bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒10‒04
ninety-two papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Nat Cell Biol. 2020 Oct;22(10): 1170-1179
      SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1-5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome-lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy-lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.
  2. Elife. 2020 Sep 29. pii: e58108. [Epub ahead of print]9
      Cancer testis antigens (CTAs) are proteins whose expression is normally restricted to the testis but anomalously activated in human cancer. In sperm, a number of CTAs support energy generation, however whether they contribute to tumor metabolism is not understood. We describe human COX6B2, a component of cytochrome c oxidase (complex IV). COX6B2 is expressed in human lung adenocarcinoma (LUAD) and expression correlates with reduced survival time. COX6B2, but not its somatic isoform COX6B1, enhances activity of complex IV, increasing oxidative phosphorylation (OXPHOS) and NAD+ generation. Consequently, COX6B2-expressing cancer cells display a proliferative advantage, particularly in low oxygen. Conversely, depletion of COX6B2 attenuates OXPHOS and collapses mitochondrial membrane potential leading to cell death or senescence. COX6B2 is both necessary and sufficient for growth of human tumor xenografts in mice. Our findings reveal a previously unappreciated, tumor specific metabolic pathway hijacked from one of the most ATP-intensive processes in the animal kingdom: sperm motility.
    Keywords:  cancer biology; human
  3. Lab Invest. 2020 Oct 02.
      Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Krasmut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-KrasG12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreERT2 mediated recombination. Hnf1b:CreERT2;KrasG12V (cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHigh mice. cKrasMod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKrasLow, cKrasMod, and cKrasHigh mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.
  4. Dev Cell. 2020 Sep 24. pii: S1534-5807(20)30713-9. [Epub ahead of print]
      Many tumors of endodermal origin are composed of highly secretory cancer cells that must adapt endoplasmic reticulum (ER) activity to enable proper folding of secreted proteins and prevent ER stress. We found that pancreatic ductal adenocarcinomas (PDACs) overexpress the myelin regulatory factor (MYRF), an ER membrane-associated transcription factor (TF) released by self-cleavage. MYRF was expressed in the well-differentiated secretory cancer cells, but not in the poorly differentiated quasi-mesenchymal cells that coexist in the same tumor. MYRF expression was controlled by the epithelial identity TF HNF1B, and it acted to fine-tune the expression of genes encoding highly glycosylated, cysteine-rich secretory proteins, thus preventing ER overload. MYRF-deficient PDAC cells showed signs of ER stress, impaired proliferation, and an inability to form spheroids in vitro, while in vivo they generated highly secretory but poorly proliferating and hypocellular tumors. These data indicate a role of MYRF in the control of ER homeostasis in highly secretory PDAC cells.
    Keywords:  ER stress; MYRF; differentiation; pancreatic cancer; stress response; transcription; tumor heterogeneity; unfolded protein response
  5. Trends Cancer. 2020 Sep 23. pii: S2405-8033(20)30236-3. [Epub ahead of print]
      Mitochondria play an essential role in cellular metabolism, generation of reactive oxygen species (ROS), and the initiation of apoptosis. These properties enable mitochondria to be crucial integrators in the pathways of tumorigenesis. An open question is to what extent variation in the mitochondrial genome (mtDNA) contributes to the biological heterogeneity observed in human tumors. In this review, we summarize our current understanding of the role of mtDNA genetics in relation to human cancers.
    Keywords:  cancer; haplotypes; mitochondria; mtDNA; oncocytoma
  6. Cancer Discov. 2020 Sep 28. pii: CD-20-0581. [Epub ahead of print]
      Nuclear pore complexes (NPCs) are the central mediators of nucleocytoplasmic transport. Increasing evidence shows that many cancer cells have increased numbers of NPCs and become addicted to the nuclear transport machinery. How reducing NPC numbers affects the physiology of normal and cancer cells and if it could be exploited for cancer therapies has not been investigated. We report that inhibition of NPC formation, a process mostly restricted to proliferating cells, causes selective cancer cell death, prevents tumor growth and induces tumor regression. While cancer cells die in response to NPC assembly inhibition, normal cells undergo a reversible cell cycle arrest that allows them to survive. Mechanistically, reducing NPC numbers results in multiple alterations contributing to cancer cell death including abnormalities in nuclear transport, catastrophic alterations in gene expression, and the selective accumulation of DNA damage. Our findings uncover the NPC formation process as a novel targetable pathway in cancer cells.
  7. Gastroenterology. 2020 Sep 29. pii: S0016-5085(20)35147-7. [Epub ahead of print]
      BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory and myofibroblastic phenotypes, however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology.METHODS: To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetical depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy various co-culture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition as well as tumor dissemination and metastasis.
