bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒05‒10
fifty-nine papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Nature. 2020 May;581(7806): 100-105
    Yamamoto K, Venida A, Yano J, Biancur DE, Kakiuchi M, Gupta S, Sohn ASW, Mukhopadhyay S, Lin EY, Parker SJ, Banh RS, Paulo JA, Wen KW, Debnath J, Kim GE, Mancias JD, Fearon DT, Perera RM, Kimmelman AC.
      Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.
  2. Cancer Res. 2020 May 06. pii: canres.1523.2019. [Epub ahead of print]
    Huang W, Navarro-Serer B, Jeong YJ, Chianchiano P, Xia L, Luchini C, Veronese N, Dowiak C, Ng T, Trujillo MA, Huang B, Pflüger MJ, Macgregor-Das AM, Lionheart G, Jones D, Fujikura K, Nguyen-Ngoc KV, Neumann NM, Groot VP, Hasanain A, van Oosten AF, Fischer SE, Gallinger S, Singhi AD, Zureikat AH, Brand RE, Gaida MM, Heinrich S, Burkhart RA, He J, Wolfgang CL, Goggins MG, Thompson ED, Roberts NJ, Ewald AJ, Wood LD.
      Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Re-expression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through non-canonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss.
  3. Proc Natl Acad Sci U S A. 2020 May 08. pii: 201921484. [Epub ahead of print]
    Somerville TDD, Xu Y, Wu XS, Maia-Silva D, Hur SK, de Almeida LMN, Preall JB, Koo PK, Vakoc CR.
      Lineage plasticity is a prominent feature of pancreatic ductal adenocarcinoma (PDA) cells, which can occur via deregulation of lineage-specifying transcription factors. Here, we show that the zinc finger protein ZBED2 is aberrantly expressed in PDA and alters tumor cell identity in this disease. Unexpectedly, our epigenomic experiments reveal that ZBED2 is a sequence-specific transcriptional repressor of IFN-stimulated genes, which occurs through antagonism of IFN regulatory factor 1 (IRF1)-mediated transcriptional activation at cooccupied promoter elements. Consequently, ZBED2 attenuates the transcriptional output and growth arrest phenotypes downstream of IFN signaling in multiple PDA cell line models. We also found that ZBED2 is preferentially expressed in the squamous molecular subtype of human PDA, in association with inferior patient survival outcomes. Consistent with this observation, we show that ZBED2 can repress the pancreatic progenitor transcriptional program, enhance motility, and promote invasion in PDA cells. Collectively, our findings suggest that high ZBED2 expression is acquired during PDA progression to suppress the IFN response pathway and to promote lineage plasticity in this disease.
    Keywords:  IRF1; ZBED2; interferon; lineage plasticity; pancreatic ductal adenocarcinoma
  4. Int Rev Cell Mol Biol. 2020 ;pii: S1937-6448(20)30058-7. [Epub ahead of print]353 ix-xiii
    Spetz J, Galluzzi L.
    Keywords:  Apoptosis; Autophagy; Immunogenic cell death; Mitochondrial outer membrane permeabilization; Mitochondrial permeability transition; Necroptosis; Parthanatos; Pyroptosis
  5. Cancers (Basel). 2020 May 04. pii: E1153. [Epub ahead of print]12(5):
    Mirabile A, Rivoltini L, Daveri E, Vernieri C, Mele R, Porcu L, Lazzari C, Bulotta A, Viganò MG, Cascinu S, Gregorc V.
      Several immunotherapy agents are the standard of care of many solid malignancies. Nevertheless, the majority of patients do not benefit from the currently available immunotherapies. It is therefore of paramount importance to identify the prognostic and predictive factors of tumor response/resistance and to design effective therapeutic strategies to overcome primary resistance and improve the efficacy of immunotherapy. The aim of this review is to underline the influence of the tumor and host metabolism on the antitumor immune response and to discuss possible strategies to improve the efficacy of available treatments by targeting the specific metabolic pathways in tumors or immune cells and by modifying patients' nutritional statuses. A systematic search of the Medline and EMBASE databases was carried out to identify scientific papers published until February 2020, which reported original research articles on the influence of tumor or host metabolism on antitumor immune response. The literature data showed the key role of glycolysis and mitochondrial oxidative phosphorylation, arginine, tryptophan, glutamine, lipid metabolism and microbiome on immune cell function. Moreover, specific nutritional behaviors, such as a low dietary intake of vitamin C, low glycemic index and alpha-linolenic acid, eicosapentenoic acid, docosahexaenoic acid, ornithine ketoglutarate, tryptophan and probiotic supplementation were associated with the potential clinical benefits from the currently available immunotherapies.
    Keywords:  cancer metabolism; immune response; immune-nutrition; immunotherapy; nutrition
  6. Int J Mol Sci. 2020 May 01. pii: E3218. [Epub ahead of print]21(9):
    Stopa KB, Kusiak AA, Szopa MD, Ferdek PE, Jakubowska MA.
      Pancreatic ductal adenocarcinoma (PDAC) causes annually well over 400,000 deaths world-wide and remains one of the major unresolved health problems. This exocrine pancreatic cancer originates from the mutated epithelial cells: acinar and ductal cells. However, the epithelia-derived cancer component forms only a relatively small fraction of the tumor mass. The majority of the tumor consists of acellular fibrous stroma and diverse populations of the non-neoplastic cancer-associated cells. Importantly, the tumor microenvironment is maintained by dynamic cell-cell and cell-matrix interactions. In this article, we aim to review the most common drivers of PDAC. Then we summarize the current knowledge on PDAC microenvironment, particularly in relation to pancreatic cancer therapy. The focus is placed on the acellular stroma as well as cell populations that inhabit the matrix. We also describe the altered metabolism of PDAC and characterize cellular signaling in this cancer.
    Keywords:  CAFs; PDAC; microenvironment; pancreatic cancer; signaling; stroma
  7. Cancer Lett. 2020 May 04. pii: S0304-3835(20)30203-2. [Epub ahead of print]
    Yamasaki A, Yanai K, Onishi H.
      Chemotherapy and immunotherapy for pancreatic ductal adenocarcinoma (PDAC) have limited success. One reason for this is thought to be the cancer microenvironment surrounding PDAC. Hypoxia is a feature of the cancer microenvironment. Under hypoxia, different various molecules and signaling pathways are activated compared with normoxia. To develop a new effective therapeutic strategy for PDAC, we need to target these hypoxic conditions to overcome PDAC. To inhibit the malignant phenotype, the cellular changes that occur under hypoxia should be elucidated. Various molecules and signaling that are activated by hypoxia may contribute to the induction of malignant phenotypes of PDAC such as proliferation, invasion, tumorigenesis, chemosensitivity, and autophagy. If we can develop therapeutic approaches to target one of these molecules or signaling pathways, we may proceed to the next therapeutic step of successfully treating refractory PDAC.
