bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒09‒27
eighty-one papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Nat Commun. 2020 09 21. 11(1): 4748
    Bailey MH, Meyerson WU, Dursi LJ, Wang LB, Dong G, Liang WW, Weerasinghe A, Li S, Kelso S, , , Saksena G, Ellrott K, Wendl MC, Wheeler DA, Getz G, Simpson JT, Gerstein MB, Ding L, .
      The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.
  2. Nat Commun. 2020 09 24. 11(1): 4837
    Li Y, Ivica NA, Dong T, Papageorgiou DP, He Y, Brown DR, Kleyman M, Hu G, Chen WW, Sullivan LB, Del Rosario A, Hammond PT, Vander Heiden MG, Chen J.
      ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
  3. Biochem Biophys Res Commun. 2020 Sep 21. pii: S0006-291X(20)31784-8. [Epub ahead of print]
    Lee SW, Commisso C.
      The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.
    Keywords:  EGFR; Glutamine; Nutrient stress; Pak; Pancreatic; Rac
  4. EMBO Rep. 2020 Sep 23. e50635
    Altea-Manzano P, Cuadros AM, Broadfield LA, Fendt SM.
      Nutrients are indispensable resources that provide the macromolecular building blocks and energy requirements for sustaining cell growth and survival. Cancer cells require several key nutrients to fulfill their changing metabolic needs as they progress through stages of development. Moreover, both cell-intrinsic and microenvironment-influenced factors determine nutrient dependencies throughout cancer progression-for which a comprehensive characterization remains incomplete. In addition to the widely studied role of genetic alterations driving cancer metabolism, nutrient use in cancer tissue may be affected by several factors including the following: (i) diet, the primary source of bodily nutrients which influences circulating metabolite levels; (ii) tissue of origin, which can influence the tumor's reliance on specific nutrients to support cell metabolism and growth; (iii) local microenvironment, which dictates the accessibility of nutrients to tumor cells; (iv) tumor heterogeneity, which promotes metabolic plasticity and adaptation to nutrient demands; and (v) functional demand, which intensifies metabolic reprogramming to fuel the phenotypic changes required for invasion, growth, or survival. Here, we discuss the influence of these factors on nutrient metabolism and dependence during various steps of tumor development and progression.
    Keywords:  cancer metabolism; diet; microenvironment; nutrients; tumor heterogeneity
  5. Cancers (Basel). 2020 Sep 21. pii: E2695. [Epub ahead of print]12(9):
    Ferri-Borgogno S, Barui S, McGee AM, Griffiths T, Singh PK, Piett CG, Ghosh B, Bhattacharyya S, Singhi A, Pradhan K, Verma A, Nagel Z, Maitra A, Gupta S.
      Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or "KAC") was generated by crossing Ptf1a-Cre; KrasG12D ("KC") mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in "KAC" as compared to "KC" mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of "KAC" mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous "KAC" mice revealed various compensatory ("escaper") mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of "escaper" mechanisms drive progression.
    Keywords:  DNA repair; SWI/SNF; mouse model; pancreatic cancer
  6. Cancer Metab. 2020 ;8 19
    Sharma N, Bhushan A, He J, Kaushal G, Bhardwaj V.
      Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant forms of cancer. Lack of effective treatment options and drug resistance contributes to the low survival among PDAC patients. In this study, we investigated the metabolic alterations in pancreatic cancer cells that do not respond to the EGFR inhibitor erlotinib. We selected erlotinib-resistant pancreatic cancer cells from MiaPaCa2 and AsPC1 cell lines. Metabolic profiling of erlotinib-resistant cells revealed a significant downregulation of glycolytic activity and reduced level of glycolytic metabolites compared to the sensitive cells. The resistant cells displayed elevated expression of the pentose phosphate pathway (PPP) enzymes involved in ROS regulation and nucleotide biosynthesis. The enhanced PPP elevated cellular NADPH/NADP+ ratio and protected the cells from reactive oxygen species (ROS)-induced damage. Inhibition of PPP using 6-aminonicotinamide (6AN) elevated ROS levels, induced G1 cell cycle arrest, and sensitized resistant cells to erlotinib. Genetic studies identified elevated PPP enzyme glucose-6-phosphate dehydrogenase (G6PD) as an important contributor to erlotinib resistance. Mechanistically, our data showed that upregulation of inhibitor of differentiation (ID1) regulates G6PD expression in resistant cells thus contributing to altered metabolic phenotype and reduced response to erlotinib. Together, our results highlight an underlying role of tumor metabolism in PDAC drug response and identify G6PD as a target to overcome drug resistance.
    Keywords:  Erlotinib resistance; Metabolic reprogramming; Pancreatic cancer
  7. Nat Metab. 2020 Sep 21.
    de Kivit S, Mensink M, Hoekstra AT, Berlin I, Derks RJE, Both D, Aslam MA, Amsen D, Berkers CR, Borst J.
      Following activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymus-derived Treg (tTreg) cells can become glycolytic in response to tumour necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3-TNFR2-activated tTreg cells is driven by PI3-kinase-mTOR signalling and supports tTreg cell identity and suppressive function. In contrast to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2hiCD4+CD25hiCD127lo effector T cells, which were FOXP3+IKZF2+, revealed an increase in glucose consumption and intracellular lactate levels, thus identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodelling, providing an additional avenue for drug targeting.
  8. Cell Metab. 2020 Sep 16. pii: S1550-4131(20)30483-6. [Epub ahead of print]
    Liu S, Dai Z, Cooper DE, Kirsch DG, Locasale JW.
      The nutritional source for catabolism in the tricarboxylic acid (TCA) cycle is a fundamental question in metabolic physiology. Limited by data and mathematical analysis, controversy exists. Using isotope-labeling data in vivo across several experimental conditions, we construct multiple models of central carbon metabolism and develop methods based on metabolic flux analysis (MFA) to solve for the preferences of glucose, lactate, and other nutrients used in the TCA cycle. We show that in nearly all circumstances, glucose contributes more than lactate as a substrate to the TCA cycle. This conclusion is verified in different animal strains from different studies and different administrations of 13C glucose, and is extended to multiple tissue types. Thus, this quantitative analysis of organismal metabolism defines the relative contributions of nutrient fluxes in physiology, provides a resource for analysis of in vivo isotope tracing data, and concludes that glucose is the major nutrient used in mammals.
    Keywords:  TCA cycle; glucose metabolism; isotope tracing; lactate; liver metabolism; metabolic flux analysis; mitochondrial metabolism; multi-tissue modeling; parameter sensitivity analysis; quantitative biology; systems biology
  9. FEBS J. 2020 Sep 22.
    González-Gualda E, Baker AG, Fruk L, Muñoz-Espín D.
      Cellular senescence is a physiological mechanism whereby a proliferating cell undergoes a stable cell cycle arrest upon damage or stress and elicits a secretory phenotype. This highly dynamic and regulated cellular state plays beneficial roles in physiology, such as during embryonic development and wound healing, but it can also result in antagonistic effects in age-related pathologies, degenerative disorders, aging and cancer. In an effort to better identify this complex state, and given that a universal marker has yet to be identified, a general set of hallmarks describing senescence has been established. However, as the senescent programme becomes more defined, further complexities, including phenotype heterogeneity, have emerged. This significantly complicates the recognition and evaluation of cellular senescence, especially within complex tissues and living organisms. To address these challenges, substantial efforts are currently being made towards the discovery of novel and more specific biomarkers, optimised combinatorial strategies, and the development of emerging detection techniques. Here, we compile such advances and present a multi-factorial guide to identify and assess cellular senescence in cell cultures, tissues and living organisms. The reliable assessment and identification of senescence is not only crucial for better understanding its underlying biology, but also imperative for the development of diagnostic and therapeutic strategies aimed at targeting senescence in the clinic.
    Keywords:  Cellular senescence; ageing; assessment; biomarkers; detection
  10. Nature. 2020 Sep 23.
    Lawson KA, Sousa CM, Zhang X, Kim E, Akthar R, Caumanns JJ, Yao Y, Mikolajewicz N, Ross C, Brown KR, Zid AA, Fan ZP, Hui S, Krall JA, Simons DM, Slater CJ, De Jesus V, Tang L, Singh R, Goldford JE, Martin S, Huang Q, Francis EA, Habsid A, Climie R, Tieu D, Wei J, Li R, Tong AHY, Aregger M, Chan KS, Han H, Wang X, Mero P, Brumell JH, Finelli A, Ailles L, Bader G, Smolen GA, Kingsbury GA, Hart T, Kung C, Moffat J.
      The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
  11. Surg Oncol. 2020 Sep 07. pii: S0960-7404(20)30381-9. [Epub ahead of print]35 285-297
    Weniger M, Moir J, Damm M, Maggino L, Kordes M, Rosendahl J, Ceyhan GO, Schorn S, .
      INTRODUCTION: The number of people aged 60 and above will rise from 46 million in 2015 to 157 in 2050 million, exceeding 30% of the population in many western countries. Consequently, the demand for oncological therapy for elderly patients will increase within the next decades. Currently, sufficient data on neoadjuvant therapy (NTx) of pancreatic cancer in elderly patients are lacking.METHODS: Data of a multinational, retrospective database were screened for patients having received preoperative FOLFIRINOX (FFx) or Gemcitabine/nab-paclitaxel (GNP) for locally advanced and borderline resectable pancreatic cancer (LAPC/BRPC) before June 2017. Data were included in an intention-to-treat-analysis and outcomes were compared between non-aged and elderly patients using a cut-off age of 63 (comparison 1) and 70 years (comparison 2).
