotropy identify 12 cancer pairs exhibiting either positive or negative genetic correlations; 25 pleiotropic loci; and 100 independent pleiotropic variants, many of which are regulatory elements and/or influence cross-tissue gene expression. Our findings demonstrate widespread pleiotropy and offer further insight into the complex genetic architecture of cross-cancer susceptibility.
    DOI:  https://doi.org/10.1038/s41467-020-18246-6
  59. Nat Rev Drug Discov. 2020 Sep 03.
    Curtin NJ, Szabo C.
      The process of poly(ADP-ribosyl)ation and the major enzyme that catalyses this reaction, poly(ADP-ribose) polymerase 1 (PARP1), were discovered more than 50 years ago. Since then, advances in our understanding of the roles of PARP1 in cellular processes such as DNA repair, gene transcription and cell death have allowed the investigation of therapeutic PARP inhibition for a variety of diseases - particularly cancers in which defects in DNA repair pathways make tumour cells highly sensitive to the inhibition of PARP activity. Efforts to identify and evaluate potent PARP inhibitors have so far led to the regulatory approval of four PARP inhibitors for the treatment of several types of cancer, and PARP inhibitors have also shown therapeutic potential in treating non-oncological diseases. This Review provides a timeline of PARP biology and medicinal chemistry, summarizes the pathophysiological processes in which PARP plays a role and highlights key opportunities and challenges in the field, such as counteracting PARP inhibitor resistance during cancer therapy and repurposing PARP inhibitors for the treatment of non-oncological diseases.
    DOI:  https://doi.org/10.1038/s41573-020-0076-6
  60. Autophagy. 2020 Aug 31.
    Zhao D, Du LL.
      The endoplasmic reticulum (ER) is a major site of protein folding. Perturbations in the folding capacity of the ER result in ER stress. ER stress triggers autophagic degradation of the ER (reticulophagy). Molecular mechanisms underlying ER stress-induced reticulophagy remain largely unknown. Our recent study identified a soluble protein, Epr1, as an autophagy receptor for ER stress-induced reticulophagy in the fission yeast Schizosaccharomyces pombe. Epr1 can interact simultaneously with Atg8 and a VAP family integral ER membrane protein, and thereby act as a bridging molecule between them. VAP family proteins contribute to reticulophagy by not only connecting Atg8 to the ER membrane through Epr1, but also by supporting the ER-plasma membrane contact. The expression of Epr1 is upregulated during ER stress in a manner dependent on the unfolded protein response (UPR) regulator Ire1. Ire1 promotes reticulophagy by upregulating Epr1.
    Keywords:  ER stress; ER-phagy; ER-plasma membrane contact; Ire1; VAP; reticulophagy; selective autophagy; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1080/15548627.2020.1816665
  61. Aging Cell. 2020 Sep 04. e13225
    Bhaskaran S, Pollock N, C Macpherson P, Ahn B, Piekarz KM, Staunton CA, Brown JL, Qaisar R, Vasilaki A, Richardson A, McArdle A, Jackson MJ, Brooks SV, Van Remmen H.
      Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1-/- mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit.
    Keywords:  CuZnSOD; aging; denervation; motor neuron; sarcopenia
    DOI:  https://doi.org/10.1111/acel.13225
  62. Dev Cell. 2020 Sep 01. pii: S1534-5807(20)30599-2. [Epub ahead of print]
    Zhan H, Bhattacharya S, Cai H, Iglesias PA, Huang CH, Devreotes PN.
      The Ras/PI3K/extracellular signal-regulated kinases (ERK) signaling network plays fundamental roles in cell growth, survival, and migration and is frequently activated in cancer. Here, we show that the activities of the signaling network propagate as coordinated waves, biased by growth factor, which drive actin-based protrusions in human epithelial cells. The network exhibits hallmarks of biochemical excitability: the annihilation of oppositely directed waves, all-or-none responsiveness, and refractoriness. Abrupt perturbations to Ras, PI(4,5)P2, PI(3,4)P2, ERK, and TORC2 alter the threshold, observations that define positive and negative feedback loops within the network. Oncogenic transformation dramatically increases the wave activity, the frequency of ERK pulses, and the sensitivity to EGF stimuli. Wave activity was progressively enhanced across a series of increasingly metastatic breast cancer cell lines. The view that oncogenic transformation is a shift to a lower threshold of excitable Ras/PI3K/ERK network, caused by various combinations of genetic insults, can facilitate the assessment of cancer severity and effectiveness of interventions.