    Keywords:  Cancer microenvironment; Hypoxia; Morphogenesis signaling; Pancreatic ductal adenocarcinoma; Therapeutic target
  8. Gastroenterology. 2020 Apr 28. pii: S0016-5085(20)30566-7. [Epub ahead of print]
    Ogawa S, Fukuda A, Matsumoto Y, Hanyu Y, Sono M, Fukunaga Y, Masuda T, Araki O, Nagao M, Yoshikawa T, Goto N, Hiramatsu Y, Tsuda M, Maruno T, Nakanishi Y, Hussein MS, Tsuruyama T, Takaori K, Uemoto S, Seno H.
      BACKGROUND & AIMS: SETDB1, a histone methyltransferase that trimethylates histone H3 on lysine 9, promotes development of several tumor types. We investigated whether SETDB1 contributes to development of pancreatic ductal adenocarcinoma (PDAC).METHODS: We performed studies with Ptf1aCre; KrasG12D; Setdb1f/f, Ptf1aCre; KrasG12D; Trp53f/+; Setdb1f/f, and Ptf1aCre; KrasG12D; Trp53f/f; Setdb1f/f mice to investigate the effects of disruption of Setdb1 in mice with activated KRAS-induced pancreatic tumorigenesis, with heterozygous or homozygous disruption of Trp53. We performed microarray analyses of whole-pancreas tissues from Ptf1aCre; KrasG12D; Setdb1f/f and Ptf1aCre; KrasG12D mice and compared their gene expression patterns. Chromatin immunoprecipitation assays were performed using acinar cells isolated from pancreata with and without disruption of Setdb1. We used human PDAC cells for SETDB1 knockdown and inhibitor experiments.
    RESULTS: Loss of SETDB1 from pancreas accelerated formation of premalignant lesions in mice with pancreata that express activated KRAS. Microarray analysis revealed upregulated expression of genes in the apoptotic pathway and genes regulated by p53 in SETDB1-deficient pancreata. Deletion of SETDB1 from pancreas prevented formation of PDACs, concomitant with increased apoptosis and upregulated expression of Trp53 in mice heterozygous for disruption of Trp53. In contrast, pancreata of mice with homozygous disruption of Trp53 had no increased apoptosis, and PDACs developed. Chromatin immunoprecipitation revealed that SETDB1 bound to the Trp53 promoter to regulate its expression. Expression of an inactivated form of SETDB1 in human PDAC cells with wild type TP53 resulted in TP53-induced apoptosis.
    CONCLUSIONS: We found that the histone methyltransferase SETDB1 is required for development of PDACs, induced by activated KRAS, in mice. SETDB1 inhibits apoptosis by regulating expression of p53. SETDB1 might be a therapeutic target for PDACs that retain p53 function.
    Keywords:  cell death; epigenetic factor; oncogene; pancreatic cancer
  9. Nat Chem Biol. 2020 May 04.
    Lorent JH, Levental KR, Ganesan L, Rivera-Longsworth G, Sezgin E, Doktorova MD, Lyman E, Levental I.
      A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.
  10. Cell Mol Gastroenterol Hepatol. 2020 May 03. pii: S2352-345X(20)30066-7. [Epub ahead of print]
    Natale CA, Li J, Pitarresi JR, Norgard RJ, Dentchev T, Capell BC, Seykora JT, Stanger BZ, Ridky TW.
      BACKGROUND AND AIMS: Female sex is associated with lower incidence and improved clinical outcomes for most cancer types, including pancreatic ductal adenocarcinoma (PDAC). The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors.METHODS: Here we used murine syngeneic tumor models and human xenografts to determine that signaling through the nonclassical estrogen receptor G Protein-Coupled Estrogen Receptor (GPER) on tumor cells inhibits PDAC.
    RESULTS: Activation of GPER with the specific, small molecule, synthetic agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), and increased tumor cell immunogenicity. Systemically administered G-1 was well tolerated in PDAC bearing mice, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. We detected GPER protein in a majority of spontaneous human PDAC tumors, independent of tumor stage.
    CONCLUSIONS: These data, coupled with the wide tissue distribution of GPER, and our previous work showing that G-1 inhibits melanoma, suggest that GPER agonists may be useful against a range of cancers which are not classically considered sex hormone responsive, and which arise in tissues outside of the reproductive system.
    Keywords:  G Protein-Coupled Estrogen Receptor; GPER; PDAC; Pancreatic Ductal Adenocarcinoma
  11. Mech Ageing Dev. 2020 May 04. pii: S0047-6374(20)30052-X. [Epub ahead of print] 111256
    Jacome-Burbano MS, Gilson E.
      Senescence is a cellular response to stress for both dividing and post-mitotic cells. Noteworthy, long-lived post-mitotic cells (collectively named LLPMCs), which can live for decades in the organism, can exhibit a distinct type of cellular aging characterized by a progressive functional decline not associated to an overt senescence phenotype. The age-related drivers of senescence and aging in LLPMCs remain largely unknown. There is evidence that an increased production of reactive oxygen species (ROS) due to dysfunctional mitochondria, coupled with an inherent inability of cellular-degradation mechanisms to remove damaged molecules, is responsible for senescence and aging in LLPMC. Although telomeric DNA shortening, by nature linked to cell division, is generally not considered as a driver of LLPMC aging and senescence, we discuss recent reports revealing the existence of age-related telomere changes in LLPMC. These findings reveal unexpected roles for telomeres in LLPMC function and invite us to consider the hypothesis of a complex telomere clock involved in both dividing and non-dividing cell aging.
    Keywords:  DNA damage; Long-lived post-mitotic cell (LLPMC); Mitochondrial dysfunction; Senescence; Telomeres
  12. Nat Rev Mol Cell Biol. 2020 May 05.
    Nakatogawa H.
      Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. Autophagosome biogenesis responds to a plethora of signals and involves unique and dynamic membrane processes. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer. Thus, not surprisingly, the elucidation of the molecular mechanisms governing autophagosome biogenesis has attracted considerable interest. Key molecules and organelles involved in autophagosome biogenesis, including autophagy-related (ATG) proteins and the endoplasmic reticulum, have been discovered, and their roles and relationships have been investigated intensely. However, several fundamental questions, such as what supplies membranes/lipids to build the autophagosome and how the membrane nucleates, expands, bends into a spherical shape and finally closes, have proven difficult to address. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying these processes on a molecular level. We now know that autophagosome biogenesis is a highly complex process, in which multiple proteins and lipids from various membrane sources, supported by the formation of membrane contact sites, cooperate with biophysical phenomena, including membrane shaping and liquid-liquid phase separation, to ensure seamless segregation of the autophagic cargo. Together, these studies pave the way to obtaining a holistic view of autophagosome biogenesis.