    RESULTS: Our in vivo findings demonstrated that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, co-culture experiments of tumor organoids and CAFs revealed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis revealed that pancreatic cancer patients with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
    CONCLUSION: Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work demonstrates that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
    Keywords:  Cancer-associated fibroblasts; extracellular matrix proteins; myofibroblasts; pancreatic ductal adenocarcinoma
  8. Proc Natl Acad Sci U S A. 2020 Sep 28. pii: 201912772. [Epub ahead of print]
      Deregulated global mRNA translation is an emerging feature of cancer cells. Oncogenic transformation in colorectal cancer (CRC) is driven by mutations in APC, KRAS, SMAD4, and TP53, known as the adenoma-carcinoma sequence (ACS). Here we introduce each of these driver mutations into intestinal organoids to show that they are modulators of global translational capacity in intestinal epithelial cells. Increased global translation resulting from loss of Apc expression was potentiated by the presence of oncogenic Kras G12D Knockdown of Smad4 further enhanced global translation efficiency and was associated with a lower 4E-BP1-to-eIF4E ratio. Quadruple mutant cells with additional P53 loss displayed the highest global translational capacity, paralleled by high proliferation and growth rates, indicating that the proteome is heavily geared toward cell division. Transcriptional reprogramming facilitating global translation included elevated ribogenesis and activation of mTORC1 signaling. Accordingly, interfering with the mTORC1/4E-BP/eIF4E axis inhibited the growth potential endowed by accumulation of multiple drivers. In conclusion, the ACS is characterized by a strongly altered global translational landscape in epithelial cells, exposing a therapeutic potential for direct targeting of the translational apparatus.
    Keywords:  colorectal cancer; driver mutations; global translation; protein synthesis
  9. Cancer Metab. 2020 ;8 22
      Abstract: Background: Rewiring of metabolism induced by oncogenic K-Ras in cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways.
    Methods: We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity.
    Results: We show that K-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism.
    Conclusions: Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.
    Keywords:  Combinatorial drug treatment; Glutamine; Glycolysis; Metabolic cancer therapy; Metabolic connectivity; Metabolic rewiring; Metabolic signature; Precision oncology
  10. Br J Pharmacol. 2020 Sep 28.
      BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is a gastrointestinal hormone released in response to nutritional intake that promotes a wide range of effects by activating the GLP-2 receptor. In addition to its intestinotrophic effects, GLP-2 has been shown to positively influence glucose metabolism under conditions of obesity, but the mechanisms behind this remain unclear. Here we investigate the molecular role of the GLP-2/GLP-2R axis in energetic metabolism, focusing on its potential modulatory effects on adipose tissue (AT).EXPERIMENTAL APPROACH: Physiological measurements (body weight, food intake, locomotor activity and energy expenditure) and metabolic studies (glucose and insulin tolerance tests) were performed on lean and obese mice treated with the protease-resistant GLP-2 analog teduglutide.
    KEY RESULTS: Acute but not chronic centrally administered teduglutide decreased food intake and weight-gain. By contrast, chronic activation of peripheral GLP-2R increased body weight-independent glucose tolerance and had anti-inflammatory effects on visceral AT. Using a gene silencing approach, we found that AT is necessary for the beneficial effects of teduglutide. Finally, teduglutide regulates the inflammatory state and acts as an anabolic signal in human adipocytes.
    CONCLUSION AND IMPLICATIONS: Overall, our data identify AT as a new clinical target for GLP-2 activity in obesity.
    Keywords:  GLP-2; adipose tissue; anti-inflammatory; obesity; teduglutide
  11. Dev Cell. 2020 Sep 18. pii: S1534-5807(20)30671-7. [Epub ahead of print]
      Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cell types, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here, we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs naive pluripotency exit, and artificially inducing abscission accelerates it. Altogether, our data indicate that a switch in the division machinery leading to faster abscission regulates pluripotency exit. Our study identifies abscission as a key cellular process coupling cell division to fate transitions.
    Keywords:  abscission; midbody; mitosis; naive pluripotency; stem cells
  12. Int J Biochem Cell Biol. 2020 Sep 25. pii: S1357-2725(20)30176-X. [Epub ahead of print]128 105859
      Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly solid tumors in the world. Aerobic glycolysis is among the characteristic features of pancreatic cancer. However, the regulatory process of aerobic glycolysis in pancreatic cancer is too complicated, and the underlying mechanism remains unexplained. Reportedly, CDK4/6 inhibitors repress breast cancer cell proliferation by modulating glucose metabolism. Here, we reveal that the CDK4/6 inhibitor, PD0332991 stabilized FBP1 to hinder aerobic glycolysis in pancreatic cancer. We also show that the CDK4/6-E2 F1 signaling pathway mediated an increase in MAGED1 expression, promoting FBP1 degradation in pancreatic cancer. We, therefore, might have identified a novel mechanism by which the CDK4/6 inhibitor, PD0332991 blocks the Warburg effect of pancreatic cancer by stabilizing FBP1.
    Keywords:  Aerobic glycolysis; CDK4/6; CDK4/6 inhibitors; FBP1; MAGED1; Pancreatic cancer
  13. Genome Med. 2020 Sep 29. 12(1): 80
      BACKGROUND: Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) comprise not just tumor cells but also a microenvironment with which the tumor cells constantly interact. Detailed characterization of the cellular composition of the tumor microenvironment is critical to the understanding of the disease and treatment of the patient. Single-cell transcriptomics has been used to study the cellular composition of different solid tumor types including PDAC. However, almost all of those studies used primary tumor tissues.METHODS: In this study, we employed a single-cell RNA sequencing technology to profile the transcriptomes of individual cells from dissociated primary tumors or metastatic biopsies obtained from patients with PDAC. Unsupervised clustering analysis as well as a new supervised classification algorithm, SuperCT, was used to identify the different cell types within the tumor tissues. The expression signatures of the different cell types were then compared between primary tumors and metastatic biopsies. The expressions of the cell type-specific signature genes were also correlated with patient survival using public datasets.