    Keywords:  ERK; PI(3,4)P2; PI(4,5)P2; PI3K; Ras; excitability; oncogenic transformation; threshold; wave
    DOI:  https://doi.org/10.1016/j.devcel.2020.08.001
  63. Small GTPases. 2020 Sep 03. 1-12
    Mysior MM, Simpson JC.
      Rho GTPases are known to play an essential role in fundamental processes such as defining cell shape, polarity and migration. As such, the majority of Rho GTPases localize and function at, or close to, the plasma membrane. However, it is becoming increasingly clear that a number of Rho family proteins are also associated with the Golgi complex, where they not only regulate events at this organelle but also more widely across the cell. Given the central location of this organelle, and the numerous membrane trafficking pathways that connect it to both the endocytic and secretory systems of cells, it is clear that the Golgi is fundamental for maintaining cellular homoeostasis. In this review, we describe these GTPases in the context of how they regulate Golgi architecture, membrane trafficking into and away from this organelle, and cell polarity and migration. We summarize the key findings that show the growing importance of the pool of Rho GTPases associated with Golgi function, namely Cdc42, RhoA, RhoD, RhoBTB1 and RhoBTB3, and we discuss how they act in concert with other key families of molecules associated with the Golgi, including Rab GTPases and matrix proteins.
    Keywords:  Cdc42; GAP; GEF; Golgi complex; Rho GTPase; RhoA; RhoBTB1; RhoBTB3; RhoD; cell polarity
    DOI:  https://doi.org/10.1080/21541248.2020.1812873
  64. Mol Cancer Ther. 2020 Sep 02. pii: molcanther.0423.2020. [Epub ahead of print]
    Dekhne AS, Hou Z, Gangjee A, Matherly LH.
      One-carbon (1C) metabolism encompasses folate-mediated 1C transfer reactions and related processes, including nucleotide and amino acid biosynthesis, antioxidant regeneration, and epigenetic regulation. 1C pathways are compartmentalized in the cytosol, mitochondria and nucleus. 1C metabolism in the cytosol has been an important therapeutic target for cancer since the inception of modern chemotherapy and "antifolates" targeting cytosolic 1C pathways continue to be a mainstay of the chemotherapy armamentarium for cancer. Recent insights into the complexities of 1C metabolism in cancer cells, including the critical role of the mitochondrial 1C pathway as a source of 1C units, glycine, reducing equivalents, and ATP, have spurred the discovery of novel compounds that target these reactions, with particular focus on 5,10-methylene tetrahydrofolate dehydrogenase 2 and serine hydroxymethyltransferase 2. In this review, we discuss key aspects of 1C metabolism, with emphasis on the importance of mitochondrial 1C metabolism to metabolic homeostasis, and its relationship to the oncogenic phenotype and therapeutic potential for cancer.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-20-0423
  65. Cancer Res. 2020 Aug 31. pii: canres.0789.2020. [Epub ahead of print]
    Chamberlin T, Clack M, Silvers C, Kuziel G, Thompson V, Johnson H, Arendt LM.
      Obesity enhances breast cancer risk in postmenopausal women and premenopausal women with genetic or familial risk factors. We have shown previously that within breast tissue, obesity increases macrophage-driven inflammation and promotes expansion of luminal epithelial cell populations which are hypothesized to be the cells of origin for the most common subtypes of breast cancer. However, it is not clear how these changes within the microenvironment of the breast alter cancer risk and tumor growth. Using a high fat diet to induce obesity, we examined preneoplastic changes associated with epithelial cell-specific loss of Trp53. Obesity significantly enhanced the incidence of tumors of diverse histotypes and increased stromal cells within the tumor microenvironment. Obesity also promoted the growth of preneoplastic lesions containing elevated numbers of luminal epithelial progenitor cells, which were surrounded by macrophages. To understand how macrophage-driven inflammation due to obesity enhances tumor formation, mice were treated with IgG or anti-F4/80 antibodies to deplete macrophages during preneoplastic growth. Unexpectedly, depletion of macrophages in obese mice enhanced mammary epithelial cell stem/progenitor activity, elevated expression of estrogen receptor alpha, and increased DNA damage in cells. Together, these results suggest that in obesity, macrophages reduce epithelial cells with DNA damage, which may limit the progression of preneoplastic breast lesions, and uncovers complex macrophage function within the evolving tumor microenvironment. Understanding how obesity alters the function of macrophages during tumor formation may lead to chemoprevention options for at-risk obese women.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0789
  66. FASEB J. 2020 Sep 03.
    Abid H, Ryan ZC, Delmotte P, Sieck GC, Lanza IR.
      Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.
    Keywords:  STAT3; interleukin-6; mitochondria; reactive oxygen species; skeletal muscle
    DOI:  https://doi.org/10.1096/fj.202000965RR
  67. Nature. 2020 Sep 02.
    Morehouse BR, Govande AA, Millman A, Keszei AFA, Lowey B, Ofir G, Shao S, Sorek R, Kranzusch PJ.
      Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides released during bacterial infection and endogenous cyclic GMP-AMP signalling during viral infection and antitumour immunity1-5. STING shares no structural homology with other known signalling proteins6-9, limiting functional analysis and preventing explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we discover functional STING homologues encoded within prokaryotic defence islands and reveal a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to c-di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple cyclic dinucleotide recognition with protein filament formation to drive TIR effector domain oligomerization and rapid NAD+ cleavage. We reconstruct the evolutionary events following acquisition of STING into metazoan innate immunity and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal conservation of a functional cGAS-STING pathway in prokaryotic bacteriophage defence.
    DOI:  https://doi.org/10.1038/s41586-020-2719-5
  68. Mol Aspects Med. 2020 Aug 29. pii: S0098-2997(20)30095-9. [Epub ahead of print] 100893
    Serhan CN, Gupta SK, Perretti M, Godson C, Brennan E, Li Y, Soehnlein O, Shimizu T, Werz O, Chiurchiù V, Azzi A, Dubourdeau M, Gupta SS, Schopohl P, Hoch M, Gjorgevikj D, Khan FM, Brauer D, Tripathi A, Cesnulevicius K, Lescheid D, Schultz M, Särndahl E, Repsilber D, Kruse R, Sala A, Haeggström JZ, Levy BD, Filep JG, Wolkenhauer O.
      Acute inflammation is a protective reaction by the immune system in response to invading pathogens or tissue damage. Ideally, the response should be localized, self-limited, and returning to homeostasis. If not resolved, acute inflammation can result in organ pathologies leading to chronic inflammatory phenotypes. Acute inflammation and inflammation resolution are complex coordinated processes, involving a number of cell types, interacting in space and time. The biomolecular complexity and the fact that several biomedical fields are involved, make a multi- and interdisciplinary approach necessary. The Atlas of Inflammation Resolution (AIR) is a web-based resource capturing an essential part of the state-of-the-art in acute inflammation and inflammation resolution research. The AIR provides an interface for users to search thousands of interactions, arranged in inter-connected multi-layers of process diagrams, covering a wide range of clinically relevant phenotypes. By mapping experimental data onto the AIR, it can be used to elucidate drug action as well as molecular mechanisms underlying different disease phenotypes. For the visualization and exploration of information, the AIR uses the Minerva platform, which is a well-established tool for the presentation of disease maps. The molecular details of the AIR are encoded using international standards. The AIR was created as a freely accessible resource, supporting research and education in the fields of acute inflammation and inflammation resolution. The AIR connects research communities, facilitates clinical decision making, and supports research scientists in the formulation and validation of hypotheses. The AIR is accessible through https://air.bio.informatik.uni-rostock.de.
    Keywords:  Acute inflammation; Disease map; Inflammation resolution; Inflammatory mediators; Molecular interaction map; Molecular switches; Pro-resolving mediators; Systems biology
    DOI:  https://doi.org/10.1016/j.mam.2020.100893
  69. J Clin Invest. 2020 Aug 31. pii: 133283. [Epub ahead of print]
    Zhu M, Deng G, Tan P, Xing C, Guan C, Jiang C, Zhang Y, Ning B, Li C, Yin B, Chen K, Zhao Y, Wang HY, Levine B, Nie G, Wang RF.
      Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.
    Keywords:  Autophagy; Cell Biology; Inflammation; Innate immunity
    DOI:  https://doi.org/10.1172/JCI133283
  70. J Cell Biol. 2020 Oct 05. pii: e202006196. [Epub ahead of print]219(10):
    Ozawa M, Hiver S, Yamamoto T, Shibata T, Upadhyayula S, Mimori-Kiyosue Y, Takeichi M.
      Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia form and found that they sporadically grew from around E-cadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also support c-lamellipodia formation by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.
    DOI:  https://doi.org/10.1083/jcb.202006196
  71. J Clin Invest. 2020 Sep 01. pii: 137554. [Epub ahead of print]
    Hatfield SM, Sitkovsky MV.
      Hypoxia/HIF-1α- and extracellular adenosine/A2-adenosine receptor-mediated immunosuppression protects tissues from collateral damage by anti-pathogen immune cells. However, this mechanism also protects cancerous tissues by inhibiting anti-tumor immune cells in hypoxic and extracellular adenosine-rich tumors that are the most resistant to current therapies. Here, we explain a conceptually novel, anti-immunosuppressive reasoning to justify strategies using respiratory hyperoxia and oxygenation agents in cancer treatment. Earlier attempts to use oxygenation of tumors as a monotherapy or to improve radiotherapy have failed because oxygenation protocols were not combined with immunotherapies of cancer. In contrast, the proposal for therapeutic use of anti-hypoxic oxygenation described here was motivated by the need to prevent the hypoxia/HIF-1α-driven accumulation of extracellular adenosine to (i) unleash anti-tumor immune cells from inhibition by intracellular cAMP and (ii) prevent immunosuppressive transcription of cAMP response element- and hypoxia response element-containing immunosuppressive gene products (e.g. TGF-β. Using oxygenation agents together with inhibitors of the A2A adenosine receptor may be required to enable the most effective cancer immunotherapy. The emerging outcomes from clinical trials of cancer patients refractory to all other treatments provide support for the molecular and immunological mechanism-based approach to cancer immunotherapy described here.
    DOI:  https://doi.org/10.1172/JCI137554
  72. Science. 2020 Sep 04. 369(6508): 1160-1161
    Hori M.
      
    DOI:  https://doi.org/10.1126/science.abb9186
  73. Cell Rep. 2020 Sep 01. pii: S2211-1247(20)31065-2. [Epub ahead of print]32(9): 108080
    Chen J, Harding SM, Natesan R, Tian L, Benci JL, Li W, Minn AJ, Asangani IA, Greenberg RA.
      The DNA-dependent pattern recognition receptor, cGAS (cyclic GMP-AMP synthase), mediates communication between the DNA damage and the immune responses. Mitotic chromosome missegregation stimulates cGAS activity; however, it is unclear whether progression through mitosis is required for cancercell-intrinsic activation of anti-tumor immune responses. Moreover, it is unknown whether cell cycle checkpoint disruption can restore responses in cancer cells that are recalcitrant to DNAdamage-induced inflammation. Here, we demonstrate that prolonged cell cycle arrest at the G2-mitosis boundary from either excessive DNA damage or CDK1 inhibition prevents inflammatory-stimulated gene expression and immune-mediated destruction of distal tumors. Remarkably, DNAdamage-induced inflammatory signaling is restored in a RIG-I-dependent manner upon concomitant disruption of p53 and the G2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage-associated molecular pattern recognition and have implications for the design of rational approaches to augment anti-tumor immune responses.
    Keywords:  ATR; DNA damage; RIG-I; anti-tumor immune response; cGAS; cell cycle checkpoint; inflammatory signaling; p53
    DOI:  https://doi.org/10.1016/j.celrep.2020.108080
  74. J Cell Biol. 2020 Nov 02. pii: e201910149. [Epub ahead of print]219(11):
    Baumann C, Zhang X, De La Fuente R.
      The polycomb group protein CBX2 is an important epigenetic reader involved in cell proliferation and differentiation. While CBX2 overexpression occurs in a wide range of human tumors, targeted deletion results in homeotic transformation, proliferative defects, and premature senescence. However, its cellular function(s) and whether it plays a role in maintenance of genome stability remain to be determined. Here, we demonstrate that loss of CBX2 in mouse fibroblasts induces abnormal large-scale chromatin structure and chromosome instability. Integrative transcriptome analysis and ATAC-seq revealed a significant dysregulation of transcripts involved in DNA repair, chromocenter formation, and tumorigenesis in addition to changes in chromatin accessibility of genes involved in lateral sclerosis, basal transcription factors, and folate metabolism. Notably, Cbx2-/- cells exhibit prominent decondensation of satellite DNA sequences at metaphase and increased sister chromatid recombination events leading to rampant chromosome instability. The presence of extensive centromere and telomere defects suggests a prominent role for CBX2 in heterochromatin homeostasis and the regulation of nuclear architecture.