  13. Cancer Discov. 2020 May 06. pii: CD-19-1242. [Epub ahead of print]
    Leibold J, Ruscetti M, Cao Z, Ho YJ, Baslan T, Zou M, Abida W, Feucht J, Han T, Barriga FM, Tsanov KM, Zamechek L, Kulick A, Amor C, Tian S, Rybczyk K, Salgado NR, Sanchez-Rivera FJ, Watson PA, de Stanchina E, Wilkinson JE, Dow LE, Abate-Shen C, Sawyers CL, Lowe SW.
      To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMMs) recapitulate features of traditional germline models and, by modeling genetic factors linked to late stage human disease, can produce tumors that are metastatic and castration resistant. A subset of tumors with p53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacological WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer.
  14. Br J Cancer. 2020 May 07.
    Robin F, Angenard G, Cano L, Courtin-Tanguy L, Gaignard E, Khene ZE, Bergeat D, Clément B, Boudjema K, Coulouarn C, Sulpice L.
      BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer worldwide, as a result of a late diagnosis and limited therapeutic options. Tumour microenvironment (or stroma) plays a key role in cancer onset and progression and constitutes an intrinsic histological hallmark of PDAC. Thus we hypothesised that relevant prognostic biomarkers and therapeutic targets can be identified in the stroma.METHODS: Laser microdissection of the stroma from freshly frozen PDAC was combined to gene expression profiling. Protein expression of candidate biomarkers was evaluated by immunohistochemistry on tissue microarrays (n = 80 tumours) and by ELISA in plasma samples (n = 51 patients).
    RESULTS: A signature made of 1256 genes that significantly discriminate the stroma from the non-tumour fibrous tissue was identified. Upregulated genes were associated with inflammation and metastasis processes and linked to NF-Kappa B and TGFβ pathways. TMA analysis validated an increased expression of SFN, ADAMTS12 and CXCL3 proteins in the stroma of PDAC. Stromal expression of SFN was further identified as an independent prognostic factor of overall (p = 0.003) and disease-free survival (DFS) (p = 0.034). SFN plasma expression was significantly associated with reduced DFS (p = 0.006).
    CONCLUSIONS: We demonstrated that gene expression changes within the stroma of PDAC correlate with tumour progression, and we identified Stratifin as a novel independent prognostic biomarker.
  15. Aging (Albany NY). 2020 May 07. 12
    Cho HJ, Yang EJ, Park JT, Kim JR, Kim EC, Jung KJ, Park SC, Lee YS.
      The selective removal of senescent cells by senolytics is suggested as a potential approach to reverse aging and extend lifespan. Using high-throughput screening with replicative senescence of human diploid fibroblasts (HDFs), we identified a novel senolytic drug R406 that showed selective toxicity in senescent cells. Using flow cytometry and caspase expression analysis, we confirmed that R406 caused apoptotic cell death along with morphological changes in senescent cells. Interestingly, R406 altered the cell survival-related molecular processes including the inhibition of phosphorylation of the focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) in senescent cells. This pattern was not observed in other known senolytic agent ABT263. Correspondingly, apoptotic cell death in senescent cells was induced by simultaneously blocking the FAK and p38 pathways. Taken together, we suggest that R406 acts as a senolytic drug by inducing apoptosis and reducing cell attachment capacity.
    Keywords:  FAK; apoptosis; cellular senescence; p38; senolytics
  16. Cell Metab. 2020 Apr 23. pii: S1550-4131(20)30186-8. [Epub ahead of print]
    Helman A, Cangelosi AL, Davis JC, Pham Q, Rothman A, Faust AL, Straubhaar JR, Sabatini DM, Melton DA.
      A drastic transition at birth, from constant maternal nutrient supply in utero to intermittent postnatal feeding, requires changes in the metabolic system of the neonate. Despite their central role in metabolic homeostasis, little is known about how pancreatic β cells adjust to the new nutritional challenge. Here, we find that after birth β cell function shifts from amino acid- to glucose-stimulated insulin secretion in correlation with the change in the nutritional environment. This adaptation is mediated by a transition in nutrient sensitivity of the mTORC1 pathway, which leads to intermittent mTORC1 activity. Disrupting nutrient sensitivity of mTORC1 in mature β cells reverts insulin secretion to a functionally immature state. Finally, manipulating nutrient sensitivity of mTORC1 in stem cell-derived β cells in vitro strongly enhances their glucose-responsive insulin secretion. These results reveal a mechanism by which nutrients regulate β cell function, thereby enabling a metabolic adaptation for the newborn.
    Keywords:  embryo; in vitro differentiation; insulin secretion; mTORC1; maturation; nutrient sensing; pancreas; stem cell-derived β cells; β cells
  17. Proc Natl Acad Sci U S A. 2020 May 05. pii: 201917174. [Epub ahead of print]
    Trentesaux C, Fraudeau M, Pitasi CL, Lemarchand J, Jacques S, Duche A, Letourneur F, Naser E, Bailly K, Schmitt A, Perret C, Romagnolo B.
      The intestinal epithelium acts as a barrier between the organism and its microenvironment, including the gut microbiota. It is the most rapidly regenerating tissue in the human body thanks to a pool of intestinal stem cells (ISCs) expressing Lgr5 The intestinal epithelium has to cope with continuous stress linked to its digestive and barrier functions. Epithelial repair is crucial to maintain its integrity, and Lgr5-positive intestinal stem cell (Lgr5+ISC) resilience following cytotoxic stresses is central to this repair stage. We show here that autophagy, a pathway allowing the lysosomal degradation of intracellular components, plays a crucial role in the maintenance and genetic integrity of Lgr5+ISC under physiological and stress conditions. Using conditional mice models lacking the autophagy gene Atg7 specifically in all intestinal epithelial cells or in Lgr5+ISC, we show that loss of Atg7 induces the p53-mediated apoptosis of Lgr5+ISC. Mechanistically, this is due to increasing oxidative stress, alterations to interactions with the microbiota, and defective DNA repair. Following irradiation, we show that Lgr5+ISC repair DNA damage more efficiently than their progenitors and that this protection is Atg7 dependent. Accordingly, we found that the stimulation of autophagy on fasting protects Lgr5+ISC against DNA damage and cell death mediated by oxaliplatin and doxorubicin treatments. Finally, p53 deletion prevents the death of Atg7-deficient Lgr5+ISC but promotes genetic instability and tumor formation. Altogether, our findings provide insights into the mechanisms underlying maintenance and integrity of ISC and highlight the key functions of Atg7 and p53.
    Keywords:  Atg7; DNA repair; autophagy; intestinal stem cells
  18. Elife. 2020 May 04. pii: e55038. [Epub ahead of print]9
    Hubert M, Larsson E, Vegesna NVG, Ahnlund M, Johansson AI, Moodie LW, Lundmark R.