    RESULTS: Our single-cell RNA sequencing analysis revealed distinct cell types in primary and metastatic PDAC tissues including tumor cells, endothelial cells, cancer-associated fibroblasts (CAFs), and immune cells. The cancer cells showed high inter-patient heterogeneity, whereas the stromal cells were more homogenous across patients. Immune infiltration varies significantly from patient to patient with majority of the immune cells being macrophages and exhausted lymphocytes. We found that the tumor cellular composition was an important factor in defining the PDAC subtypes. Furthermore, the expression levels of cell type-specific markers for EMT+ cancer cells, activated CAFs, and endothelial cells significantly associated with patient survival.
    CONCLUSIONS: Taken together, our work identifies significant heterogeneity in cellular compositions of PDAC tumors and between primary tumors and metastatic lesions. Furthermore, the cellular composition was an important factor in defining PDAC subtypes and significantly correlated with patient outcome. These findings provide valuable insights on the PDAC microenvironment and could potentially inform the management of PDAC patients.
    Keywords:  Cellular heterogeneity; Pancreatic cancer; Pancreatic cancer subtypes; Single-cell RNA sequencing
  14. Nature. 2020 Sep 30.
      Blood vessels support tumours by providing nutrients and oxygen, while also acting as conduits for the dissemination of cancer1. Here we use mouse models of breast and lung cancer to investigate whether endothelial cells also have active 'instructive' roles in the dissemination of cancer. We purified genetically tagged endothelial ribosomes and their associated transcripts from highly and poorly metastatic tumours. Deep sequencing revealed that metastatic tumours induced expression of the axon-guidance gene Slit2 in endothelium, establishing differential expression between the endothelial (high Slit2 expression) and tumoural (low Slit2 expression) compartments. Endothelial-derived SLIT2 protein and its receptor ROBO1 promoted the migration of cancer cells towards endothelial cells and intravasation. Deleting endothelial Slit2 suppressed metastatic dissemination in mouse models of breast and lung cancer. Conversely, deletion of tumoural Slit2 enhanced metastatic progression. We identified double-stranded RNA derived from tumour cells as an upstream signal that induces expression of endothelial SLIT2 by acting on the RNA-sensing receptor TLR3. Accordingly, a set of endogenous retroviral element RNAs were upregulated in metastatic cells and detected extracellularly. Thus, cancer cells co-opt innate RNA sensing to induce a chemotactic signalling pathway in endothelium that drives intravasation and metastasis. These findings reveal that endothelial cells have a direct instructive role in driving metastatic dissemination, and demonstrate that a single gene (Slit2) can promote or suppress cancer progression depending on its cellular source.
  15. Sci Signal. 2020 Sep 29. pii: eaba3300. [Epub ahead of print]13(651):
      T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP-such as in response to inflammation, sepsis, and cancer-disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.
  16. Nature. 2020 Sep 30.
      The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.
  17. J Biol Chem. 2020 Oct 01. pii: jbc.RA120.014884. [Epub ahead of print]
      Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected seven-week old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared to controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-L-arginine, in tumor-bearing mice compared to control mice. Compared to healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.
    Keywords:  cachexia; cancer; metabolomics; methylarginines; mitochondria; protein synthesis; skeletal muscle
  18. Nat Cell Biol. 2020 Oct;22(10): 1252-1263
      Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
  19. JCI Insight. 2020 Sep 29. pii: 135544. [Epub ahead of print]
      Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired PDX and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between Area Under the Curve value of organoid drug dose-response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50-94% of the relative abundance of all N-glycans detected in each of the model. Lastly, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicles (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis, and discover that four EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically-actionable serologic biomarkers.
    Keywords:  Cancer; Glycobiology; Oncology
  20. JCI Insight. 2020 Sep 29. pii: 140495. [Epub ahead of print]
      Somatic KRAS mutations are highly prevalent in many human cancers. In addition, a distinct spectrum of germline KRAS mutations cause developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than the mutant proteins found in cancer. We generated mice harboring conditional KrasLSL-P34R and KrasLSL-T58I "knock in" alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of RASopathy disorders, and these mice also exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects despite causing a similar spectrum of hematologic diseases. These novel mouse strains are robust models for investigating the consequences of endogenous hyperactive K-Ras signaling in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
    Keywords:  Genetic diseases; Genetics; Mouse models; Signal transduction
  21. PLoS Genet. 2020 Sep 28. 16(9): e1009053
      Autophagy is a fundamental process responsible for degradation and recycling of intracellular contents. In the budding yeast, non-selective macroautophagy and microautophagy of the endoplasmic reticulum (ER) are caused by ER stress, the circumstance where aberrant proteins accumulate in the ER. The more recent study showed that protein aggregation in the ER initiates ER-selective macroautophagy, referred to as ER-phagy; however, the mechanisms by which ER stress induces ER-phagy have not been fully elucidated. Here, we show that the expression levels of ATG39, encoding an autophagy receptor specific for ER-phagy, are significantly increased under ER-stressed conditions. ATG39 upregulation in ER stress response is mediated by activation of its promoter, which is positively regulated by Snf1 AMP-activated protein kinase (AMPK) and negatively by Mig1 and Mig2 transcriptional repressors. In response to ER stress, Snf1 promotes nuclear export of Mig1 and Mig2. Our results suggest that during ER stress response, Snf1 mediates activation of the ATG39 promoter and consequently facilitates ER-phagy by negatively regulating Mig1 and Mig2.