    DOI:  https://doi.org/10.1083/jcb.201910149
  75. Neoplasma. 2020 Sep 04. pii: 200408N372. [Epub ahead of print]
    Eid M, Ostřížková L, Kunovský L, Brančíková D, Kala Z, Hlavsa J, Janeček P, Kosíková I, Blažková M, Slabý O, Mayer J.
      Pancreatic ductal adenocarcinoma (PDAC) is now the 11th most common cancer and in 2018 there were 458,918 new cases worldwide. In the Czech Republic, a total of 2,173 patients were diagnosed in 2015, ranking the second in incidence worldwide. In contrast to other malignancies, recent research has not brought any major breakthrough in the treatment of PDAC and hence the prognosis remains very serious. Radical resection is the only curative approach, but after the initiation of the standard pathological evaluation of the resected tissue, according to the Leeds protocol, 80% of the resections are R1 (resections with microscopically positive margins). The results of studies in patients with borderline resectable or locally advanced PDAC prefer neoadjuvant chemotherapy or chemoradiotherapy. This approach leads to a higher number of radical R0 resections and better survival. For neoadjuvant treatment in patients with primarily resectable PDAC, most results come from retrospective analysis or phase II trials. However, recently, data from three randomized clinical trials with neoadjuvant therapy for resectable PDAC were presented. These results support the use of chemotherapy or chemoradiotherapy prior to surgery. In the trials published to date, there are differences in chemotherapeutic regimens, cytostatic doses, and the definition of resectability. Thus, up-front resection with adjuvant chemotherapy is still the standard of care and a well-designed randomized trial using neoadjuvant therapy is now necessary.
    DOI:  https://doi.org/10.4149/neo_2020_200408N372
  76. Proc Natl Acad Sci U S A. 2020 Sep 03. pii: 202008789. [Epub ahead of print]
    Kelley EG, Butler PD, Ashkar R, Bradbury R, Nagao M.
      The elastic and viscous properties of biological membranes play a vital role in controlling cell functions that require local reorganization of the membrane components as well as dramatic shape changes such as endocytosis, vesicular trafficking, and cell division. These properties are widely acknowledged to depend on the unique composition of lipids within the membrane, yet the effects of lipid mixing on the membrane biophysical properties remain poorly understood. Here, we present a comprehensive characterization of the structural, elastic, and viscous properties of fluid membranes composed of binary mixtures of lipids with different tail lengths. We show that the mixed lipid membrane properties are not simply additive quantities of the single-component analogs. Instead, the mixed membranes are more dynamic than either of their constituents, quantified as a decrease in their bending modulus, area compressibility modulus, and viscosity. While the enhanced dynamics are seemingly unexpected, we show that the measured moduli and viscosity for both the mixed and single-component bilayers all scale with the area per lipid and collapse onto respective master curves. This scaling links the increase in dynamics to mixing-induced changes in the lipid packing and membrane structure. More importantly, the results show that the membrane properties can be manipulated through lipid composition the same way bimodal blends of surfactants, liquid crystals, and polymers are used to engineer the mechanical properties of soft materials, with broad implications for understanding how lipid diversity relates to biomembrane function.
    Keywords:  elasticity; phospholipid membrane; structure; viscosity
    DOI:  https://doi.org/10.1073/pnas.2008789117
  77. Mol Cell. 2020 Aug 19. pii: S1097-2765(20)30550-5. [Epub ahead of print]
    Ong JY, Torres JZ.
      Cell division requires the assembly and organization of a microtubule spindle for the proper separation of chromosomes in mitosis and meiosis. Phase separation is an emerging paradigm for understanding spatial and temporal regulation of a variety of cellular processes, including cell division. Phase-separated condensates have been recently discovered at many structures during cell division as a possible mechanism for properly localizing, organizing, and activating proteins involved in cell division. Here, we review how these condensates play roles in regulating microtubule density and organization and spindle assembly and function and in activating some of the key players in cell division. We conclude with perspectives on areas of future research for this exciting and rapidly advancing field.