      Caveolae are bulb-shaped invaginations of the plasma membrane (PM) that undergo scission and fusion at the cell surface and are enriched in specific lipids. However, the influence of lipid composition on caveolae surface stability is not well described or understood. Accordingly, we inserted specific lipids into the cell PM via membrane fusion and studied their acute effects on caveolae dynamics. We demonstrate that sphingomyelin stabilizes caveolae to the cell surface, while cholesterol and glycosphingolipids drive caveolae scission from the PM. Whilst all three lipids accumulated specifically in caveolae, cholesterol and sphingomyelin were actively sequestered, whereas glycosphingolipids diffused freely. The ATPase EHD2 restricts lipid diffusion and counteracts lipid-induced scission. We propose that specific lipid accumulation in caveolae generates an intrinsically unstable domain prone to scission if not restrained by EHD2 at the caveolae neck. This work provides a mechanistic link between caveolae and their ability to sense the PM lipid composition.
    Keywords:  biochemistry; cell biology; chemical biology
  19. JAMA. 2020 May 05. 323(17): 1663-1665
    Berwick DM.
  20. Aging (Albany NY). 2020 May 05. 12
    Hernandez A, Truckenbrod L, Federico Q, Campos K, Moon B, Ferekides N, Hoppe M, D'Agostino D, Burke S.
      The ability to switch between glycolysis and ketosis promotes survival by enabling metabolism through fat oxidation during periods of fasting. Carbohydrate restriction or stress can also elicit metabolic switching. Keto-adapting from glycolysis is delayed in aged rats, but factors mediating this age-related impairment have not been identified. We measured metabolic switching between glycolysis and ketosis, as well as glycogen dynamics, in young and aged rats undergoing time-restricted feeding (TRF) with a standard diet or a low carbohydrate ketogenic diet (KD). TRF alone reversed markers of insulin-related metabolic deficits and accelerated metabolic switching in aged animals. A KD+TRF, however, provided additive benefits on these variables. Remarkably, the ability to keto-adapt was not related to glycogen levels and KD-fed rats showed an enhanced elevation in glucose following epinephrine administration. This study provides new insights into the mechanisms of keto-adaptation demonstrating the utility of dietary interventions to treat metabolic impairments across the lifespan.
    Keywords:  diet; glucose; intermittent fasting; keto-adaptation; ketogenic diet
  21. Elife. 2020 May 05. pii: e55185. [Epub ahead of print]9
    Lim AR, Rathmell WK, Rathmell JC.
      Breakthroughs in anti-tumor immunity have led to unprecedented advances in immunotherapy, yet it is now clear that the tumor microenvironment (TME) restrains immunity. T cells must substantially increase nutrient uptake to mount a proper immune response and failure to obtain sufficient nutrients or engage the appropriate metabolic pathways can alter or prevent effector T cell differentiation and function. The TME, however, can be metabolically hostile due to insufficient vascular exchange and cancer cell metabolism that leads to hypoxia, depletion of nutrients, and accumulation of waste products. Further, inhibitory receptors present in the TME can inhibit T cell metabolism and alter T cell signaling both directly and through release of extracellular vesicles such as exosomes. This review will discuss the metabolic changes that drive T cells into different stages of their development and how the TME imposes barriers to the metabolism and activity of tumor infiltrating lymphocytes.
    Keywords:  cancer; cancer biology; immunology; immunometabolism; immunotherapy; inflammation; t cells; tumor microenvironment; tumor-infiltrating lymphocytes
  22. Trends Cell Biol. 2020 Apr 28. pii: S0962-8924(20)30075-1. [Epub ahead of print]
    Shapira SN, Christofk HR.
      Adult tissue stem cells mediate organ homeostasis and regeneration and thus are continually making decisions about whether to remain quiescent, proliferate, or differentiate into mature cell types. These decisions often integrate external cues, such as energy balance and the nutritional status of the organism. Metabolic substrates and byproducts that regulate epigenetic and signaling pathways are now appreciated to have instructive rather than bystander roles in regulating cell fate decisions. In this review, we highlight recent literature focused on how metabolites and dietary manipulations can impact cell fate decisions, with a focus on the regulation of adult tissue stem cells.
    Keywords:  adult stem cells; diet; differentiation; metabolism; metabolomics
  23. Front Cell Dev Biol. 2020 ;8 239
    Bakula D, Scheibye-Knudsen M.
      Maintaining mitochondrial health is emerging as a keystone in aging and associated diseases. The selective degradation of mitochondria by mitophagy is of particular importance in keeping a pristine mitochondrial pool. Indeed, inherited monogenic diseases with defects in mitophagy display complex multisystem pathologies but particularly progressive neurodegeneration. Fortunately, therapies are being developed that target mitophagy allowing new hope for treatments for previously incurable diseases. Herein, we describe mitophagy and associated diseases, coin the term mitophaging and describe new small molecule interventions that target different steps in the mitophagic pathway. Consequently, several age-associated diseases may be treated by targeting mitophagy.
    Keywords:  aging; autophagy; interventions; mitophaging; mitophagy; monogenic disorders
  24. Cell. 2020 Apr 30. pii: S0092-8674(20)30336-6. [Epub ahead of print]181(3): 748-748.e1
    Holland LKK, Nielsen IØ, Maeda K, Jäättelä M.
      In addition to their well-defined recycling function, lysosomes act as metabolic signaling hubs that adjust cellular metabolism according to the availability of nutrients and growth factors by regulating metabolic kinases and transcription factors on their surface. Moreover, lysosomal hydrolases and ions released to cytosol or extracellular space have recently emerged as important regulators of various cellular processes from cell death to cell division. To view this SnapShot, open or download the PDF.
  25. iScience. 2020 Apr 13. pii: S2589-0042(20)30246-7. [Epub ahead of print]23(5): 101061
    Monteiro L, Da Silva L, Lipinski B, Fauvet F, Vigneron A, Puisieux A, Martinez P.
      Despite advances in single-cell and molecular techniques, it is still unclear how to best quantify phenotypic heterogeneity in cancer cells that evolved beyond normal, known classifications. We present an approach to phenotypically characterize cells based on their activities rather than static classifications. We validated the detectability of specific activities (epithelial-mesenchymal transition, glycolysis) in single cells, using targeted RT-qPCR analyses and in vitro inductions. We analyzed 50 established activity signatures as a basis for phenotypic description in public data and computed cell-cell distances in 28,513 cells from 85 patients and 8 public datasets. Despite not relying on any classification, our measure correlated with standard diversity indices in populations of known structure. We identified bottlenecks as phenotypic diversity reduced upon colorectal cancer initiation. This suggests that focusing on what cancer cells do rather than what they are can quantify phenotypic diversity in universal fashion, to better understand and predict intra-tumor heterogeneity dynamics.