    RESULTS: Of 165 patients receiving NTx, 76 and 33 were older than 63 and 70 years. Baseline characteristics revealed that elderly patients preferably undergo GNP (comparison 1: p = 0.063; comparison2: p = 0.005), with less cycles of NTx (comparison 1: p = 0.057). Whereas reductions of NTx dosage was more common in elderly patients in comparison 1 (p = 0.003), resection rates (p = 0.575; p = 1.000) and median survival (p = 0.406; p = 0.499) were not different. Whereas resected patients showed no differences in survival (p = 0.328; p = 0.132), patients aged >70 years showed a decreased progression-free survival (p = 0.019).
    CONCLUSION: Elderly patients treated with NTx show encouragingly high resection rates. If comorbidities allow for FFx or GNP, elderly patients with LAPC/BRPC can offered NTx with the prospect of survival comparable to younger patients.
    Keywords:  Advanced age; Neoadjuvant chemotherapy; Survival
  12. Trends Cell Biol. 2020 Sep 23. pii: S0962-8924(20)30170-7. [Epub ahead of print]
    Nakao M, Tanaka H, Koga T.
      Cellular senescence is a state of permanent cell cycle arrest accompanied by unique secretory actions, which influences tissue formation, tumor suppression and aging in vivo. Recent evidences suggest that metabolic and epigenomic reprogram cooperatively creates phenotypic differences of senescent cells, which may provide new clues to control aging processes.
    Keywords:  cellular senescence; epigenome; gene regulation; metabolism; senescence-associated secretory phenotype
  13. Science. 2020 Sep 25. pii: eabc5809. [Epub ahead of print]369(6511):
    McKenna MJ, Sim SI, Ordureau A, Wei L, Harper JW, Shao S, Park E.
      Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.
  14. Oncogene. 2020 Sep 21.
    Li LY, Chen XS, Wang KS, Guan YD, Ren XC, Cao DS, Sun XY, Li AX, Tao YG, Zhang Y, Yin MZ, Wang XL, Wu MH, Yang JM, Cheng Y.
      Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.
  15. J Biol Chem. 2020 Sep 25. pii: jbc.RA120.013893. [Epub ahead of print]
    Onodera T, Momose I, Adachi H, Yamazaki Y, Sawa R, Ohba SI, Kawada M.
      Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as glutathione and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in glutathione; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.
    Keywords:  cancer therapy; chemical biology; drug discovery; drug screening; redox regulation
  16. Mol Metab. 2020 Sep 18. pii: S2212-8778(20)30159-9. [Epub ahead of print] 101085
    Mittenbühler MJ, Saedler K, Nolte H, Kern L, Zhou J, Qian SB, Meder L, Ullrich RT, Brüning JC, Wunderlich FT.
      OBJECTIVE: Single nucleotide polymorphisms in the FTO gene encoding an m6Am and an m6A demethylase are associated with obesity. Moreover, recent studies have linked a dysregulation of m6A modifications and its machinery, including FTO, to the development of several forms of cancers. However, the functional role of hepatic FTO in metabolism and the development and progression of hepatocellular carcinoma (HCC), a proteotypic obesity-associated cancer, remains unclear. Thus, we aimed at uncovering the role of hepatic FTO in metabolism and in the initiation and progression of HCC in vivo.METHODS: We have generated mice with hepatic FTO deficiency (FTOL-KO). The effect of hepatic FTO on metabolism was investigated by extensive metabolic phenotyping. To determine the impact of hepatic FTO on HCC development, FTOL-KO and Ctrl mice were subjected to long-term Diethylnitrosamine (DEN)-induced HCC-development and the tumor initiation phase was examined via a short-term DEN protocol.
    RESULTS: In long-term DEN experiments, FTOL-KO mice exhibit increased HCC burden compared to Ctrl mice. In the tumor initiation phase, Ctrl mice display a dynamic regulation of FTO upon induction of liver damage, while this response is abrogated in FTO-deficient mice. Proteomic analyses revealed that liver damage-induced increases in FTO expression reduces CUL4A protein abundance. Functionally, simultaneous knockdown of Cul4a reverses the increased hepatocyte proliferation observed upon loss of FTO.
    CONCLUSION: Collectively, our study demonstrates that hepatic FTO is dispensable for the control of energy homeostasis and glucose metabolism. However, we show a protective function of FTO in liver carcinogenesis and suggest the FTO-dependent dynamic mRNA demethylation of Cul4a in the initiation of HCC development contributes to this effect.
    Keywords:  Cul4a; FTO; Hepatocellular Carcinoma; m(6)A
  17. Cancer Res. 2020 Sep 23. pii: canres.0360.2020. [Epub ahead of print]
    Jeong GY, Park MK, Choi HJ, An HW, Park YU, Choi HJ, Park J, Kim HY, Son T, Lee H, Min KW, Oh YH, Lee JY, Kong G.
      Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In breast cancer patients, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. In vivo, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer.
  18. Nat Commun. 2020 09 24. 11(1): 4828
    Kidiyoor GR, Li Q, Bastianello G, Bruhn C, Giovannetti I, Mohamood A, Beznoussenko GV, Mironov A, Raab M, Piel M, Restuccia U, Matafora V, Bachi A, Barozzi S, Parazzoli D, Frittoli E, Palamidessi A, Panciera T, Piccolo S, Scita G, Maiuri P, Havas KM, Zhou ZW, Kumar A, Bartek J, Wang ZQ, Foiani M.
      ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.
  19. N Engl J Med. 2020 09 24. 383(13): 1207-1217
    Hong DS, Fakih MG, Strickler JH, Desai J, Durm GA, Shapiro GI, Falchook GS, Price TJ, Sacher A, Denlinger CS, Bang YJ, Dy GK, Krauss JC, Kuboki Y, Kuo JC, Coveler AL, Park K, Kim TW, Barlesi F, Munster PN, Ramalingam SS, Burns TF, Meric-Bernstam F, Henary H, Ngang J, Ngarmchamnanrith G, Kim J, Houk BE, Canon J, Lipford JR, Friberg G, Lito P, Govindan R, Li BT.
      BACKGROUND: No therapies for targeting KRAS mutations in cancer have been approved. The KRAS p.G12C mutation occurs in 13% of non-small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRASG12C.METHODS: We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1.
    RESULTS: A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma.
    CONCLUSIONS: Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 number, NCT03600883.).
  20. J Clin Endocrinol Metab. 2020 Sep 21. pii: dgaa673. [Epub ahead of print]
    Hasan B, Nayfeh T, Alzuabi M, Wang Z, Kuchkuntla AR, Prokop LJ, Newman CB, Murad MH, Rajjo TI.
      BACKGROUND: Excess adipose tissue is associated with an abnormal lipid profile that may improve with weight reduction. In this meta-analysis, we aimed to estimate the magnitude of change in lipid parameters associated with weight loss in adults who are overweight or obese.METHODS: We searched MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, and Scopus from 2013 to September, 2018. We included randomized-controlled trials (RCTs) that evaluated interventions to treat adult obesity (lifestyle, pharmacologic and surgical) with ≥6-month follow-up.
    RESULTS: We included 73 RCTs with moderate-to-low risk of bias, enrolling 32,496 patients (mean age, 48.1 years; weight, 101.6 kg; and body mass index [BMI], 36.3 kg/m 2). Lifestyle interventions (diet, exercise, or both), pharmacotherapy, and bariatric surgery were associated with reduced triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) concentrations and increased high-density lipoprotein cholesterol (HDL-C) at 6 and 12 months. Data below are for changes in lipid parameters after 12 months of the intervention with 95% confidence intervals (CIs). Following lifestyle interventions, per 1 kg of weight lost, TGs were reduced by -4.0 mg/dL (CI, -5.24 to -2.77), LDL-C was reduced by -1.28 mg/dL (CI, -2.19 to -0.37), and HDL-C increased by 0.46 mg/dL (CI, 0.20-0.71). Following pharmacologic interventions, per 1 kg of weight lost, TGs were reduced by -1.25 mg/dL (CI, -2.94 to 0.43), LDL-C was reduced by -1.67 mg/dL (CI, -2.28 to -1.06), and HDL-C increased by 0.37 mg/dL (CI, 0.23-0.52). Following bariatric surgery, per 1 kg of weight lost, TGs were reduced by -2.47 mg/dL (CI, -3.14 to -1.80), LDL-C was reduced by -0.33 mg/dL (CI, -0.77 to 0.10), and HDL-C increased by 0.42 mg/dL (CI, 0.37-0.47). Low-carbohydrate diets resulted in reductions in TGs and increases in HDL-C, whereas low-fat diets resulted in reductions in TGs and LDL-C and increases in HDL-C. Results were consistent across malabsorptive and restrictive surgery.
    CONCLUSIONS: Weight loss in adults is associated with statistically significant changes in serum lipids. The reported magnitude of improvement can help in setting expectations, inform shared decision making, and facilitate counseling.
    Keywords:  Weight loss; meta-analysis; obesity; serum lipids; systematic review
  21. Cancer Metab. 2020 ;8 20
    Tait-Mulder J, Hodge K, Sumpton D, Zanivan S, Vazquez A.