    Keywords:  cell division; membraneless compartments; microtubule-based spindle; phase separation; phase-separated condensates
    DOI:  https://doi.org/10.1016/j.molcel.2020.08.007
  78. Mol Biol Cell. 2020 Sep 02. mbcE18010013
    Brown M, Dainty S, Strudwick N, Mihai AD, Watson JN, Dendooven R, Paton AW, Paton JC, Schröder M.
      Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signalling network known as the unfolded protein response (UPR). Here we characterise how ER stress and the UPR inhibit insulin signalling. We find that ER stress inhibits insulin signalling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesised insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signalling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible FV2E dimerisation domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signalling events in the UPR, such as activation of the JNK MAP kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signalling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesised insulin receptors in ER-stressed cells.
    DOI:  https://doi.org/10.1091/mbc.E18-01-0013
  79. Mol Cell. 2020 Aug 27. pii: S1097-2765(20)30553-0. [Epub ahead of print]
    Huang YH, Hilal T, Loll B, Bürger J, Mielke T, Böttcher C, Said N, Wahl MC.
      Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.
    Keywords:  Nus factors; RNA polymerase pausing and backtracking, RNA chaperone; co-transcriptional RNA folding and processing; ribosomal RNA transcription; single-particle cryo-electron microscopy; structural biology; transcription (anti-)termination; transcription regulation
    DOI:  https://doi.org/10.1016/j.molcel.2020.08.010
  80. Mol Cancer Res. 2020 Sep 01. pii: molcanres.0439.2020. [Epub ahead of print]
    Honselmann KC, Finetti P, Birnbaum DJ, Monsalve CS, Wellner UF, Begg SKS, Nakagawa A, Hank T, Li A, Goldsworthy MA, Sharma H, Bertucci F, Birnbaum D, Tai E, Ligorio M, Ting DT, Schilling O, Biniossek ML, Bronsert P, Ferrone CR, Keck T, Mino-Kenudson M, Lillemoe KD, Warshaw AL, Fernandez-Del Castillo C, Liss AS.
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic reaction, warranting intense cancer-stroma communication. In this study we interrogated the contribution of the BET family of chromatin adaptors to the crosstalk between PDAC cells and the tumor stroma. Short-term treatment of orthotopic xenograft tumors with CPI203, a small molecule inhibitor of BET proteins, resulted in broad changes in the expression of genes encoding components of the extracellular matrix (matrisome) in both cancer and stromal cells. Remarkably, more than half of matrisome genes were expressed by cancer cells. In vitro co-cultures of PDAC cells and cancer associated fibroblasts (CAFs) demonstrated that matrisome expression was regulated by BET-dependent cancer-CAF crosstalk. Disrupting this crosstalk in vivo resulted in diminished growth of orthotopic patient-derived xenograft tumors, reduced proliferation of cancer cells, and changes in collagen structure consistent with that of patients who experienced better survival. Examination of matrisome gene expression in publicly available data sets of 573 PDAC tumors identified a 65-gene signature that was able to distinguish long- and short-term PDAC survivors. Importantly, the expression of genes predictive of short-term survival was diminished in the cancer cells of orthotopic xenograft tumors of mice treated with CPI203. Taken together, these results demonstrate that inhibiting the activity BET proteins results in transcriptional and structural differences in the matrisome are associated with better patient survival. Implications: These studies highlight the biological relevance of the matrisome program in PDAC and suggest targeting of epigenetically driven tumor-stroma crosstalk as a potential therapeutic avenue.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0439
  81. J Exp Med. 2020 Dec 07. pii: e20200261. [Epub ahead of print]217(12):
    Law EK, Levin-Klein R, Jarvis MC, Kim H, Argyris PP, Carpenter MA, Starrett GJ, Temiz NA, Larson LK, Durfee C, Burns MB, Vogel RI, Stavrou S, Aguilera AN, Wagner S, Largaespada DA, Starr TK, Ross SR, Harris RS.
      The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.
    DOI:  https://doi.org/10.1084/jem.20200261
  82. Cell Syst. 2020 Apr 22. 10(4): 323-332.e8
    Satas G, Zaccaria S, Mon G, Raphael BJ.