    Keywords:  Biological Sciences; Cancer; Cancer Systems Biology; Mathematical Biosciences
  26. Nat Rev Endocrinol. 2020 May 06.
    Pipatpolkai T, Usher S, Stansfeld PJ, Ashcroft FM.
      The ATP-sensitive potassium channel (KATP channel) couples blood levels of glucose to insulin secretion from pancreatic β-cells. KATP channel closure triggers a cascade of events that results in insulin release. Metabolically generated changes in the intracellular concentrations of adenosine nucleotides are integral to this regulation, with ATP and ADP closing the channel and MgATP and MgADP increasing channel activity. Activating mutations in the genes encoding either of the two types of KATP channel subunit (Kir6.2 and SUR1) result in neonatal diabetes mellitus, whereas loss-of-function mutations cause hyperinsulinaemic hypoglycaemia of infancy. Sulfonylurea and glinide drugs, which bind to SUR1, close the channel through a pathway independent of ATP and are now the primary therapy for neonatal diabetes mellitus caused by mutations in the genes encoding KATP channel subunits. Insight into the molecular details of drug and nucleotide regulation of channel activity has been illuminated by cryo-electron microscopy structures that reveal the atomic-level organization of the KATP channel complex. Here we review how these structures aid our understanding of how the various mutations in the genes encoding Kir6.2 (KCNJ11) and SUR1 (ABCC8) lead to a reduction in ATP inhibition and thereby neonatal diabetes mellitus. We also provide an update on known mutations and sulfonylurea therapy in neonatal diabetes mellitus.
  27. Cell Death Dis. 2020 May 04. 11(5): 310
    Oizel K, Tait-Mulder J, Fernandez-de-Cossio-Diaz J, Pietzke M, Brunton H, Lilla S, Dhayade S, Athineos D, Blanco GR, Sumpton D, Mackay GM, Blyth K, Zanivan SR, Meiser J, Vazquez A.
      Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.
  28. Trends Immunol. 2020 Apr 30. pii: S1471-4906(20)30071-5. [Epub ahead of print]
    Chazaud B.
      Inflammation is usually considered as harmful; however, it is also necessary for tissue recovery after injury. Macrophages exert immune and nonimmune functions throughout this process. During skeletal muscle regeneration, they mount an inflammatory response while exerting trophic roles on muscle and mesenchymal stem cells. Proinflammatory macrophages shift to being anti-inflammatory, triggering the resolution of inflammation. Studies have highlighted that during this shift, a crosstalk ensues, integrating cues for resolution, efferocytosis, cellular metabolism, and signaling pathways. During the restorative phase, macrophages dampen inflammation while promoting stem cell differentiation, angiogenesis, and matrix remodeling. Since blunting the inflammatory phase can be detrimental for muscle regeneration, we suggest that rather than fighting inflammation, it should be allowed to operate and resolve, thus allowing for tissue recovery.
    Keywords:  efferocytosis; inflammatory response; macrophage; resolution of inflammation; skeletal muscle regeneration; tissue injury
  29. Proc Natl Acad Sci U S A. 2020 May 08. pii: 201919394. [Epub ahead of print]
    Seo BR, Chen X, Ling L, Song YH, Shimpi AA, Choi S, Gonzalez J, Sapudom J, Wang K, Andresen Eguiluz RC, Gourdon D, Shenoy VB, Fischbach C.
      Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.
    Keywords:  3D fibrous matrix mechanics; adipose-derived stem cells; collagen microarchitecture; mechanosignaling; myofibroblast differentiation
  30. Nat Metab. 2020 Apr;2(4): 351-363
    van Veen JE, Kammel LG, Bunda PC, Shum M, Reid MS, Massa MG, Arneson D, Park JW, Zhang Z, Joseph AM, Hrncir H, Liesa M, Arnold AP, Yang X, Correa SM.
      Estrogen receptor a (ERa) signaling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERa is enriched in populations showing sex biased expression of reprimo (Rprm), tachykinin 1 (Tac1), and prodynorphin (Pdyn). Female biased expression of Tac1 and Rprm is patterned by ERa-dependent repression during male development, whereas male biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERa positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together these findings reveal a role for Rprm in temperature regulation and ERa in the masculinization of neuron populations that underlie energy expenditure.
  31. J Mol Med (Berl). 2020 May 07.
    Karakas D, Ozpolat B.
      Eukaryotic elongation factor-2 kinase (eEF2K), an atypical member of alpha-kinase family, is highly overexpressed in breast, pancreatic, brain, and lung cancers, and associated with poor survival in patients. eEF2K promotes cell proliferation, survival, and aggressive tumor characteristics, leading to tumor growth and progression. While initial studies indicated that eEF2K acts as a negative regulator of protein synthesis by suppressing peptide elongation phase, later studies demonstrated that it has multiple functions and promotes cell cycle, angiogenesis, migration, and invasion as well as induction of epithelial-mesenchymal transition through induction of integrin β1, SRC/FAK, PI3K/AKT, cyclin D1, VEGF, ZEB1, Snail, and MMP-2. Under stress conditions such as hypoxia and metabolic distress, eEF2K is activated by several signaling pathways and slows down protein synthesis and helping cells to save energy and survive. In vivo therapeutic targeting of eEF2K by genetic methods inhibits tumor growth in various tumor models, validating it as a potential molecular target. Recent studies suggest that eEF2K plays a role in tumor microenvironment cells by monocyte chemoattractant protein-1 (MCP-1) and accumulation of tumor-associated macrophages. Due to its clinical significance and the pivotal role in tumorigenesis and progression, eEF2K is considered as an important therapeutic target in solid tumors. However, currently, there is no specific and potent inhibitor for translation into clinical studies. Here, we aim to systematically review current knowledge regarding eEF2K in tumor biology, microenvironment, and development of eEF2K targeted inhibitors and therapeutics.
    Keywords:  Eukaryotic elongation factor-2 kinase; Gene regulation; Protein synthesis; Therapy; Tumor microenvironment; eEF2K
  32. Cancer Res. 2020 May 04. pii: canres.0506.2020. [Epub ahead of print]
    Han L, Long Q, Li S, Xu Q, Zhang B, Dou X, Qian M, Jiramongkol Y, Guo J, Cao L, Chin YE, Lam EW, Jiang J, Sun Y.
      Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP), which is implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathological activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells.
  33. Nat Commun. 2020 May 04. 11(1): 2184
    Mudumbi KC, Czapiewski R, Ruba A, Junod SL, Li Y, Luo W, Ngo C, Ospina V, Schirmer EC, Yang W.
      Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.
  34. Nat Commun. 2020 May 08. 11(1): 2315
    Zapp C, Obarska-Kosinska A, Rennekamp B, Kurth M, Hudson DM, Mercadante D, Barayeu U, Dick TP, Denysenkov V, Prisner T, Bennati M, Daday C, Kappl R, Gräter F.