      Background: Mitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor, it is not clear how formate induces pyrimidine synthesis.Methods: Here we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis.
    Results: We discover that formate induces phosphorylation of carbamoyl phosphate synthetase (CAD), which is known to increase CAD enzymatic activity. Mechanistically, formate induces mechanistic target of rapamycin complex 1 (mTORC1) activity as quantified by phosphorylation of its targets S6, 4E-BP1, S6K1 and CAD. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. Furthermore, we show that the formate-dependent induction of mTOR signalling and CAD phosphorylation is dependent on an increase in purine synthesis.
    Conclusions: We conclude that formate activates mTORC1 and induces pyrimidine synthesis via the mTORC1-dependent phosphorylation of CAD.
  22. Sci Adv. 2020 Sep;pii: eabb0205. [Epub ahead of print]6(39):
    Gambardella G, Staiano L, Moretti MN, De Cegli R, Fagnocchi L, Di Tullio G, Polletti S, Braccia C, Armirotti A, Zippo A, Ballabio A, De Matteis MA, di Bernardo D.
      Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.
  23. Diabetologia. 2020 Sep 22.
    Chareyron I, Christen S, Moco S, Valsesia A, Lassueur S, Dayon L, Wollheim CB, Santo Domingo J, Wiederkehr A.
      AIMS/HYPOTHESIS: In islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress.METHODS: Respiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters.
    RESULTS: At early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%, p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%, p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus-secretion coupling as assessed by cytosolic calcium signalling, was restored.
    CONCLUSION/INTERPRETATION: We propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress. Graphical abstract.
    Keywords:  Beta cells; Calcium; Human islets; Metabolomics; Mitochondria
  24. Cancer Cell. 2020 Sep 15. pii: S1535-6108(20)30426-8. [Epub ahead of print]
    Gabitova-Cornell L, Surumbayeva A, Peri S, Franco-Barraza J, Restifo D, Weitz N, Ogier C, Goldman AR, Hartman TR, Francescone R, Tan Y, Nicolas E, Shah N, Handorf EA, Cai KQ, O'Reilly AM, Sloma I, Chiaverelli R, Moffitt RA, Khazak V, Fang CY, Golemis EA, Cukierman E, Astsaturov I.
      Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor β signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.
    Keywords:  TGF-β signaling; cholesterol metabolism; epithelial-to-mesenchymal transition; pancreatic cancer
  25. J Biol Chem. 2020 Sep 21. pii: jbc.RA120.013565. [Epub ahead of print]
    Meguro S, Zhuang X, Kirisako H, Nakatogawa H.
      In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by post-translational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.
    Keywords:  Atg36; Hrr25; Pex3; Saccharomyces cerevisiae; autophagy; peroxisome; pexophagy; proteasome; protein degradation; protein phosphorylation
  26. J Biol Chem. 2020 Sep 25. pii: jbc.RA120.014790. [Epub ahead of print]
    Amraei R, Alwani T, Ho RX, Aryan Z, Wang S, Rahimi N.
      Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin and lipopolysaccharide (LPS). Manipulating the IκB kinaseβ (IKKβ) activity coupled with in vivo and in vitro kinase assays demonstrated that IKKb is a key serine/threonine kinase activated by autophagy stimuli and catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase (AMPK), which leads to phosphorylation of major pro-autophagy proteins, ULK1 and Beclin-1 (BECN1), increased LC3-II levels and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer, and suggest that IGPR-1 may serve as a promising therapeutic target.
    Keywords:  AMP-activated kinase (AMPK); IGPR-1; Serine phosphorylation of IGPR-1; autophagy; cell adhesion molecule; cell surface receptor; cell-cell interaction; immunoglobulin-like domain; nutrient deprivation; post-translational modification (PTM); serine/threonine protein kinase
  27. Biochim Biophys Acta Mol Cell Res. 2020 Sep 16. pii: S0167-4889(20)30215-9. [Epub ahead of print] 118857
    Peng X, Yang L, Ma Y, Li X, Yang S, Li Y, Wu B, Tang S, Zhang F, Zhang B, Liu J, Li H.
      Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKβ subunits (CA-IKKβ) on autophagy and endosomal system interactions. We confirm that CA-IKKβ induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKβ inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKβ also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKβ drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.
    Keywords:  Amphisomes; Autophagy; Extracellular vesicles; IKKβ; Multivesicular bodies; Tumor cell
  28. Nat Protoc. 2020 Sep 25.
    Scherer M, Nazarov PV, Toth R, Sahay S, Kaoma T, Maurer V, Vedeneev N, Plass C, Lengauer T, Walter J, Lutsik P.
      DNA methylation profiling offers unique insights into human development and diseases. Often the analysis of complex tissues and cell mixtures is the only feasible option to study methylation changes across large patient cohorts. Since DNA methylomes are highly cell type specific, deconvolution methods can be used to recover cell type-specific information in the form of latent methylation components (LMCs) from such 'bulk' samples. Reference-free deconvolution methods retrieve these components without the need for DNA methylation profiles of purified cell types. Currently no integrated and guided procedure is available for data preparation and subsequent interpretation of deconvolution results. Here, we describe a three-stage protocol for reference-free deconvolution of DNA methylation data comprising: (i) data preprocessing, confounder adjustment using independent component analysis (ICA) and feature selection using DecompPipeline, (ii) deconvolution with multiple parameters using MeDeCom, RefFreeCellMix or EDec and (iii) guided biological inference and validation of deconvolution results with the R/Shiny graphical user interface FactorViz. Our protocol simplifies the analysis and guides the initial interpretation of DNA methylation data derived from complex samples. The harmonized approach is particularly useful to dissect and evaluate cell heterogeneity in complex systems such as tumors. We apply the protocol to lung cancer methylomes from The Cancer Genome Atlas (TCGA) and show that our approach identifies the proportions of stromal cells and tumor-infiltrating immune cells, as well as associations of the detected components with clinical parameters. The protocol takes slightly >3 d to complete and requires basic R skills.
  29. Autophagy. 2020 Sep 20. 1-2
    Miceli C, Roccio F, Penalva-Mousset L, Morel E, Codogno P, Dupont N.
      The kidney, similar to many other organs, has to face shear stress induced by biological fluids. How epithelial kidney cells respond to shear stress is poorly understood. Recently we showed in vitro and in vivo that proximal tubule epithelial cells use lipophagy to fuel mitochondria with fatty acids. Lipophagy is stimulated by a primary cilium-dependent signaling that converges at AMP kinase. AMP kinase is a central signaling hub to trigger lipophagy and also to stimulate mitochondrial biogenesis. These two pathways contribute to generate ATP needed to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis. These findings demonstrate the role of the primary cilium and selective macroautophagy/autophagy to integrate shear stress and to sustain the execution of a specific cellular program.
    Keywords:  macroautophagy; metabolism; nephrology; oxidative phosphorylation
  30. Cell Metab. 2020 Sep 11. pii: S1550-4131(20)30484-8. [Epub ahead of print]
    Omori S, Wang TW, Johmura Y, Kanai T, Nakano Y, Kido T, Susaki EA, Nakajima T, Shichino S, Ueha S, Ozawa M, Yokote K, Kumamoto S, Nishiyama A, Sakamoto T, Yamaguchi K, Hatakeyama S, Shimizu E, Katayama K, Yamada Y, Yamazaki S, Iwasaki K, Miyoshi C, Funato H, Yanagisawa M, Ueno H, Imoto S, Furukawa Y, Yoshida N, Matsushima K, Ueda HR, Miyajima A, Nakanishi M.
      Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
    Keywords:  NASH; aging; p16Ink4a; senescence; single-cell transcriptomics
  31. Science. 2020 Sep 24. pii: eabc4209. [Epub ahead of print]
    Kampjut D, Sazanov LA.
      Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here we present cryo-EM structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3-2.5 Å. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during "open" to "closed" state transitions of the enzyme. In the induced deactive state, the "open" conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.
  32. Oncogene. 2020 Sep 25.
    Peng H, Wang Y, Luo W.
      Metabolic reprogramming fulfils increased nutrient demands and regulates numerous oncogenic processes in tumors, leading to tumor malignancy. Branched-chain amino acids (BCAAs, i.e., valine, leucine, and isoleucine) function as nitrogen donors to generate macromolecules such as nucleotides and are indispensable for human cancer cell growth. The cell-autonomous and non-autonomous roles of altered BCAA metabolism have been implicated in cancer progression and the key proteins in the BCAA metabolic pathway serve as possible prognostic and diagnostic biomarkers in human cancers. Here we summarize how BCAA metabolic reprogramming is regulated in cancer cells and how it influences cancer progression.
  33. Nat Chem Biol. 2020 Sep 21.
    Tamura T, Fujisawa A, Tsuchiya M, Shen Y, Nagao K, Kawano S, Tamura Y, Endo T, Umeda M, Hamachi I.
      Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.
  34. Cell Div. 2020 ;15 11
    Mitchell E, Mellor CEL, Purba TS.