      A small number of somatic mutations drive the development of cancer, but all somatic mutations are markers of the evolutionary history of a tumor. Prominent methods to construct phylogenies from single-cell sequencing data use single-nucleotide variants (SNVs) as markers but fail to adequately account for copy-number aberrations (CNAs), which can overlap SNVs and result in SNV losses. Here, we introduce SCARLET, an algorithm that infers tumor phylogenies from single-cell DNA sequencing data while accounting for both CNA-driven loss of SNVs and sequencing errors. SCARLET outperforms existing methods on simulated data, with more accurate inference of the order in which mutations were acquired and the mutations present in individual cells. Using a single-cell dataset from a patient with colorectal cancer, SCARLET constructs a tumor phylogeny that is consistent with the observed CNAs and suggests an alternate origin for the patient's metastases. SCARLET is available at: github.com/raphael-group/scarlet.
    DOI:  https://doi.org/10.1016/j.cels.2020.04.001
  83. Nat Commun. 2020 Sep 02. 11(1): 4411
    Hablitz LM, Plá V, Giannetto M, Vinitsky HS, Stæger FF, Metcalfe T, Nguyen R, Benrais A, Nedergaard M.
      The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on time-of-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm.
    DOI:  https://doi.org/10.1038/s41467-020-18115-2
  84. Nat Genet. 2020 Sep;52(9): 898-907
    Caravagna G, Heide T, Williams MJ, Zapata L, Nichol D, Chkhaidze K, Cross W, Cresswell GD, Werner B, Acar A, Chesler L, Barnes CP, Sanguinetti G, Graham TA, Sottoriva A.
      Most cancer genomic data are generated from bulk samples composed of mixtures of cancer subpopulations, as well as normal cells. Subclonal reconstruction methods based on machine learning aim to separate those subpopulations in a sample and infer their evolutionary history. However, current approaches are entirely data driven and agnostic to evolutionary theory. We demonstrate that systematic errors occur in the analysis if evolution is not accounted for, and this is exacerbated with multi-sampling of the same tumor. We present a novel approach for model-based tumor subclonal reconstruction, called MOBSTER, which combines machine learning with theoretical population genetics. Using public whole-genome sequencing data from 2,606 samples from different cohorts, new data and synthetic validation, we show that this method is more robust and accurate than current techniques in single-sample, multiregion and longitudinal data. This approach minimizes the confounding factors of nonevolutionary methods, thus leading to more accurate recovery of the evolutionary history of human cancers.
    DOI:  https://doi.org/10.1038/s41588-020-0675-5
  85. Cell Metab. 2020 Sep 01. pii: S1550-4131(20)30412-5. [Epub ahead of print]32(3): 479-497.e9
    Antonicka H, Lin ZY, Janer A, Aaltonen MJ, Weraarpachai W, Gingras AC, Shoubridge EA.
      We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
    Keywords:  BioID proximity interactions; functional modules; mitochondrial protein proximity map; mitochondrial translation initiation; organellar contact sites; sub-mitochondrial organization
    DOI:  https://doi.org/10.1016/j.cmet.2020.07.017
  86. Trends Cell Biol. 2020 Aug 29. pii: S0962-8924(20)30149-5. [Epub ahead of print]
    Shellard A, Mayor R.
      Directional cell migration normally relies on a variety of external signals, such as chemical, mechanical, or electrical, which instruct cells in which direction to move. Many of the major molecular and physical effects derived from these cues are now understood, leading to questions about whether directional cell migration is alike or distinct under these different signals, and how cells might be directed by multiple simultaneous cues, which would be expected in complex in vivo environments. In this review, we compare how different stimuli are spatially distributed, often as gradients, to direct cell movement and the mechanisms by which they steer cells. A comparison of the downstream effectors of directional cues suggests that different external signals regulate a common set of components: small GTPases and the actin cytoskeleton, which implies that the mechanisms downstream of different signals are likely to be closely related and underlies the idea that cell migration operates by a common set of physical principles, irrespective of the input.
    Keywords:  chemotaxis; durotaxis; galvanotaxis; gradients; haptotaxis; migration
    DOI:  https://doi.org/10.1016/j.tcb.2020.08.002
  87. Elife. 2020 Sep 04. pii: e57837. [Epub ahead of print]9
    Tao L, Lemoff A, Wang G, Zarek C, Lowe A, Yan N, Reese TA.