      As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.
  35. Nat Commun. 2020 May 08. 11(1): 2266
    Liu Z, Yang Y, Gu A, Xu J, Mao Y, Lu H, Hu W, Lei QY, Li Z, Zhang M, Cai Y, Wen W.
      The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. Here we show that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. We propose that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes.
  36. JCI Insight. 2020 May 05. pii: 136283. [Epub ahead of print]
    Hauffe R, Stein V, Chudoba C, Flore T, Rath M, Ritter K, Schell M, Wardelmann K, Deubel S, Kopp JF, Schwarz M, Kappert K, Blüher M, Schwerdtle T, Kipp AP, Kleinridders A.
      Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of diabetic patients are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of the IR with a reduction of mRNA expression of the selenoproteins Txnrd3, Sephs2, and Gpx3. Consistently, GPX3 is also decreased in adipose tissue of insulin resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation and elevated IR expression in WAT. Again, IR expression correlates positively with Gpx3 expression, a phenotype which is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduces IR expression in 3T3-L1 preadipocytes and insulin sensitivity. Overall our data identify GPx3 as a novel regulator of IR expression and insulin sensitivity in adipose tissue.
    Keywords:  Adipose tissue; Endocrinology; Insulin signaling; Metabolism; Obesity
  37. Nat Commun. 2020 May 07. 11(1): 2246
    Kim D, Park G, Huuhtanen J, Lundgren S, Khajuria RK, Hurtado AM, Muñoz-Calleja C, Cardeñoso L, Gómez-García de Soria V, Chen-Liang TH, Eldfors S, Ellonen P, Hannula S, Kankainen M, Bruck O, Kreutzman A, Salmenniemi U, Lönnberg T, Jerez A, Itälä-Remes M, Myllymäki M, Keränen MAI, Mustjoki S.
      Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.
  38. Dev Cell. 2020 Apr 23. pii: S1534-5807(20)30269-0. [Epub ahead of print]
    Gabay Yehezkely R, Zaffryar-Eilot S, Kaganovsky A, Fainshtain Malka N, Aviram R, Livneh I, Hasson P.
      Integration of extracellular matrix (ECM)-derived cues into transcriptional programs is essential primarily in rapidly morphing environments, such as regenerating tissues. Here, we demonstrate that lysyl oxidase (Lox), known for its ECM-modifying activities, primarily collagen crosslinking, also directly regulates transcription factor (TF) localization. Using genetic and pharmacological strategies, we highlight an intracellular role for Lox in myogenic progenitors essential for muscle regeneration. We show that Lox interacts with, and directly oxidizes, vestigial-like 3 (Vgll3), a transcriptional co-activator acting with Mef2 and transcriptional enhancer factor (TEF) TFs. This enzymatic activity is required for Vgll3 cytoplasmic-to-nuclear translocation in regulation of myogenic differentiation. Our work highlights an additional mechanism for TF subcellular localization facilitating integration of ECM organization with transcriptional output during myogenic differentiation. Modulating this integration mechanism could affect the balance between ECM organization and cell differentiation and serve as a basis for novel therapeutic strategies targeting fibrotic pathologies.
    Keywords:  Vgll; extracellular matrix; lysyl oxidase; mouse; muscle regeneration; myogenesis; nuclear translocation; satellite cells; transcriptional regulation
  39. Proc Natl Acad Sci U S A. 2020 May 05. pii: 201913767. [Epub ahead of print]
    Woronoff G, Nghe P, Baudry J, Boitard L, Braun E, Griffiths AD, Bibette J.
      Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.
    Keywords:  adaptation; droplet-based microfluidics; genetic rewiring; single-cell metabolism
  40. Cancer Res. 2020 May 05. pii: canres.2052.2019. [Epub ahead of print]
    Wu F, Zhang C, Zhao C, Wu H, Teng Z, Jiang T, Wang Y.
      Aberrant activation of the Hedgehog (HH) signaling pathway underlines the initiation and progression of a multitude of cancers. The effectiveness of the leading drugs vismodegib (GDC-0449) and sonidegib (LDE225), both Smoothened (SMO) antagonists, is compromised by acquisition of mutations that alter pathway components, notably secondary mutations in SMO and amplification of GLI2, a transcriptional mediator at the end of the pathway. Pharmacological blockade of GLI2 activity could ultimately overcome these diversified refractory mechanisms, which would also be effective in a broader spectrum of primary tumors than current SMO antagonists. To this end, we conducted a high-content screen directly analyzing the ciliary translocation of GLI2, a key event for GLI2 activation in HH signal transduction. Several prostaglandin compounds were shown to inhibit accumulation of GLI2 within the primary cilium (PC). In particular, prostaglandin E1 (PGE1), an FDA-approved drug, is a potent GLI2 antagonist that overcame resistance mechanisms of both SMO mutagenesis and GLI2 amplification. Consistent with a role in HH pathway regulation, EP4 receptor localized to the PC. Mechanistically, PGE1 inhibited HH signaling through the EP4 receptor, enhancing cAMP-PKA activity, which promoted phosphorylation and degradation of GLI2 via the ubiquitination pathway. PGE1 also effectively inhibited the growth of drug refractory human medulloblastoma (MB) xenografts. Together, these results identify PGE1 and other prostaglandins as potential templates for complementary therapeutic development to circumvent resistance to current generation SMO antagonists in use in the clinic.
  41. Mol Cancer Ther. 2020 May 05. pii: molcanther.1060.2019. [Epub ahead of print]
    Davis AA, Iams WT, Chan D, Oh MS, Lentz RW, Peterman N, Robertson A, Shah A, Srivas R, Wilson TJ, Lambert NJ, George PS, Wong B, Wood HW, Close JC, Tezcan A, Nesmith K, Tezcan H, Chae YK.
      Treatment response assessment for patients with advanced solid tumors is complex and existing methods require greater precision. Current guidelines rely on imaging, which has known limitations, including the time required to show a deterministic change in target lesions. Serial changes in whole-genome (WG) circulating tumor DNA (ctDNA) were used to assess response or resistance to treatment early in the treatment course. 96 patients with advanced cancer were prospectively enrolled (91 analyzed and 5 excluded), and blood was collected before and after initiation of a new, systemic treatment. Plasma cell-free DNA libraries were prepared for either WG or WG bisulfite sequencing. Longitudinal changes in the fraction of ctDNA were quantified to retrospectively identify molecular progression (MP) or major molecular response (MMR). Study endpoints were concordance with first follow-up imaging (FFUI) and stratification of progression-free survival (PFS) and overall survival (OS). Patients with MP (n=13) had significantly shorter PFS (median 62d vs. 310d) and OS (255d vs. not reached). Sensitivity for MP to identify clinical progression was 54% and specificity was 100%. MP calls were from samples taken a median of 28d into treatment and 39d before FFUI. Patients with MMR (n=27) had significantly longer PFS and OS compared to those with neither call (n=51). These results demonstrated that ctDNA changes early after treatment initiation inform response to treatment and correlate with long-term clinical outcomes. Once validated, molecular response assessment can enable early treatment change minimizing side effects and costs associated with additional cycles of ineffective treatment.