  22. Mol Metab. 2020 Sep 28. pii: S2212-8778(20)30156-3. [Epub ahead of print] 101082
      OBJECTIVE: The human adaptive fasting response enables survival during periods of caloric deprivation. A crucial component of the fasting response is the shift from glucose metabolism to utilization of lipids, underscoring the importance of adipose tissue as the central lipid-storing organ. The objective of this study was to investigate the response of adipose tissue to a prolonged fast in humans.METHODS: We performed RNA sequencing of subcutaneous adipose tissue samples longitudinally collected during a 10-day, 0-calorie fast in humans. We further investigated observed transcriptional signatures utilizing cultured human monocytes and Thp1 cells. We examined the cellularity of adipose tissue biopsies with transmission electron microscopy and tested for associated changes in relevant inflammatory mediators in the systemic circulation by ELISA assays of longitudinally collected blood samples.
    RESULTS: Coincident with the expected shift away from glucose utilization and lipid storage, we demonstrated downregulation of pathways related to glycolysis, oxidative phosphorylation, and lipogenesis. The canonical lipolysis pathway was also downregulated, whereas fasting drove alternative lysosomal paths to lipid digestion. Unexpectedly, the dominant induced pathways were associated with immunity and inflammation, although this only became evident at the 10-day time point. Among the most augmented transcripts were those associated with macrophage identity and function, such as members of the erythroblast transformation-specific (ETS) transcription factor family. Key components of the macrophage transcriptional signal in fasting adipose tissue were recapitulated with induced expression of two of the ETS transcription factors via cultured macrophages, SPIC and SPI1. The inflammatory signal was further reflected by an increase in systemic inflammatory mediators.
    CONCLUSIONS: Collectively, this study demonstrates an unexpected role of metabolic inflammation in the human adaptive fasting response.
    Keywords:  Adipose tissue; Fasting; Human; Inflammation; Macrophage; SPIC
  23. PLoS Genet. 2020 Sep 28. 16(9): e1008704
      ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.
  24. Elife. 2020 Sep 29. pii: e54707. [Epub ahead of print]9
      Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.
    Keywords:  S. cerevisiae; biochemistry; chemical biology; molecular biophysics; structural biology
  25. Cancers (Basel). 2020 Sep 30. pii: E2819. [Epub ahead of print]12(10):
      The deregulation of the oxidative metabolism in cancer, as shown by the increased aerobic glycolysis and impaired oxidative phosphorylation (Warburg effect), is coordinated by genetic changes leading to the activation of oncogenes and the loss of oncosuppressor genes. The understanding of the metabolic deregulation of cancer cells is necessary to prevent and cure cancer. In this review, we illustrate and comment the principal metabolic and molecular variations of cancer cells, involved in their anomalous behavior, that include modifications of oxidative metabolism, the activation of oncogenes that promote glycolysis and a decrease of oxygen consumption in cancer cells, the genetic susceptibility to cancer, the molecular correlations involved in the metabolic deregulation in cancer, the defective cancer mitochondria, the relationships between the Warburg effect and tumor therapy, and recent studies that reevaluate the Warburg effect. Taken together, these observations indicate that the Warburg effect is an epiphenomenon of the transformation process essential for the development of malignancy.
    Keywords:  Warburg effect; glycolysis; oncogenes; oxidative metabolism; tumor therapy
  26. Am J Physiol Endocrinol Metab. 2020 Sep 28.
      Obesity and insulin resistance (IR) are associated with endoplasmic reticulum (ER) stress and mitochondrial dysfunction in several tissues. Although for many years mitochondrial and ER function were studied separately, these organelles also connect to produce interdependent functions. Communication occurs at mitochondria-associated ER membranes (MAM) and regulates lipid and calcium homeostasis, apoptosis, and the exchange of adenine nucleotides, among other things. Recent evidence suggests that MAMs contribute to organelle, cellular, and systemic metabolism. In obesity and IR models, metabolic tissues such as the liver, skeletal muscle, pancreas, and adipose tissue present alterations in MAM structure or function. The purpose of this mini-review is to highlight the MAM disruptions that occur in each tissue during obesity and IR and its relationship with glucose homeostasis and IR. We also discuss the current controversy that surrounds MAMs' role in the development of insulin resistance.
    Keywords:  MAMs; endoplasmic reticulum; glucose metabolism; insulin resistance; mitochondria
  27. Cancer Res. 2020 Oct 01. pii: canres.0524.2020. [Epub ahead of print]
      T cell exhaustion in cancer is linked to poor clinical outcomes and evidence suggests T cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TILs. Environmental stress produces epigenome remodeling events within tumor-infiltrating lymphocytes resulting from loss of the histone methyltransferase EZH2. Using a multi-omics approach, we have defined a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest manipulation of T cell EZH2 within the context of cellular therapies may yield lymphocytes which are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults.
  28. J Physiol. 2020 Oct 02.
      Under conditions of hypoxia, most eukaryotic cells can shift their primary metabolic strategy from predominantly mitochondrial respiration towards increased glycolysis to maintain ATP levels. This hypoxia-induced reprogramming of metabolism is key to satisfying cellular energetic requirements during acute hypoxic stress. At a transcriptional level, this metabolic switch can be regulated by several pathways including the hypoxia inducible factor-1α (HIF-1α) which induces an increased expression of glycolytic enzymes. While this increase in glycolytic flux is beneficial for maintaining bioenergetic homeostasis during hypoxia, the pathways mediating this increase can also be exploited by cancer cells to promote tumor survival and growth, an area which has been extensively studied. It has recently become appreciated that increased glycolytic metabolism in hypoxia may also have profound effects on cellular physiology in hypoxic immune and endothelial cells. Therefore, understanding the mechanisms central to mediating this reprogramming are of importance from both physiologic and pathophysiologic standpoints. In this review, we highlight the role of HIF-1α in the regulation of hypoxic glycolysis and its implications for physiological processes such as angiogenesis and immune cell effector function. Abstract figure legend Schematic outlining the functional physiological consequences of enhanced glycolytic metabolism in endothelial cells and immune cells. This article is protected by copyright. All rights reserved.