      Reactive oxygen species (ROS) are by-products of cellular respiration that can promote oxidative stress and damage cellular proteins and lipids. One canonical role of ROS is to defend the cell against invading bacterial and viral pathogens. Curiously, some viruses, including herpesviruses, thrive despite the induction of ROS, suggesting that ROS are beneficial for the virus. However, the underlying mechanisms remain unclear. Here, we found that ROS impaired interferon response during murine herpesvirus infection and that the inhibition occurred downstream of cytoplasmic DNA sensing. We further demonstrated that ROS suppressed the type I interferon response by oxidizing Cysteine 147 on murine stimulator of interferon genes (STING), an ER-associated protein that mediates interferon response after cytoplasmic DNA sensing. This inhibited STING polymerization and activation of downstream signaling events. These data indicate that redox regulation of Cysteine 147 of mouse STING, which is equivalent to Cysteine 148 of human STING, controls interferon production. Together, our findings reveal that ROS orchestrates anti-viral immune responses, which can be exploited by viruses to evade cellular defenses.
    Keywords:  STING; herpesvirus; immunology; infectious disease; inflammation; interferon; microbiology; mouse; reactive oxygen species; redox regulation
    DOI:  https://doi.org/10.7554/eLife.57837
  88. Aging Cell. 2020 Aug 29. e13216
    Gutman D, Lidzbarsky G, Milman S, Gao T, Sin-Chan P, Gonzaga-Jauregui C, , Deelen J, Shuldiner AR, Barzilai N, Atzmon G.
      Centenarians (exceptionally long-lived individuals-ELLI) are a unique segment of the population, exhibiting long human lifespan and healthspan, despite generally practicing similar lifestyle habits as their peers. We tested disease-associated mutation burden in ELLI genomes by determining the burden of pathogenic variants reported in the ClinVar and HGMD databases using data from whole exome sequencing (WES) conducted in a cohort of ELLI, their offspring, and control individuals without antecedents of familial longevity (n = 1879), all descendent from the founder population of Ashkenazi Jews. The burden of pathogenic variants did not differ between the three groups. Additional analyses of variants subtypes and variant effect predictor (VEP) biotype frequencies did not reveal a decrease of pathogenic or loss-of-function (LoF) variants in ELLI and offspring compared to the control group. Case-control pathogenic variants enrichment analyses conducted in ELLI and controls also did not identify significant differences in any of the variants between the groups and polygenic risk scores failed to provide a predictive model. Interestingly, cancer and Alzheimer's disease-associated variants were significantly depleted in ELLI compared to controls, suggesting slower accumulation of mutation. That said, polygenic risk score analysis failed to find any predictive variants among the functional variants tested. The high similarity in the burden of pathogenic variation between ELLI and individuals without familial longevity supports the notion that extension of lifespan and healthspan in ELLI is not a consequence of pathogenic variant depletion but rather a result of other genomic, epigenomic, or potentially nongenomic properties.
    DOI:  https://doi.org/10.1111/acel.13216
  89. J Cell Physiol. 2020 Sep 03.
    Chen W, Wang L, You W, Shan T.
      Myokines are muscle-derived cytokines and chemokines that act extensively on organs and exert beneficial metabolic functions in the whole-body through specific signal networks. Myokines as mediators provide the conceptual basis for a whole new paradigm useful for understanding how skeletal muscle communicates with other organs. In this review, we summarize and discuss classes of myokines and their physiological functions in mediating the regulatory roles of skeletal muscle on other organs and the regulation of the whole-body energy metabolism. We review the mechanisms involved in the interaction between skeletal muscle and nonmuscle organs through myokines. Moreover, we clarify the connection between exercise, myokines and disease development, which may contribute to the understanding of a potential mechanism by which physical inactivity affects the process of metabolic diseases via myokines. Based on the current findings, myokines are important factors that mediate the effect of skeletal muscle on other organ functions and whole-body metabolism.
    Keywords:  adipokine; cross talk; exercise; interaction; myokine; skeletal muscle
    DOI:  https://doi.org/10.1002/jcp.30033
  90. J Biol Chem. 2020 Sep 02. pii: jbc.RA120.013121. [Epub ahead of print]
    Biswas D, Dao KT, Mercer A, Cowie AM, Duffley L, El Hiani Y, Kienesberger PC, Pulinilkunnil T.
      Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs is cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FA) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblot and UPLC MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKAs effect on insulin-induced AKT phosphorylation. This study provides evidence for FA mediated regulation of BCAA catabolizing enzymes, BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function.
    Keywords:  BCKA; Insulin signaling; amino acid; cardiomyocyte; cardiomyocytes; insulin resistance; protein translation; skeletal muscle; skeletal muscle metabolism; translation
    DOI:  https://doi.org/10.1074/jbc.RA120.013121