      Background: XMU-MP-1 is an inhibitor of the Hippo pathway kinases MST1/2 and has been shown to promote the downstream activation of the pro-proliferative, pro-regenerative and anti-apoptotic transcriptional regulator YAP1. We tested whether XMU-MP-1 can activate YAP1 in a model human mini-organ, namely the hair follicle, to determine whether it can be pharmacologically exploited to promote regeneration in the hair follicle as a novel strategy to treat pathological hair loss disorders.Results: XMU-MP-1 treatment inhibited MOB1 phosphorylation but did not increase active YAP1 in the hair follicle. Rather than promote proliferation, XMU-MP-1 serendipitously decreased the number of Ki-67+, EdU+ and phospho histone H3+ hair matrix keratinocytes and antagonised the cytotoxic effects of paclitaxel.
    Conclusions: XMU-MP-1 perturbs epithelial cell cycle progression in a model human mini-organ. This may arise as an off-target effect, especially when XMU-MP-1 has been described to strongly inhibit 21 additional kinases beyond MST1/2. Therefore, whilst these effects may be dependent on tissue context, researchers should exercise caution when interpreting the effects of XMU-MP-1, especially in tissues with actively proliferating cell populations.
    Keywords:  Alopecia; Aurora B; Cell cycle; Chemotherapy; Hair follicle; Hippo; MST1/2; Proliferation; XMU-MP-1; YAP1
  35. Mitochondrion. 2020 Sep 17. pii: S1567-7249(20)30180-X. [Epub ahead of print]55 54-63
    Hwang SY, Kuk MU, Kim JW, Lee YH, Lee YS, Choy HE, Park SC, Park JT.
      Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration.
    Keywords:  ATM inhibition; Metabolic reprogrammer; Mitochondria; P53; Senescence alleviation
  36. Cancer Sci. 2020 Sep 24.
    Fujiwara N, Shibutani S, Sakai Y, Watanabe T, Kitabayashi I, Oshima H, Oshima M, Hoshida H, Akada R, Usui T, Ohama T, Sato K.
      Protein Phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells. In this study, we show that PP6 protein levels increase in response to pharmacologic and genetic inhibition of autophagy. PP6 associates with autophagic adaptor protein p62/SQSTM1 and is degraded in a p62-dependent manner. Accordingly, protein levels of PP6 and p62 fluctuate in concert under different physiological and pathophysiological conditions. Our data reveal that PP6 is regulated by p62-dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy.
    Keywords:  autophagy; cell biology; p62/SQSTM1; protein phosphatase 2A; protein phosphatase 6
  37. Life Sci. 2020 Sep 18. pii: S0024-3205(20)31206-6. [Epub ahead of print]261 118453
    de Souza Teixeira AA, Lira FS, Rosa-Neto JC.
      Aging is associated with gradual decline in numerous physiological processes, including a reduction in metabolic functions and immunological system. The circadian rhythm plays a vital role in health, and prolonged clock disruptions are associated with chronic diseases. The relationships between clock genes, aging, and immunosenescence are not well understood. Inflammation is an immune response triggered in living organisms in response to the danger associated with pathogens and injury. The term 'inflammaging' has been used to describe the chronic low-grade-inflammation that develops with advancing age and predicts susceptibility to age-related pathologies. Equilibrium between pro-and anti-inflammatory cytokines is needed for healthy aging and longevity. Sedentary and poor nutrition style life indices a disruption in circadian rhythm promoting an increase in pro-inflammatory factors or leads for chronic low-grade inflammation. Moreover, signals mediated by pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6, might accentuate of the muscle loss during aging. Circadian clock is important to maintain the physiological functions, as maintenance of immune system. A strategy for imposes rhythmicity in the physiological systems may be adopted of exercise training routine. The lifelong regular practice of physical exercise decelerates the processes of aging, providing better quality and prolongation of life. Thus, in this review, we will focus on how aging affects circadian rhythms and its relationship to inflammatory processes (inflammaging), as well as the role of physical exercise as a regulator of the circadian rhythm, promoting aging with rhythmicity.
    Keywords:  Aging; Circadian rhythms; Clock genes; Immunosenescence; Inflammaging
  38. Cell Calcium. 2020 Sep 11. pii: S0143-4160(20)30130-5. [Epub ahead of print]92 102288
    Tomar D, Elrod JW.
      Calcium (Ca2+) is known to stimulate mitochondrial bioenergetics through the modulation of TCA cycle dehydrogenases and electron transport chain (ETC) complexes. This is hypothesized to be an essential pathway of energetic control to meet cellular ATP demand. While regulatory mechanisms of mitochondrial calcium uptake have been reported, it remains unknown if metabolite flux itself feedsback to regulate mitochondrial calcium (mCa2+) uptake. This hypothesis was recently tested by Nemani et al. (Sci. Signal. 2020) where the authors report that TCA cycle substrate flux regulates the mitochondrial calcium uniporter channel gatekeeper, mitochondrial calcium uptake 1 (MICU1), gene transcription in an early growth response protein 1 (EGR1) dependent fashion. They posit this is a regulatory feedback mechanism to control ionic homeostasis and mitochondrial bioenergetics with changing fuel availability. Here, we provide a historical overview of mitochondrial calcium exchange and comprehensive appraisal of these results in the context of recent literature and discuss possible regulatory pathways of mCa2+ uptake and mitochondrial bioenergetics.
    Keywords:  Calcium; Energetics; MCU; MICU1; MPC; Mitochondria; OXPHOS; TCA cycle; TCA substrates
  39. Elife. 2020 Sep 22. pii: e56686. [Epub ahead of print]9
    Gitschlag BL, Tate AT, Patel MR.
      Cooperation and cheating are widespread evolutionary strategies. While cheating confers an advantage to individual entities within a group, competition between groups favors cooperation. Selfish or cheater mitochondrial DNA (mtDNA) proliferates within hosts while being selected against at the level of host fitness. How does environment shape cheater dynamics across different selection levels? Focusing on food availability, we address this question using heteroplasmic Caenorhabditis elegans. We find that the proliferation of selfish mtDNA within hosts depends on nutrient status stimulating mtDNA biogenesis in the developing germline. Interestingly, mtDNA biogenesis is not sufficient for this proliferation, which also requires the stress-response transcription factor FoxO/DAF-16. At the level of host fitness, FoxO/DAF-16 also prevents food scarcity from accelerating the selection against selfish mtDNA. This suggests that the ability to cope with nutrient stress can promote host tolerance of cheaters. Our study delineates environmental effects on selfish mtDNA dynamics at different levels of selection.
    Keywords:  C. elegans; cheating; evolutionary biology; genetics; genomics; heteroplasmy; metabolism; mitochondria; multilevel selection; nutrient availability
  40. Nat Rev Endocrinol. 2020 Sep 22.
    Fritzen AM, Lundsgaard AM, Kiens B.
      Both the consumption of a diet rich in fatty acids and exercise training result in similar adaptations in several skeletal muscle proteins. These adaptations are involved in fatty acid uptake and activation within the myocyte, the mitochondrial import of fatty acids and further metabolism of fatty acids by β-oxidation. Fatty acid availability is repeatedly increased postprandially during the day, particularly during high dietary fat intake and also increases during, and after, aerobic exercise. As such, fatty acids are possible signalling candidates that regulate transcription of target genes encoding proteins involved in muscle lipid metabolism. The mechanism of signalling might be direct or indirect targeting of peroxisome proliferator-activated receptors by fatty acid ligands, by fatty acid-induced NAD+-stimulated activation of sirtuin 1 and/or fatty acid-mediated activation of AMP-activated protein kinase. Lactate might also have a role in lipid metabolic adaptations. Obesity is characterized by impairments in fatty acid oxidation capacity, and individuals with obesity show some rigidity in increasing fatty acid oxidation in response to high fat intake. However, individuals with obesity retain improvements in fatty acid oxidation capacity in response to exercise training, thereby highlighting exercise training as a potential method to improve lipid metabolic flexibility in obesity.
  41. Nat Commun. 2020 09 24. 11(1): 4841
    Kocher HM, Basu B, Froeling FEM, Sarker D, Slater S, Carlin D, deSouza NM, De Paepe KN, Goulart MR, Hughes C, Imrali A, Roberts R, Pawula M, Houghton R, Lawrence C, Yogeswaran Y, Mousa K, Coetzee C, Sasieni P, Prendergast A, Propper DJ.
      Pre-clinical models have shown that targeting pancreatic stellate cells with all-trans-retinoic-acid (ATRA) reprograms pancreatic stroma to suppress pancreatic ductal adenocarcinoma (PDAC) growth. Here, in a phase Ib, dose escalation and expansion, trial for patients with advanced, unresectable PDAC (n = 27), ATRA is re-purposed as a stromal-targeting agent in combination with gemcitabine-nab-paclitaxel chemotherapy using a two-step adaptive continual re-assessment method trial design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D, primary outcome) is the FDA/EMEA approved dose of gemcitabine-nab-paclitaxel along-with ATRA (45 mg/m2 orally, days 1-15/cycle). Dose limiting toxicity (DLT) is grade 4 thrombocytopenia (n = 2). Secondary outcomes show no detriment to ATRA pharmacokinetics.. Median overall survival for RP2D treated evaluable population, is 11.7 months (95%CI 8.6-15.7 m, n = 15, locally advanced (2) and metastatic (13)). Exploratory pharmacodynamics studies including changes in diffusion-weighted (DW)-MRI measured apparent diffusion coefficient after one cycle, and, modulation of cycle-specific serum pentraxin 3 levels over various cycles indicate stromal modulation. Baseline stromal-specific retinoid transport protein (FABP5, CRABP2) expression may be predicitve of response. Re-purposing ATRA as a stromal-targeting agent with gemcitabine-nab-paclitaxel is safe and tolerable. This combination will be evaluated in a phase II randomized controlled trial for locally advanced PDAC. Clinical trial numbers: EudraCT: 2015-002662-23; NCT03307148. Trial acronym: STARPAC.