    Keywords:  HIF; glycolysis; hypoxia; metabolism
  29. J Hepatol. 2020 Sep 25. pii: S0168-8278(20)33664-3. [Epub ahead of print]
      BACKGROUND&AIM: IL-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signalling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6, making it difficult to delineate cell type-specific effects of IL-6 type cytokines. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signalling.METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signalling in distinct liver cell types: hepatic stellate cells (HSCs), cholangiocytes (CCs)/liver progenitor cells (LPCs) or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model.
    RESULTS: Hepatocyte-specific gp130 activation led to the up-regulation of innate immune system components, including acute-phase-proteins. Consequently, we observed peripheral mobilisation and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of HSC or CC/LPC-specific transgenic gp130 signalling.
    CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NFκB activation. We therefore conclude that gp130 engagement e.g. by IL-6 trans-signalling represents a safe-guard mechanism in innate immunity.
    Keywords:  IL-6; acute-phase response; gp130; innate immunity
  30. Cancer Res. 2020 Sep 30. pii: canres.0852.2019. [Epub ahead of print]
      Chromosomal instability (CIN) comprises continual gain and loss of chromosomes or parts of chromosomes and occurs in the majority of cancers, often conferring poor prognosis. Due to a scarcity of functional studies and poor understanding of how genetic or gene expression landscapes connect to specific CIN mechanisms, causes of CIN in most cancer types remain unknown. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynaecological malignancy in the Western world, with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosomal aberrations and knowledge of causative mechanisms would represent an important step towards combating this disease. Here we perform the first in-depth functional characterization of mechanisms driving CIN in HGSC in seven cell lines that accurately recapitulate HGSC genetics. Multiple mechanisms co-existed to drive CIN in HGSC, including elevated microtubule dynamics and DNA replication stress that can be partially rescued to reduce CIN by low doses of paclitaxel and nucleoside supplementation, respectively. Distinct CIN mechanisms indicated relationships with HGSC-relevant therapy including Poly (ADP-Ribose) Polymerase (PARP) inhibition and microtubule-targeting agents. Comprehensive genomic and transcriptomic profiling revealed deregulation of various genes involved in genome stability but were not directly predictive of specific CIN mechanisms, underscoring the importance of functional characterization to identify causes of CIN. Overall, we show that HGSC CIN is complex and suggest that specific CIN mechanisms could be used as functional biomarkers to indicate appropriate therapy.
  31. Elife. 2020 Sep 28. pii: e61287. [Epub ahead of print]9
      APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here, we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.
    Keywords:  cancer biology; chromosomes; gene expression; human
  32. Nat Metab. 2020 Sep 28.
      Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.
  33. Nat Commun. 2020 09 30. 11(1): 4907
      Global alterations in the metabolic network provide substances and energy to support tumor progression. To fuel these metabolic processes, extracellular matrix (ECM) plays a dominant role in supporting the mass transport and providing essential nutrients. Here, we report a fibrinogen and thrombin based coagulation system to construct an artificial ECM (aECM) for selectively cutting-off the tumor metabolic flux. Once a micro-wound is induced, a cascaded gelation of aECM can be triggered to besiege the tumor. Studies on cell behaviors and metabolomics reveal that aECM cuts off the mass transport and leads to a tumor specific starvation to inhibit tumor growth. In orthotopic and spontaneous murine tumor models, this physical barrier also hinders cancer cells from distant metastasis. The in vivo gelation provides an efficient approach to selectively alter the tumor mass transport. This strategy results in a 77% suppression of tumor growth. Most importantly, the gelation of aECM can be induced by clinical operations such as ultrasonic treatment, surgery or radiotherapy, implying this strategy is potential to be translated into a clinical combination regimen.
  34. Cell Chem Biol. 2020 Sep 23. pii: S2451-9456(20)30377-9. [Epub ahead of print]
      Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.
    Keywords:  DUBs; chemical probe; deubiquitinating enzymes; drug discovery; proteomics
  35. Trends Mol Med. 2020 Sep 23. pii: S1471-4914(20)30217-3. [Epub ahead of print]
      Cells may naturally proceed or be forced to transition to a state with a radically lower biological age, that is, be rejuvenated. Examples are the conversion of somatic cells to induced pluripotent stem cells and rejuvenation of the germline with each generation. We posit that these processes converge to the same 'ground zero', the mid-embryonic state characterized by the lowest biological age where both organismal life and aging begin. It may also be related to the phylotypic state. The ground zero model clarifies the relationship between aging, development, rejuvenation, and de-differentiation, which are distinct throughout life. By extending the rejuvenation phase during early embryogenesis and editing the genome, it may be possible to achieve the biological age at the ground zero lower than that achieved naturally.