  42. Nat Cell Biol. 2020 May 04.
    Huang Y, Wang H, Hao Y, Lin H, Dong M, Ye J, Song L, Wang Y, Li Q, Shan B, Jiang Y, Li H, Shao Z, Kroemer G, Zhang H, Bai L, Jin T, Wang C, Ma Y, Cai Y, Ding C, Liu S, Pan Y, Jiang W, Zhou R.
      PTEN is a dual-specificity phosphatase that is frequently mutated in human cancer, and its deficiency in cancer has been associated with therapy resistance and poor survival. Although the intrinsic tumour-suppressor function of PTEN has been well established, evidence of its role in the tumour immune microenvironment is lacking. Here, we show that chemotherapy-induced antitumour immune responses and tumour suppression rely on myeloid-cell PTEN, which is essential for chemotherapy-induced activation of the NLRP3 inflammasome and antitumour immunity. PTEN directly interacts with and dephosphorylates NLRP3 to enable NLRP3-ASC interaction, inflammasome assembly and activation. Importantly, supplementation of IL-1β restores chemotherapy sensitivity in mouse myeloid cells with a PTEN deficiency. Clinically, chemotherapy-induced IL-1β production and antitumour immunity in patients with cancer is correlated with PTEN expression in myeloid cells, but not tumour cells. Our results demonstrate that myeloid PTEN can determine chemotherapy responsiveness by promoting NLRP3-dependent antitumour immunity and suggest that myeloid PTEN might be a potential biomarker to predict chemotherapy responses.
  43. EMBO Rep. 2020 May 03. e48927
    Govindarajan S, Verheugen E, Venken K, Gaublomme D, Maelegheer M, Cloots E, Gysens F, De Geest BG, Cheng TY, Moody DB, Janssens S, Drennan M, Elewaut D.
      CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids.
    Keywords:  ER stress; NKT cells; antigen-presenting cells; lipid antigens; neutral lipid
  44. Nat Cell Biol. 2020 May 04.
    Li F, Huangyang P, Burrows M, Guo K, Riscal R, Godfrey J, Lee KE, Lin N, Lee P, Blair IA, Keith B, Li B, Simon MC.
      The crosstalk between deregulated hepatocyte metabolism and cells within the tumour microenvironment, as well as the consequent effects on liver tumorigenesis, are not completely understood. We show here that hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression. FBP1 is universally silenced in both human and murine liver tumours. Hepatocyte-specific Fbp1 deletion results in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype. Depleting senescent HSCs by 'senolytic' treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, the subsequent development of the senescence-associated secretory phenotype and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a critical crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth.
  45. J Cell Biol. 2020 Jul 06. pii: e201902059. [Epub ahead of print]219(7):
    Petzoldt AG, Götz TWB, Driller JH, Lützkendorf J, Reddy-Alla S, Matkovic-Rachid T, Liu S, Knoche E, Mertel S, Ugorets V, Lehmann M, Ramesh N, Beuschel CB, Kuropka B, Freund C, Stelzl U, Loll B, Liu F, Wahl MC, Sigrist SJ.
      At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.
  46. Pancreatology. 2020 Apr 22. pii: S1424-3903(20)30143-5. [Epub ahead of print]
    Krapf SA, Lund J, Lundkvist M, Dale MG, Nyman TA, Thoresen GH, Kase ET.
      BACKGROUND: /Objectives: We aimed to metabolically compare healthy primary human pancreatic epithelial cells (hPEC) to a pancreatic cancer cell line (PANC-1) and explore the effect on energy metabolism of exposing primary human myotubes to conditioned medium from hPEC and PANC-1 cells.METHODS: Differences in metabolism were examined with radiolabeled glucose, oleic acid and lactic acid, and by qPCR. Mass spectrometry-based proteomics was used to study global protein secretion from the two cell types. Pathway analyses were performed.
    RESULTS: PANC-1 cells tended to have higher glucose uptake, production of lactic acid, and glucose oxidation compared to hPEC cells. PANC-1 cells had higher uptake but lower oxidation of oleic acid, and mitochondrial reserve capacity from oleic acid was lower in PANC-1 cells. These differences in energy metabolism were reflected by differences in gene expressions and pathway analyses of the secretome. Conditioned medium from PANC-1 cells attenuated oleic acid oxidation in primary human myotubes.
    CONCLUSIONS: Metabolic characterization of the PANC-1 cells revealed a glycolytic phenotype since they had an active glucose oxidation. Furthermore, PANC-1 cells showed a lower oleic acid oxidation and secreted a high amount of proteins into conditioned medium that also induced a reduced oleic acid oxidation in myotubes.
    Keywords:  Energy metabolism; Myotubes; Pancreatic cell models; Proteomics
  47. Aging (Albany NY). 2020 May 08. 12
    Ma Z, Lou S, Jiang Z.
      High levels of the imprinted gene pleckstrin homology like domain family A member 2 (PHLDA2) correlate with tumor progression in several malignancies. Here, we investigated the effects of PHDLDA2 expression in CRC through assays of cellular proliferation, invasion, migration, and apoptosis. We also screened for possible mechanisms of action. Our results show that PHLDA2 was upregulated in CRC tissues. Knockdown of PHLDA2 inhibited cellular proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) in vitro. Knockout of PHLDA2 promoted cellular apoptosis, in part by activating autophagy. PHLDA2 knockout also inhibited tumorigenesis and expression of KI67 protein in vivo. The effects of PHLDA2 on autophagy and EMT were mediated in part via the PI3K/AKT signaling pathway. Taken together, these results suggest that downregulation of PHLDA2 inhibits tumor growth and PI3K, thereby promoting autophagy and inhibiting EMT, in part through the PI3K/AKT/mTOR and PI3K/AKT/GSK-3β signaling pathways.
    Keywords:  PHLDA2; apoptosis; autophagy; colorectal cancer; tumorigenesis
  48. Nat Commun. 2020 May 08. 11(1): 2282
    Gonçalves SM, Duarte-Oliveira C, Campos CF, Aimanianda V, Ter Horst R, Leite L, Mercier T, Pereira P, Fernández-García M, Antunes D, Rodrigues CS, Barbosa-Matos C, Gaifem J, Mesquita I, Marques A, Osório NS, Torrado E, Rodrigues F, Costa S, Joosten LA, Lagrou K, Maertens J, Lacerda JF, Campos A, Brown GD, Brakhage AA, Barbas C, Silvestre R, van de Veerdonk FL, Chamilos G, Netea MG, Latgé JP, Cunha C, Carvalho A.