  42. Nat Immunol. 2020 Sep 21.
    Li P, Lu M, Shi J, Gong Z, Hua L, Li Q, Lim B, Zhang XH, Chen X, Li S, Shultz LD, Ren G.
      Acquisition of a lipid-laden phenotype by immune cells has been defined in infectious diseases and atherosclerosis but remains largely uncharacterized in cancer. Here, in breast cancer models, we found that neutrophils are induced to accumulate neutral lipids upon interaction with resident mesenchymal cells in the premetastatic lung. Lung mesenchymal cells elicit this process through repressing the adipose triglyceride lipase (ATGL) activity in neutrophils in prostaglandin E2-dependent and -independent manners. In vivo, neutrophil-specific deletion of genes encoding ATGL or ATGL inhibitory factors altered neutrophil lipid profiles and breast tumor lung metastasis in mice. Mechanistically, lipids stored in lung neutrophils are transported to metastatic tumor cells through a macropinocytosis-lysosome pathway, endowing tumor cells with augmented survival and proliferative capacities. Pharmacological inhibition of macropinocytosis significantly reduced metastatic colonization by breast tumor cells in vivo. Collectively, our work reveals that neutrophils serve as an energy reservoir to fuel breast cancer lung metastasis.
  43. Trends Mol Med. 2020 Sep 17. pii: S1471-4914(20)30190-8. [Epub ahead of print]
    Saeb-Parsy K, Martin JL, Summers DM, Watson CJE, Krieg T, Murphy MP.
      Advances in surgical procedures, technology, and immune suppression have transformed organ transplantation. However, the metabolic changes that occur during organ retrieval, storage, and implantation have been relatively neglected since the developments many decades ago of cold storage organ preservation solutions. In this review we discuss how the metabolic changes that occur within the organ during transplantation, particularly those associated with mitochondria, may contribute to the outcome. We show how a better understanding of these processes can lead to changes in surgical practice and the development of new drug classes to improve the function and longevity of transplanted grafts, while increasing the pool of organs available for transplantation.
    Keywords:  ischemia–reperfusion injury; metabolism.; mitochondria; transplantation
  44. Ageing Res Rev. 2020 Sep 16. pii: S1568-1637(20)30291-9. [Epub ahead of print] 101156
    Uyar B, Palmer D, Kowald A, Escobar HM, Barrantes I, Möller S, Akalin A, Fuellen G.
      Single-cell gene expression (transcriptomics) data are becoming robust and abundant, and are increasingly used to track organisms along their life-course. This allows investigation into how aging affects cellular transcriptomes, and how changes in transcriptomes may underlie aging, including chronic inflammation (inflammaging), immunosenescence and cellular senescence. We compiled and tabulated aging-related single-cell datasets published to date, collected and discussed relevant findings, and inspected some of these datasets ourselves. We specifically note insights that cannot (or not easily) be based on bulk data. For example, in some datasets, the fraction of cells expressing p16 (CDKN2A), one of the most prominent markers of cellular senescence, was reported to increase, in addition to its upregulated mean expression over all cells. Moreover, we found evidence for inflammatory processes in most datasets, some of these driven by specific cells of the immune system. Further, single-cell data are specifically useful to investigate whether transcriptional heterogeneity (also called noise or variability) increases with age, and many (but not all) studies in our review report an increase in such heterogeneity. Finally, we demonstrate some stability of marker gene expression patterns across closely similar studies and suggest that single-cell experiments may hold the key to provide detailed insights whenever interventions (countering aging, inflammation, senescence, disease, etc.) are affecting cells depending on cell type.
    Keywords:  Aging; Biomarkers; Cellular senescence; Inflammaging; Single-cell sequencing; Transcriptional heterogeneity
  45. Cancer Cell. 2020 Sep 14. pii: S1535-6108(20)30427-X. [Epub ahead of print]
    Ros S, Wright AJ, D'Santos P, Hu DE, Hesketh RL, Lubling Y, Georgopoulou D, Lerda G, Couturier DL, Razavi P, Pelossof R, Batra AS, Mannion E, Lewis DY, Martin A, Baird RD, Oliveira M, de Boo LW, Linn SC, Scaltriti M, Rueda OM, Bruna A, Caldas C, Brindle KM.
      PIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance.
    Keywords:  FDG-PET; FOXM1; MRI; PI3K alpha inhibition; biomarker; breast cancer; hexokinase 2; hyperpolarized [1-(13)C]pyruvate; lactate dehydrogenase; treatment response
  46. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31170-0. [Epub ahead of print]32(12): 108181
    Canesin G, Di Ruscio A, Li M, Ummarino S, Hedblom A, Choudhury R, Krzyzanowska A, Csizmadia E, Palominos M, Stiehm A, Ebralidze A, Chen SY, Bassal MA, Zhao P, Tolosano E, Hurley L, Bjartell A, Tenen DG, Wegiel B.
      Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.
    Keywords:  DNA G-quadruplexes; c-myc; heme; heme oxygenase-1; hemopexin; metastases; prostate cancer
  47. Cell Death Dis. 2020 Sep 24. 11(9): 798
    Chen Q, Sun X, Luo X, Wang J, Hu J, Feng Y.
      Cellular senescence is a stress response of human cells that removes potentially harmful cells by initiating cell cycle arrest. Inducing senescence of tumor cells may be an effective tumor-inhibiting strategy. In this study we found that PIK3R3 could inhibit the cell senescence of colorectal cancer cells and promote cell proliferation through the p53/p21 signal pathway. PIK3R3 could bind to p53 and inhibit the binding of p53 to the p21 gene promoter region, and thus affecting the transcriptional activity of p21 gene. Our study has provided new evidence of the role of PIK3R3 in p53 regulation and inhibition of PIK3R3 may be one of the potential targets of tumor therapy.
  48. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 202011785. [Epub ahead of print]
    Sens P.
      Cell crawling requires the generation of intracellular forces by the cytoskeleton and their transmission to an extracellular substrate through specific adhesion molecules. Crawling cells show many features of excitable systems, such as spontaneous symmetry breaking and crawling in the absence of external cues, and periodic and propagating waves of activity. Mechanical instabilities in the active cytoskeleton network and feedback loops in the biochemical network of activators and repressors of cytoskeleton dynamics have been invoked to explain these dynamical features. Here, I show that the interplay between the dynamics of cell-substrate adhesion and linear cellular mechanics is sufficient to reproduce many nonlinear dynamical patterns observed in spreading and crawling cells. Using an analytical formalism of the molecular clutch model of cell adhesion, regulated by local mechanical forces, I show that cellular traction forces exhibit stick-slip dynamics resulting in periodic waves of protrusion/retraction and propagating waves along the cell edge. This can explain spontaneous symmetry breaking and polarization of spreading cells, leading to steady crawling or bipedal motion, and bistability, where persistent cell motion requires a sufficiently strong transient external stimulus. The model also highlights the role of membrane tension in providing the long-range mechanical communication across the cell required for symmetry breaking.
    Keywords:  cell motility; membrane tension; stick–slip; symmetry breaking
  49. EMBO Rep. 2020 Sep 25. e50202
    Hara Y, Yanatori I, Tanaka A, Kishi F, Lemasters JJ, Nishina S, Sasaki K, Hino K.
      Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.
    Keywords:  hepatocellular carcinoma; iron chelator; mitochondria; mitochondrial ferritin; mitophagy
  50. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 202001966. [Epub ahead of print]
    Fernandez Lahore G, Raposo B, Lagerquist M, Ohlsson C, Sabatier P, Xu B, Aoun M, James J, Cai X, Zubarev RA, Nandakumar KS, Holmdahl R.
      It has proven difficult to identify the underlying genes in complex autoimmune diseases. Here, we use forward genetics to identify polymorphisms in the vitamin D receptor gene (Vdr) promoter, controlling Vdr expression and T cell activation. We isolated these polymorphisms in a congenic mouse line, allowing us to study the immunomodulatory properties of VDR in a physiological context. Congenic mice overexpressed VDR selectively in T cells, and thus did not suffer from calcemic effects. VDR overexpression resulted in an enhanced antigen-specific T cell response and more severe autoimmune phenotypes. In contrast, vitamin D3-deficiency inhibited T cell responses and protected mice from developing autoimmune arthritis. Our observations are likely translatable to humans, as Vdr is overexpressed in rheumatic joints. Genetic control of VDR availability codetermines the proinflammatory behavior of T cells, suggesting that increased presence of VDR at the site of inflammation might limit the antiinflammatory properties of its ligand.
    Keywords:  T cells; genetics; inflammation; vitamin D; vitamin D receptor
  51. Nat Commun. 2020 09 21. 11(1): 4766
    Chakravarti D, Hu B, Mao X, Rashid A, Li J, Li J, Liao WT, Whitley EM, Dey P, Hou P, LaBella KA, Chang A, Wang G, Spring DJ, Deng P, Zhao D, Liang X, Lan Z, Lin Y, Sarkar S, Terranova C, Deribe YL, Blutt SE, Okhuysen P, Zhang J, Vilar E, Nielsen OH, Dupont A, Younes M, Patel KR, Shroyer NF, Rai K, Estes MK, Wang YA, Bertuch AA, DePinho RA.
      Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.
  52. Nat Protoc. 2020 Sep 23.
    Tang Y, Zhao Y, Lin W.
      Formaldehyde (FA) is the simplest active carbonyl species that can be spontaneously produced in the body and plays important roles in human cognitive ability and spatial memory. However, excessive intake of FA may cause a series of diseases, including cancer, diabetes, heart and liver diseases and various neuropathies. Hence, the exploration of sensitive and fast detection methods for FA is crucial to understand and diagnose these diseases. Recently, fluorescent probes have been increasingly employed as powerful tools for detecting a broad range of different small molecules due to their high selectivity, rapid response, convenient operation and relatively non-invasive nature. Thus, we have developed two naphthalimide-based fluorescent probes for detecting FA in cells and in lysosomes. Compared with other FA fluorescent probes, these two probes have several advantages, including high sensitivity and selectivity, excellent two-photon properties and high signal-to-noise ratio. In this protocol, we provide detailed procedures for the synthesis of the two probes; characterization of their sensitivity, selectivity and stability in solution; and representative application procedures for detecting FA in living cells and mouse liver tissue slices. The protocol requires ~88 h to synthesize the probes, ~24 h to characterize the probes in solution and ~25 h to carry out the biological fluorescence imaging experiments in cells and liver tissue slices.
  53. Cells. 2020 Sep 22. pii: E2140. [Epub ahead of print]9(9):
    Andrade-Tomaz M, de Souza I, Rocha CRR, Gomes LR.
      The cell cycle involves a network of proteins that modulate the sequence and timing of proliferation events. Unregulated proliferation is the most fundamental hallmark of cancer; thus, changes in cell cycle control are at the heart of malignant transformation processes. Several cellular processes can interfere with the cell cycle, including autophagy, the catabolic pathway involved in degradation of intracellular constituents in lysosomes. According to the mechanism used to deliver cargo to the lysosome, autophagy can be classified as macroautophagy (MA), microautophagy (MI), or chaperone-mediated autophagy (CMA). Distinct from other autophagy types, CMA substrates are selectively recognized by a cytosolic chaperone, one-by-one, and then addressed for degradation in lysosomes. The function of MA in cell cycle control, and its influence in cancer progression, are already well-established. However, regulation of the cell cycle by CMA, in the context of tumorigenesis, has not been fully addressed. This review aims to present and debate the molecular mechanisms by which CMA can interfere in the cell cycle, in the context of cancer. Thus, cell cycle modulators, such as MYC, hypoxia-inducible factor-1 subunit alpha (HIF-1α), and checkpoint kinase 1 (CHK1), regulated by CMA activity will be discussed. Finally, the review will focus on how CMA dysfunction may impact the cell cycle, and as consequence promote tumorigenesis.
    Keywords:  MYC; autophagy; cancer; chaperone-mediated autophagy (CMA), cell cycle; checkpoints; hypoxia-inducible factor-1 subunit alpha (HIF-1α), checkpoint kinase 1 (CHK1)
  54. Cell Res. 2020 Sep 21.
    Wei D, Zhan W, Gao Y, Huang L, Gong R, Wang W, Zhang R, Wu Y, Gao S, Kang T.
      Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.
  55. Elife. 2020 Sep 24. pii: e62514. [Epub ahead of print]9
    Mei L, Espinosa de Los Reyes S, Reynolds MJ, Leicher R, Liu S, Alushin GM.
      The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show single piconewton forces applied solely to F-actin enhance binding by the human version of the essential cell-cell adhesion protein αE-catenin, but not its homolog vinculin. Cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin's C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin's C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through a-catenin.
    Keywords:  cell biology; chicken; human; molecular biophysics; structural biology
  56. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 202010928. [Epub ahead of print]
    Niu L, Li Y, Zong P, Liu P, Shui Y, Chen B, Wang ZW.
      Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2 A peptide acting to release free Gβγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.
    Keywords:  BK channel; PCDR-1; melatonin; melatonin receptor; sleep
  57. Nat Metab. 2020 Sep 21.
    Linke V, Overmyer KA, Miller IJ, Brademan DR, Hutchins PD, Trujillo EA, Reddy TR, Russell JD, Cushing EM, Schueler KL, Stapleton DS, Rabaglia ME, Keller MP, Gatti DM, Keele GR, Pham D, Broman KW, Churchill GA, Attie AD, Coon JJ.
      Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.
  58. Mech Ageing Dev. 2020 Sep 16. pii: S0047-6374(20)30140-8. [Epub ahead of print] 111344
    Cohen AA, Legault V, Fülöp T.
      Are diseases caused by aging? What are the mechanisms of aging? Do all species age? These hotly debated questions revolve around a unitary definition of aging. Because we use the word "aging" so frequently, both colloquially and scientifically, we rarely pause to consider whether this word maps to an underlying biological phenomenon, or whether it is simply a grab-bag of diverse phenomena linked more by our mental associations than by any underlying biology. Here, we consider how the presence of the colloquial word "aging" generates a cognitive bias towards supposing there is a unitary biological phenomenon. We ask what kind of evidence would support or refute that idea, and subsequently show clear evidence at multiple levels that aging is not a unitary phenomenon. In particular, the known aging pathways lead to heterogeneous outputs, not a single coordinated phenomenon. From levels ranging from cellular/molecular to clinical to demographic to evolutionary, we show how the supposition that aging is a unitary phenomenon can mislead and distract us from asking the best questions. For major sub-disciplines of aging biology, we show how going beyond the notion of unitary aging can hone the paradigm and help advance the pace of discovery.
    Keywords:  Bow-tie; Complex systems; Information theory; Linguistics; Neural network; Paradigm; Philosophy of science; Sapir-Whorf
  59. Aging Clin Exp Res. 2020 Sep 23.
    Herbst A, Lee CC, Vandiver AR, Aiken JM, McKenzie D, Hoang A, Allison D, Liu N, Wanagat J.
      BACKGROUND: Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells.AIMS: Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages.
    METHODS: We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency.
    RESULTS: Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach.
    DISCUSSION/CONCLUSIONS: The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.
    Keywords:  Biomarker; Deletion; Mitochondria; MtDNA; Mutation; Sarcopenia
  60. Curr Opin Cell Biol. 2020 Sep 19. pii: S0955-0674(20)30111-3. [Epub ahead of print]68 28-36
    Fenton AR, Jongens TA, Holzbaur ELF.
      Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole.
    Keywords:  Cytoskeleton; Fission; Fusion; Mitochondria; Morphology; Transport
  61. Carcinogenesis. 2020 Sep 21. pii: bgaa102. [Epub ahead of print]
    Zou D, Day R, Cocadiz JA, Parackal S, Mitchell W, Black MA, Lawrence B, Fitzgerald S, Print C, Jackson C, Guilford P.
      Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized ddPCR assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42-548 days (average of ten time points/patient). 29/30 (97%) patients had detectable ctDNA compared to 83% whose tumors were CEA-positive (>5ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) vs 6 (30%), respectively; P-value=0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value=0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value =0.07). In addition, 28 further colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to two years. Clinical relapse was observed in 6/28 (21%) patients. 4/6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.
  62. Nat Metab. 2020 Sep 21.
    Franco F, Jaccard A, Romero P, Yu YR, Ho PC.
      Current immunotherapies yield remarkable clinical outcomes by boosting the power of host immunity in cancer cell elimination and viral clearance. However, after prolonged antigen exposure, CD8+ T cells differentiate into a special differentiation state known as T-cell exhaustion, which poses one of the major hurdles to antiviral and antitumor immunity during chronic viral infection and tumour development. Growing evidence indicates that exhausted T cells undergo metabolic insufficiency with altered signalling cascades and epigenetic landscapes, which dampen effector immunity and cause poor responsiveness to immune-checkpoint-blockade therapies. How metabolic stress affects T-cell exhaustion remains unclear; therefore, in this Review, we summarize current knowledge of how T-cell exhaustion occurs, and discuss how metabolic insufficiency and prolonged stress responses may affect signalling cascades and epigenetic reprogramming, thus locking T cells into an exhausted state via specialized differentiation programming.
  63. Molecules. 2020 Sep 22. pii: E4343. [Epub ahead of print]25(18):
    Jové M, Mota-Martorell N, Pradas I, Galo-Licona JD, Martín-Gari M, Obis È, Sol J, Pamplona R.
      Lipids were determinants in the appearance and evolution of life. Recent studies disclose the existence of a link between lipids and animal longevity. Findings from both comparative studies and genetics and nutritional interventions in invertebrates, vertebrates, and exceptionally long-lived animal species-humans included-demonstrate that both the cell membrane fatty acid profile and lipidome are a species-specific optimized evolutionary adaptation and traits associated with longevity. All these emerging observations point to lipids as a key target to study the molecular mechanisms underlying differences in longevity and suggest the existence of a lipidome profile of long life.
    Keywords:  fatty acids; lipidomics; longevity; membrane unsaturation; peroxidation index
  64. Curr Opin Cell Biol. 2020 Sep 22. pii: S0955-0674(20)30109-5. [Epub ahead of print]68 37-44
    Papalazarou V, Machesky LM.