    Keywords:  aging; biological age; development; ground zero; lifespan; rejuvenation
  36. Cancer Res. 2020 Sep 30. pii: canres.2199.2020. [Epub ahead of print]
      Lipid rafts are tightly packed, cholesterol- and sphingolipid-enriched microdomains within the plasma membrane that play important roles in many pathophysiological processes. Rafts have been strongly implicated as master regulators of signal transduction in cancer, where raft compartmentalization can promote transmembrane receptor oligomerization, shield proteins from enzymatic degradation, and act as scaffolds to enhance intracellular signaling cascades. Cancer cells have been found to exploit these mechanisms to initiate oncogenic signaling and promote tumor progression. This review highlights the roles of lipid rafts within the metastatic cascade, specifically within tumor angiogenesis, cell adhesion, migration, EMT, and transendothelial migration. Additionally, the interplay between lipid rafts and different modes of cancer cell death, including necrosis, apoptosis, and anoikis will be described. The clinical role of lipid raft-specific proteins caveolin and flotillin in assessing patient prognosis and evaluating metastatic potential of various cancers will be presented. Collectively, elucidation of the complex roles of lipid rafts and raft components within the metastatic cascade may be instrumental for therapeutic discovery to curb pro-metastatic processes.
  37. Biomedicines. 2020 Sep 26. pii: E379. [Epub ahead of print]8(10):
      Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive tumor malignancy worldwide, mainly due to uncontrolled metastasis. Among the numerous molecules deregulated in PDAC, different members of the Akt pathways are of great importance because they are involved in tumor cell proliferation, migration, and invasion. We have recently demonstrated that Vav1, ectopically expressed in solid tumors, is capable of down-modulating expression and/or activation of specific Akt isoforms in breast cancer cells. By using pancreatic cell lines expressing different basal levels of Vav1, we demonstrated here that Vav1 down-regulates the expression of Akt2, known to correlate with tumor metastases and resistance to therapy. In particular, while the silencing of Vav1 is sufficient to induce Akt2, its up-modulation reduces Akt2 levels only when Vav1 accumulates inside the nucleus of PDAC cells. Moreover, in PDAC tissues, we revealed that high nuclear levels of Vav1 correlate with low Akt2 expression. Although we cannot demonstrate the mechanisms involved, our results provide new insights into the role of Vav1 in PDAC and, as targeting specific members of the Akt family is a promising therapeutic chance in solid tumors, they suggest that Vav1, by down-modulating Akt2, has potential as a molecular target in PDAC.
    Keywords:  Vav1; pancreatic ductal adenocarcinoma; targeting Akt pathways
  38. Mol Biol Cell. 2020 Oct 01. 31(21): 2289-2297
      Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.
  39. Support Care Cancer. 2020 Sep 29.
      BACKGROUND: Selecting study endpoints in prospective cancer cachexia trials remains poorly defined. The aim of this study was to further evaluate associations in changes in weight, body composition, functional outcomes, and patient-reported outcomes (PROs) in patients with metastatic cancer.METHODS: We completed a 2-year (2016-2018) observational study in patients with metastatic solid cancer and ECOG performance status 0 to 2 while receiving chemotherapy and/or immunotherapy. We completed assessments at study enrollment and 3 months from enrollment. We analyzed longitudinal changes in weight and body composition using validated methods. Functional assessments included the 6-Min Walk Test, Timed Up and Go Test, and Short Physical Performance Battery. PROs included the Functional Assessment of Anorexia/Cachexia Therapy and Functional Assessment of Cancer Therapy Fatigue. We analyzed changes in body composition and functional assessment using paired t tests. Additionally, we utilized linear regression models to assess relationships between changes in body composition and function outcomes and PROs, adjusting for age and sex.
    RESULTS: A total of 57 patients completed baseline assessments, but 19 patients did not complete 3-month assessments (5 died, 1 hospice, 13 withdrew). Of the 38 patients with complete data, the mean age was 61.8 years and 47% were female. Metastatic cancer types included 71% gastrointestinal, 13% lung, and 8% gynecologic. Half received chemotherapy, 16% immunotherapy, and 34% a combination. From enrollment to 3 months, we did not observe a change in weight or skeletal muscle but did find an increase in total adipose tissue (16.9 ± 52.4 cm2, 95% CI - 33.79-0.63; p = 0.059; ~ 1.5 pounds). We did not observe any association with changes in weight with any functional outcomes or PROs. However, greater losses in skeletal muscle were associated with greater declines in physical function (6-Min Walk Test [B = 0.04, p = 0.01], Short Physical Performance Battery [B = 2.44, p < 0.01]).
    CONCLUSIONS: Patients with metastatic cancer receiving cancer-directed therapy may not experience a change in body weight. However, we found an association between losses in skeletal muscle and greater declines in physical function. Therefore, when selecting study endpoints, prospective cancer cachexia studies may consider selecting changes in body composition over weight.
    Keywords:  Cancer cachexia; Functional status; Quality of life
  40. J Cachexia Sarcopenia Muscle. 2020 Oct 02.
      BACKGROUND: Cachexia is a major cause of morbidity in pancreatic ductal adenocarcinoma (PDAC) patients. Our purpose was to understand the impact of PDAC-induced cachexia on brain metabolism in PDAC xenograft studies, to gain new insights into the causes of cachexia-induced morbidity. Changes in mouse and human plasma metabolites were characterized to identify underlying causes of brain metabolic changes.METHODS: We quantified metabolites, detected with high-resolution 1 H magnetic resonance spectroscopy, in the brain and plasma of normal mice (n = 10) and mice bearing cachexia (n = 10) or non-cachexia (n = 9) inducing PDAC xenografts as well as in human plasma obtained from normal individuals (n = 24) and from individuals with benign pancreatic disease (n = 20) and PDAC (n = 20). Statistical significance was defined as a P value ≤0.05.