      In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.
  49. J Immunol. 2020 May 06. pii: ji2000172. [Epub ahead of print]
    Chen YT, Kung JT.
      BCR-mediated tonic signaling is an indispensable requirement for the survival of follicular B (FOB) cells and Burkitt lymphoma (BL) cells. FOB cells of the I-A12% mutant mouse express unfolded protein response and are extremely short lived. Among the myriad molecules activated by unfolded protein response in I-A12% B cells, Xbp1s singularly "hijacked" p110 from p85:p110 heterodimeric PI3K, thereby abating BCR tonic signaling, resulting in their extremely short lifespan. Long-lived normal FOB cells became short lived upon ectopic Xbp1s expression. The proapoptotic Xbp1s role in FOB cells starkly contrasts with its antithetical prosurvival function in plasma cells. Also, tonic signaling and clonal expansion, two important functions mediated by the same BCR, operate in independent and distinct manners. Furthermore, concerning the development of new therapeutic treatment of drug-refractory BL patients, our finding of Xbp1s-mediated rapid death of BL cells brings forth a conceptual advancement based on blocking PI3K heterodimer formation rather than inhibition of PI3K enzyme activity.
  50. Cell Rep. 2020 May 05. pii: S2211-1247(20)30546-5. [Epub ahead of print]31(5): 107597
    Camps J, Breuls N, Sifrim A, Giarratana N, Corvelyn M, Danti L, Grosemans H, Vanuytven S, Thiry I, Belicchi M, Meregalli M, Platko K, MacDonald ME, Austin RC, Gijsbers R, Cossu G, Torrente Y, Voet T, Sampaolesi M.
      Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.
    Keywords:  GDF10; adipogenesis; interstitial stromal cells; muscular dystrophy; single-cell transcriptomics; skeletal muscle
  51. J Hepatol. 2020 May 03. pii: S0168-8278(20)30275-0. [Epub ahead of print]
    Schwerbel K, Kamitz A, Krahmer N, Hallahan N, Jähnert M, Gottmann P, Lebek S, Schallschmidt T, Arends D, Schumacher F, Kleuser B, Haltenhof T, Heyd F, Gancheva S, Broman KW, Roden M, Joost HG, Chadt A, Al-Hasani H, Vogel H, Jonas W, Schürmann A.
      BACKGROUND & AIMS: The cause of fatty liver is multifactorial, including genetic and environmental factors. Currently, only a few genetic variants explain the heritability of the disease. QTL analysis of mouse strains enables the identification of genes causing complex human diseases. In a backcross of New Zealand obese (NZO) and C57BL/6J (B6) mice we identified the QTL Ltg/NZO on chromosome 18, which associates with increased liver triglycerides.METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injection of specific microRNAs in B6 mice. Pulldown coupled with mass-spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2.
    RESULTS: Through positional cloning, we identified two immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 increased hepatic fat content 3 to 4-fold. In the liver of low-fat diet fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased association of LC3B with lipid droplets and decreased triglyceride storage.
    CONCLUSION: IFGGA2 interacts with ATGL and protects from hepatic steatosis presumably by enhancing LC3B binding to lipid droplets.
    Keywords:  fatty liver; immunity-related GTPases; miRNA; positional cloning
  52. J Cell Biol. 2020 Jul 06. pii: e201905160. [Epub ahead of print]219(7):
    Chen Z, Wang ZH, Zhang G, Bleck CKE, Chung DJ, Madison GP, Lindberg E, Combs C, Balaban RS, Xu H.
      Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we demonstrate that mitochondrial fission, together with the lack of mtDNA replication, segregate mtDNA into individual organelles in the Drosophila early germarium. After mtDNA segregation, mtDNA transcription begins, which activates respiration. Mitochondria harboring wild-type genomes have functional electron transport chains and propagate more vigorously than mitochondria containing deleterious mutations in hetreoplasmic cells. Therefore, mtDNA expression acts as a stress test for the integrity of mitochondrial genomes and sets the stage for replication competition. Our observations support selective inheritance at the organelle level through a series of developmentally orchestrated mitochondrial processes. We also show that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. These two mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis.
  53. Immunity. 2020 Apr 26. pii: S1074-7613(20)30164-3. [Epub ahead of print]
    Ohkura N, Yasumizu Y, Kitagawa Y, Tanaka A, Nakamura Y, Motooka D, Nakamura S, Okada Y, Sakaguchi S.
      The contribution of FOXP3-expressing naturally occurring regulatory T (Treg) cells to common polygenic autoimmune diseases remains ambiguous. Here, we characterized genome-wide epigenetic profiles (CpG methylation and histone modifications) of human Treg and conventional T (Tconv) cells in naive and activated states. We found that single-nucleotide polymorphisms (SNPs) associated with common autoimmune diseases were predominantly enriched in CpG demethylated regions (DRs) specifically present in naive Treg cells but much less enriched in activation-induced DRs common in Tconv and Treg cells. Naive Treg cell-specific DRs were largely included in Treg cell-specific super-enhancers and closely associated with transcription and other epigenetic changes in naive and effector Treg cells. Thus, naive Treg cell-specific CpG hypomethylation had a key role in controlling Treg cell-specific gene transcription and epigenetic modification. The results suggest possible contribution of altered function or development of natural Treg cells to the susceptibility to common autoimmune diseases.
    Keywords:  CD25; CTLA-4; DNA methylation; FoxP3; SNPs; autoimmune diseases; epigenome; genetic susceptibility; regulatory T cells; super-enhancer
  54. Oncoimmunology. 2020 ;9(1): 1741267
    Baruch EN, Ortenberg R, Avivi C, Anafi L, Dick-Necula D, Stossel C, Moshkovits Y, Itzhaki O, Besser MJ, Schachter J, Barshack I, Markel G.
      Omics analyses often result in dozens to hundreds of potential targets, requiring validation for their biological relevance. Current high-throughput functional investigation methods are frequently labor-intensive, expensive, and display low reproducibility. The Immune Co-Culture Cell Microarray (ICCM) is a formalin-fixed paraffin-embedded cell block microarray based on co-cultures of patient-derived tumor-infiltrating lymphocytes and their autologous melanoma cells. Each ICCM slide represents the same experiment and can be stained using standard immunohistochemistry and immunofluorescence techniques. Functional dynamics assessment of both proteins and microRNAs using ICCM stained slides demonstrated similar findings to flow cytometry assays and to previously published patient-derived biopsy reports.
    Keywords:  Immunotherapy; functional protein expression; immunological cytotoxicity test; melanoma; omics validation