      The Arp2/3 complex orchestrates the formation of branched actin networks at the interface between the cytoplasm and membranes. Although it is widely appreciated that these networks are useful for scaffolding, creating pushing forces and delineating zones at the membrane interface, it has only recently come to light that branched actin networks are mechanosensitive, giving them special properties. Here, we discuss recent advances in our understanding of how Arp2/3-generated actin networks respond to load forces and thus allow cells to create pushing forces in responsive and tuneable ways to effect cellular processes such as migration, invasion, phagocytosis, adhesion and even nuclear and DNA damage repair.
    Keywords:  Actin; Arp2/3 complex; Cell migration; Endocytosis; Mechanosensing; Metabolism
  65. Eur J Cancer. 2020 Sep 22. pii: S0959-8049(20)30436-6. [Epub ahead of print]139 51-58
    Carrato A, Vieitez JM, Benavides M, Rodriguez-Garrote M, Castillo A, Ogalla GD, Bermejo LG, Ruiz de Mena I, Guillén-Ponce C, Aranda E, .
      BACKGROUND: Although occasioned through different mechanisms, the potential neurotoxicity and also haematological toxicity of nab-paclitaxel and oxaliplatin-based chemotherapy regimen were studied in this trial, which aimed to determine the maximum-tolerated dose (MTD) and to evaluate safety and efficacy of the combination in a sequential regimen of nab-paclitaxel, gemcitabine (GEM) and modified FOLFOX (mFOLFOX) in untreated patients with metastatic pancreatic ductal adenocarcinoma (PDAC).MATERIALS AND METHODS: Treatment consisted of nab-paclitaxel (125/100 mg/m2) plus GEM (1000/800 mg/m2) on days 1, 8 and 15, followed by mFOLFOX (oxaliplatin [85/75/65 mg/m2], 5-FU bolus [400/300/200 mg/m2], 5-FU infusion [2400/2000/1600 mg/m2]) on day 28, of a 42-day cycle. Patients were enrolled at the highest dose level with a subsequent 3 + 3 dose de-escalation plan.
    RESULTS: Eleven patients (median age = 61, 64% with performance status [PS] = 1) were eligible. All patients received the highest dose level. No de-escalation was needed. A dose-limiting toxicity was reported, an upper gastrointestinal haemorrhage. The MTD was nab-paclitaxel 125 mg/m2, GEM 1000 mg/m2, oxaliplatin 85 mg/m2, 5-FU bolus 400 mg/m2 and 5-FU infusion 2400 mg/m2. Common all-grade toxicities were neutropenia (73%), anaemia (55%), thrombocytopenia (55%) and asthenia (55%). Other relevant toxicities were paraesthesia (46%), nausea (36%), dysesthesia (27%) and pyrexia (27%). Objective response rate was 50% and disease control rate was 80%.
    CONCLUSIONS: The regimen of nab-paclitaxel plus GEM followed by mFOLFOX showed favourable safety and tolerability profiles with significant anti-tumor activity. More data are being achieved in a randomised phase II trial, to confirm efficacy rates and dismiss long-term neurotoxicity concerns regarding the sequencing of nab-paclitaxel and oxaliplatin.
    Keywords:  Dose de-escalation; FOLFOX; Gemcitabine; Nab-paclitaxel; Pancreatic ductal adenocarcinoma; Phase I; Sequential treatment
  66. Clin Cancer Res. 2020 Sep 21. pii: clincanres.1042.2020. [Epub ahead of print]
    Tsang ES, Topham JT, Karasinska JM, Lee MK, Williamson LM, Mendis S, Denroche RE, Jang GH, Kalloger SE, Moore RA, Mungall AJ, Bathe OF, Tang PA, Notta F, Wilson JM, Laskin J, O'Kane GM, Knox JJ, Goodwin RA, Loree JM, Jones SJ, Marra MA, Gallinger S, Schaeffer DF, Renouf DJ.
      PURPOSE: With the rising incidence of early-onset pancreatic cancer (EOPC), molecular characteristics that distinguish early onset pancreatic ductal adenocarcinoma (PDAC) tumors from those arising at a later age are not well understood.EXPERIMENTAL DESIGN: We performed bioinformatic analysis of genomic and transcriptomic data generated from 269 advanced (metastatic or locally advanced) and 277 resectable PDAC tumor samples. Patient samples were stratified into EOPC (age of onset ≤55 years; n=117), intermediate (age of onset 55-70 years; n=264) and average (age of onset ≥70 years; n=165) groups. Frequency of somatic mutations affecting genes commonly implicated in PDAC, as well as gene expression patterns, were compared between EOPC and all other groups.
    RESULTS: EOPC tumors showed significantly lower frequency of somatic SNV/indels in CDKN2A (p=0.0017), and were more likely to achieve bi-allelic mutation of CDKN2A through homozygous copy loss as opposed to heterozygous copy loss coupled with a loss-of-function SNV/indel mutation, the latter of which was more common for tumors with later ages of onset (p=1.5e-4). Transcription factor FOXC2 was significantly up-regulated in EOPC tumors (p=0.032). Genes significantly correlated with FOXC2 in PDAC samples were enriched for gene sets related to epithelial-mesenchymal transition (EMT) and included VIM (p=1.8e-8), CDH11 (p=6.5e-5) and CDH2 (p=2.4e-2).
    CONCLUSIONS: Our comprehensive analysis of sequencing data generated from a large cohort of PDAC patient samples highlights a distinctive pattern of bi-allelic CDKN2A mutation in EOPC tumors. Increased expression of FOXC2 in EOPC, with the correlation between FOXC2 and EMT pathways, represent novel molecular characteristics of EOPC.
  67. Cancer Discov. 2020 Sep 25. pii: CD-20-0336. [Epub ahead of print]
    Chen M, Chen X, Li S, Pan X, Gong Y, Zheng J, Xu J, Zhao C, Zhang Q, Zhang S, Qi L, Wang Z, Shi K, Ding BS, Xue Z, Chen L, Yang S, Wang Y, Niu T, Dai L, Lowe SW, Chen C, Liu Y.
      Chromosome copy number variations are a hallmark of cancers. Among them, the prevalent chromosome 17p deletions are associated with poor prognosis and can promote tumorigenesis more than TP53 loss. Here we utilize multiple functional genetic strategies and identify a new 17p tumor suppressor gene, PHD finger protein 23 (PHF23). Its deficiency impairs B cell differentiation and promotes immature B lymphoblastic malignancy. Mechanistically, we demonstrate that PHF23, an H3K4me3 reader, directly binds the SIN3-HDAC complex through its N-terminus and represses its deacetylation activity on H3K27ac. Thus, the PHF23-SIN3-HDAC (PSH) complex coordinates these two major active histone markers for the activation of downstream TSGs and differentiation-related genes. Further, dysregulation of the PSH complex is essential for the development and maintenance of PHF23 deficient and 17p deleted tumors. Hence, our study reveals a novel epigenetic regulatory mechanism that contributes to the pathology of 17p-deleted cancers and suggests novel susceptibility to this disease.
  68. Autophagy. 2020 Sep 25.
    Zhou C, Liang Y, Zhou L, Yan Y, Liu N, Zhang R, Huang Y, Wang M, Tang Y, Ali DW, Wang Y, Michalak M, Chen XZ, Tang J.
      Pancreatic cancer is one of the most aggressive tumors associated with a poor clinical prognosis, weakly effective therapeutic options. Therefore, there is a strong impetus to discover new therapeutic targets in pancreatic cancer. In the present study, we first demonstrated that TSPAN1 is upregulated in pancreatic cancer and that TSPAN1 depletion decreases pancreatic cancer cell proliferation in vitro and in vivo. TSPAN1 expression was correlated with poor overall survival of pancreatic cancer patients. Moreover, we demonstrated that TSPAN1 is a novel positive regulator of macroautophagy/autophagy characterized by decreased LC3-II and SQSTM1/p62 expressions, inhibited puncta formation of GFP-LC3 and autophagic vacuoles. We also demonstrated that tspan1 mutation impaired autophagy in the zebrafish model. Furthermore, we showed that TSPAN1 promoted autophagy maturation via direct binding to LC3 by two conserved LIR motifs. Mutations in the LIR motifs of TSPAN1 resulted in a loss of the ability to induce autophagy and promote pancreatic cancer proliferation. Second, we discovered two conservative TCF/LEF binding elements present in the promoter region of the TSPAN1 gene, which was further verified through luciferase activity and ChIP assays. Furthermore, TSPAN1 was upregulated by FAM83A through the canonical WNT-CTNNB1 signaling pathway. We further demonstrated that both TSPAN1 and FAM83A are both direct targets of MIR454 (microRNA 454). Additionally, we revealed the role of MIR454-FAM83A-TSPAN1 in the proliferation of pancreatic cancer cells in vitro and in vivo. Our findings suggest that components of the MIR454-FAM83A-TSPAN1 axis may be valuable prognosis markers or therapeutic targets for pancreatic cancer.
    Keywords:   MIR454 ; FAM83A; WNT-CTNNB1; autophagy; pancreatic cancer; tetraspanin 1
  69. Autophagy. 2020 Sep 22. 1-15
    Pantoom S, Konstantinidis G, Voss S, Han H, Hofnagel O, Li Z, Wu YW.
      Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation. Abbreviations : ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle's balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5'-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5'-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type.
    Keywords:  ATG12–ATG5-ATG16L1 complex; ATG16L1; RAB33B; RAB33B-ATG16L1 complex; autophagosome formation; autophagy; crystal structure
  70. Methods Mol Biol. 2021 ;2221 291-299
    Molstad DHH, Bradley EW.
      Musculoskeletal pain contributes significantly to chronic pain experienced by adults and to health care use. This chapter details several methods to evaluate pain and physical activity in mice that can be applied to preclinical orthopedic models. These methods include the von Frey filament assay that measures mechanical allodynia, open-field activity assays for evaluation of ambulation, and incapacitance measurements to determine static weight bearing.
    Keywords:  Evoked pain; Functional assays; Incapacitance; Mechanical allodynia; Open-field activity assays; Static weight bearing; von Frey
  71. J Exp Med. 2021 Jan 04. pii: e20192378. [Epub ahead of print]218(1):
    Dalal PJ, Sullivan DP, Weber EW, Sacks DB, Gunzer M, Grumbach IM, Heller Brown J, Muller WA.
      Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.
  72. Exp Dermatol. 2020 Sep 23.
    Runel G, Cario M, Lopez-Ramirez N, Malbouyres M, Ruggiero F, Bernard L, Puisieux A, Caramel J, Chlasta J, Masse I.
      Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin aging is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin aging. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through aging in these models, can be a new biomarker of skin aging, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin aging in vivo.
    Keywords:  3D model; Biomechanics; Medaka fish; dermis; electron microscopy
  73. Ageing Res Rev. 2020 Sep 21. pii: S1568-1637(20)30310-X. [Epub ahead of print] 101175
    Hamrick MW, Stranahan AM.
      Inquiry into relationships between energy metabolism and brain function requires a uniquely interdisciplinary mindset, and implementation of anti-aging lifestyle strategies based on this work also involves consistent mental and physical discipline. Dr. Mark P. Mattson embodies both of these qualities, based on the breadth and depth of his work on neurobiological responses to energetic stress, and on his own diligent practice of regular exercise and caloric restriction. Dr. Mattson created a neurotrophic niche in his own laboratory, allowing trainees to grow their skills, form new connections, and eventually migrate, forming their own labs while remaining part of the extended lab family. In this historical review, we highlight Dr. Mattson's many contributions to understanding neurobiological responses to physical exercise and dietary restriction, with an emphasis on the mechanisms that may underlie neuroprotection in ageing and age-related disease. On the occasion of Dr. Mattson's retirement from the National Institute on Aging, we highlight his foundational work on metabolism and neuroplasticity by reviewing the context for these findings and considering their impact on future research on the neuroscience of aging.
    Keywords:  Aging; Caloric restriction; Exercise; Hippocampus; Intermittent fasting; Mitochondria
  74. J Biol Chem. 2020 Sep 25. pii: jbc.RA120.013987. [Epub ahead of print]
    Liu Q, Yang X, Long G, Hu Y, Gu Z, Boisclair YR, Long Q.
      Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, non-alcohol fatty liver disease (NAFLD) and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation and increased mitochondrial outer membrane permeability (MOMP). Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.
    Keywords:  endoplasmic reticulum stress (ER stress); endoplasmic-reticulum-associated protein degradation (ERAD); liver; mitochondrial disease; mitochondrial permeability transition (MPT)
  75. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31166-9. [Epub ahead of print]32(12): 108177
    Boteva L, Nozawa RS, Naughton C, Samejima K, Earnshaw WC, Gilbert N.
      Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.
    Keywords:  chromatin; chromosome; common fragile sites; condensation; condensin; genome stability; replication; replication stress
  76. Aging (Albany NY). 2020 Sep 23. 12
    Zhou S, Yan Y, Chen X, Zeng S, Wei J, Wang X, Gong Z, Xu Z.
      The purpose of this study was to identify a vital gene signature that has prognostic value for pancreatic cancer based on gene expression datasets from the Cancer Genome Atlas and Gene Expression Omnibus. A total of 34 genes were obtained by the univariate analysis, which were significantly associated with the overall survival of PC patients. After further analysis, Anillin (ANLN) and Histone H1c (HIST1H1C) were identified and considered to be the most significant prognostic genes among the 34 genes. A prognostic model based on these two genes was constructed, and successfully distinguished pancreatic cancer survival into high-risk and low-risk groups in the training set and testing set. Subsequently, independent predictive factors, including the age, margin condition and risk score, were then employed to construct the nomogram model. The area under curve for the nomogram model was 0.826 at 0.5 years and 0.726 at 1 year, and the C-index of the nomogram model was 0.664 higher than the others variables alone. These findings have indicated that high expression of ANLN and HIST1H1C predicted poor outcomes for patients with pancreatic cancer. The nomogram model based on the expression of two genes could be valuable for the guidance of clinical treatment.
    Keywords:  ANLN; HIST1H1C; overall survival; pancreatic cancer; prognosis
  77. Nat Commun. 2020 09 21. 11(1): 4740
    Ballhausen A, Przybilla MJ, Jendrusch M, Haupt S, Pfaffendorf E, Seidler F, Witt J, Hernandez Sanchez A, Urban K, Draxlbauer M, Krausert S, Ahadova A, Kalteis MS, Pfuderer PL, Heid D, Stichel D, Gebert J, Bonsack M, Schott S, Bläker H, Seppälä T, Mecklin JP, Ten Broeke S, Nielsen M, Heuveline V, Krzykalla J, Benner A, Riemer AB, von Knebel Doeberitz M, Kloor M.
      The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.
  78. Cancer Prev Res (Phila). 2020 Sep 21. pii: canprevres.0322.2020. [Epub ahead of print]
    Ranade A, Bhatt A, Page R, Limaye S, Crook T, Akolkar D, Patil D.
      We have previously shown that Circulating Ensembles of Tumor Associated Cell (C-ETACs) are a systemic hallmark of cancer based on analysis of blood samples from 16,134 individuals including 10,625 asymptomatic individuals and 5,509 diagnosed cases of cancer. C-ETACs were ubiquitously (90%) detected across all cancer-types and were rare (3.6%) among the asymptomatic population. Consequently, we hypothesized that asymptomatic individuals with detectable C-ETACs would have a definitively elevated risk of developing cancer as compared to individuals without C-ETACs. In the present manuscript we present one-year follow-up data of the asymptomatic cohort which shows that C-ETAC positive individuals have a 230-fold (p<0.00001) higher 1-year cancer risk as compared to individuals where C-ETAC were undetectable. Simultaneously, we also expanded the study to include 4,419 symptomatic individuals, suspected of cancer, prior to undergoing an invasive biopsy for diagnosis. C-ETACs were detected in 4,101 (92.8%) out of these 4,419 cases where cancer was eventually confirmed. We conclude that detection of C-ETACs can identify patients at risk of cancer and can be reliably used to stratify asymptomatic individuals with an elevated 1-year risk of cancer.
  79. J Cell Physiol. 2020 Sep 22.
    Zhang B.
      Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR/Cas9-based genome editing has been becoming one of the most promising tools for treating human genetic diseases, including cardiovascular diseases, neuro-disorders, and cancers. As the quick modification of the CRISPR/Cas9 system, including delivery system, CRISPR/Cas9-based gene therapy has been extensively studied in preclinic and clinic treatments. CRISPR/Cas genome editing is also a robust tool to create animal genetic models for studying and treating human genetic disorders, particularly diseases associated with point mutations. However, significant challenges also remain before CRISPR/Cas technology can be routinely employed in the clinic for treating different genetic diseases, which include toxicity and immune response of treated cells to CRISPR/Cas component, highly throughput delivery method, and potential off-target impact. The off-target effect is one of the major concerns for CRISPR/Cas9 gene therapy, more research should be focused on limiting this impact by designing high specific gRNAs and using high specificity of Cas enzymes. Modifying the CRISPR/Cas9 delivery method not only targets a specific tissue/cell but also potentially limits the off-target impact.
    Keywords:  CRISPR/Cas9; animal model; gene therapy; genetic disease; genetic disorder; genome editing
  80. J Biol Chem. 2020 Sep 25. 295(39): 13458-13473
    Sies H.
      My interest in biological chemistry proceeded from enzymology in vitro to the study of physiological chemistry in vivo Investigating biological redox reactions, I identified hydrogen peroxide (H2O2) as a normal constituent of aerobic life in eukaryotic cells. This finding led to developments that recognized the essential role of H2O2 in metabolic redox control. Further research included studies on GSH, toxicological aspects (the concept of "redox cycling"), biochemical pharmacology (ebselen), nutritional biochemistry and micronutrients (selenium, carotenoids, flavonoids), and the concept of "oxidative stress." Today, we recognize that oxidative stress is two-sided. It has its positive side in physiology and health in redox signaling, "oxidative eustress," whereas at higher intensity, there is damage to biomolecules with potentially deleterious outcome in pathophysiology and disease, "oxidative distress." Reflecting on these developments, it is gratifying to witness the enormous progress in redox biology brought about by the science community in recent years.
    Keywords:  Redox regulation; carotenoid; ebselen; flavonoid; glutathione; hydrogen peroxide; micronutrients; oxidative eustress; oxidative stress; redox regulation; selenium; singlet oxygen