    RESULTS: The brain metabolic signature of cachexia-inducing PDAC was characterized by a significant depletion of choline of -27% and -21% as well as increases of glutamine of 13% and 9% and formate of 21% and 14%, relative to normal controls and non-cachectic tumour-bearing mice, respectively. Good to moderate correlations with percent weight change were found for choline (r = 0.70), glutamine (r = -0.58), and formate (r = -0.43). Significant choline depletion of -38% and -30%, relative to normal controls and non-cachectic tumour-bearing mice, respectively, detected in the plasma of cachectic mice likely contributed to decreased brain choline in cachectic mice. Similarly, relative to normal controls and patients with benign disease, choline levels in human plasma samples of PDAC patients were significantly lower by -12% and -20% respectively. A comparison of plasma metabolites from PDAC patients with and without weight loss identified significant changes in glutamine metabolism.
    CONCLUSIONS: Disturbances in metabolites of the choline/cholinergic and glutamine/glutamate/glutamatergic neurotransmitter pathways may contribute to morbidity. Metabolic normalization may provide strategies to reduce morbidity. The human plasma metabolite changes observed may lead to the development of companion diagnostic markers to detect PDAC and PDAC-induced cachexia.
    Keywords:  Brain and plasma 1H MR spectroscopy; Cachexia; Human pancreatic cancer xenografts; Human plasma; Metabolites
  41. Am J Physiol Cell Physiol. 2020 Sep 30.
      We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase α1 expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase α isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase α isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the α1 isoform, but not the α2 or α3 isoform. Imaging analyses showed the cellular redistribution of α1 and α3, but not α2. Moreover, U18666A led to redistribution of α1 to late endosomes/lysosomes, while the proteasome inhibitor blocked α1 reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase α subunit by cholesterol is isoform-specific and α1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in α1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in α2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different α isoforms, we found that Na/K-ATPase, Src and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex is indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.
    Keywords:  Cholesterol; Na/K-ATPase; Src-binding domain; caveolin-1; isoforms
  42. Nat Rev Genet. 2020 Sep 28.
      Contrary to the long-held view that most humans harbour only identical mitochondrial genomes, deep resequencing has uncovered unanticipated extreme genetic variation within mitochondrial DNA (mtDNA). Most, if not all, humans contain multiple mtDNA genotypes (heteroplasmy); specific patterns of variants accumulate in different tissues, including cancers, over time; and some variants are preferentially passed down or suppressed in the maternal germ line. These findings cast light on the origin and spread of mtDNA mutations at multiple scales, from the organelle to the human population, and challenge the conventional view that high percentages of a mutation are required before a new variant has functional consequences.
  43. J Cell Biol. 2020 Nov 02. pii: e202001063. [Epub ahead of print]219(11):
      How rapid cell multiplication leads to cell differentiation in developing tissues is still enigmatic. This question is central to morphogenesis, cell number control, and homeostasis. Self-renewal epidermoid epithelia are continuously exposed to mutagens and are the most common target of cancer. Unknown mechanisms commit rapidly proliferating cells to post-mitotic terminal differentiation. We have over-activated or inhibited the endogenous DNA damage response (DDR) pathways by combinations of activating TopBP1 protein, specific shRNAs, or chemical inhibitors for ATR, ATM, and/or DNA-PK. The results dissect and demonstrate that these signals control keratinocyte differentiation in proliferating cells independently of actual DNA damage. The DDR limits keratinocyte multiplication upon hyperproliferative stimuli. Moreover, knocking down H2AX, a common target of the DDR pathways, inhibits the epidermoid phenotype. The results altogether show that the DDR is required to maintain the balance proliferation differentiation and suggest that is part of the squamous program. We propose a homeostatic model where genetic damage is automatically and continuously cleansed by cell-autonomous mechanisms.
  44. Elife. 2020 Sep 28. pii: e56006. [Epub ahead of print]9
      Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mice revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases in order to sustain neuronal migration.
    Keywords:  mouse; neuroscience
  45. Cancer Metastasis Rev. 2020 Oct 02.
      Environment surrounding tumours are now recognized to play an important role in tumour development and progression. Among the cells found in the tumour environment, adipocytes from adipose tissue establish a vicious cycle with cancer cells to promote cancer survival, proliferation, metastasis and treatment resistance. This cycle is particularly of interest in the context of obesity, which has been found as a cancer risk factor. Cancers cells can reprogram adipocyte physiology leading to an "activated" phenotype characterized by delipidation and secretion of inflammatory adipokines. The adipocyte secretions then influence tumour growth and metastasis which has been mainly attributed to interleukin 6 (IL-6) or leptin but also to the release of fatty acids which are able to change cancer cell metabolism and signalling pathways. The aim of this review is to report recent advances in the understanding of the molecular mechanisms linking adipose tissue with cancer progression in order to propose new therapeutic strategies based on pharmacological or nutritional intervention.
    Keywords:  Adipocyte; Adipokines; Cancer; Exosome; Fatty acid; Metabolism
  46. J Biol Chem. 2020 Oct 02. pii: jbc.RA120.014704. [Epub ahead of print]
      CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro CARM1 knockout mice are perinatally lethal and display a reduced fetal size, while PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of wild-type MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.
    Keywords:  Arginine Methylation; CARM1; PRMT6; epigenetics; histone methylation; post-transcriptional regulation; post-translational modification (PTM); transcription coactivator
  47. Nat Genet. 2020 Sep 28.
      Epidemiological studies have identified many environmental agents that appear to significantly increase cancer risk in human populations. By analyzing tumor genomes from mice chronically exposed to 1 of 20 known or suspected human carcinogens, we reveal that most agents do not generate distinct mutational signatures or increase mutation burden, with most mutations, including driver mutations, resulting from tissue-specific endogenous processes. We identify signatures resulting from exposure to cobalt and vinylidene chloride and link distinct human signatures (SBS19 and SBS42) with 1,2,3-trichloropropane, a haloalkane and pollutant of drinking water, and find these and other signatures in human tumor genomes. We define the cross-species genomic landscape of tumors induced by an important compendium of agents with relevance to human health.
  48. Oncogene. 2020 Sep 28.
      Hypoxia-inducible factor 1 (HIF1) signaling pathway plays a key role in cancer progression by enhancing glycolysis through activating the transcription of glycolytic genes. JMJD2D, a histone demethylase that specifically demethylates H3K9me2/3, can promote colorectal cancer (CRC) progression. However, it is unknown whether JMJD2D could promote CRC progression by enhancing glycolysis through activating HIF1 signaling pathway. In this study, we found that downregulation of JMJD2D inhibited the glycolysis in CRC cells through suppressing HIF1 signaling pathway to downregulate glycolytic gene expression. Restoring HIF1 signaling by enforced expression of HIF1α in JMJD2D-knockdown CRC cells partially recovered CRC cell glycolysis, proliferation, migration, invasion, xenograft growth, and metastasis, suggesting that JMJD2D promotes CRC progression by enhancing glycolysis through activating HIF1 signaling pathway. JMJD2D activated HIF1 signaling pathway through three different mechanisms: JMJD2D cooperated with the transcription factor SOX9 to enhance mTOR expression and then to promote HIF1α translation; JMJD2D cooperated with the transcription factor c-Fos to enhance HIF1β transcription; JMJD2D interacted and cooperated with HIF1α to enhance the expression of glycolytic gene. The demethylase-defective mutant of JMJD2D could not induce the expression of mTOR, HIF1α, HIF1β, and glycolytic genes, suggesting that the demethylase activity of JMJD2D is important for glycolysis through activating HIF1 signaling. Clinically, a highly positive correlation between the expression of JMJD2D and mTOR, HIF1β, and several glycolytic genes in human CRC specimens was identified. Collectively, our study reveals an important role of JMJD2D in CRC progression by enhancing glycolysis through activating HIF1 signaling pathway.
  49. J Cachexia Sarcopenia Muscle. 2020 Sep 28.
      BACKGROUND: Our evolutionary history is defined, in part, by our ability to survive times of nutrient scarcity. The outcomes of the metabolic and behavioural adaptations during starvation are highly efficient macronutrient allocation, minimization of energy expenditure, and maximized odds of finding food. However, in different contexts, caloric deprivation is met with vastly different physiologic and behavioural responses, which challenge the primacy of energy homeostasis.METHODS: We conducted a literature review of scientific studies in humans, laboratory animals, and non-laboratory animals that evaluated the physiologic, metabolic, and behavioural responses to fasting, starvation, protein-deficient or essential amino acid-deficient diets, and cachexia. Studies that investigated the changes in ingestive behaviour, locomotor activity, resting metabolic rate, and tissue catabolism were selected as the focus of discussion.
    RESULTS: Whereas starvation responses prioritize energy balance, both protein malnutrition and cachexia present existential threats that induce unique adaptive programmes, which can exacerbate the caloric insufficiency of undernutrition. We compare and contrast the behavioural and metabolic responses and elucidate the mechanistic pathways that drive state-dependent alterations in energy seeking and partitioning.
    CONCLUSIONS: The evolution of energetically inefficient metabolic and behavioural responses to protein malnutrition and cachexia reveal a hierarchy of metabolic priorities governed by discrete regulatory networks.
    Keywords:  Cachexia; Evolution; Metabolism; Protein malnutrition; Starvation
  50. Cell. 2020 Sep 28. pii: S0092-8674(20)31142-9. [Epub ahead of print]
      Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.
    Keywords:  circulating tumor DNA; immune checkpoint inhibition; immunotherapy; liquid biopsy; non-small cell lung cancer; response classification
  51. Nat Commun. 2020 Oct 02. 11(1): 4627
      Animals have evolved responses to low oxygen conditions to ensure their survival. Here, we have identified the C. elegans zinc finger transcription factor PQM-1 as a regulator of the hypoxic stress response. PQM-1 is required for the longevity of insulin signaling mutants, but surprisingly, loss of PQM-1 increases survival under hypoxic conditions. PQM-1 functions as a metabolic regulator by controlling oxygen consumption rates, suppressing hypoxic glycogen levels, and inhibiting the expression of the sorbitol dehydrogenase-1 SODH-1, a crucial sugar metabolism enzyme. PQM-1 promotes hypoxic fat metabolism by maintaining the expression of the stearoyl-CoA desaturase FAT-7, an oxygen consuming, rate-limiting enzyme in fatty acid biosynthesis. PQM-1 activity positively regulates fat transport to developing oocytes through vitellogenins under hypoxic conditions, thereby increasing survival rates of arrested progeny during hypoxia. Thus, while pqm-1 mutants increase survival of mothers, ultimately this loss is detrimental to progeny survival. Our data support a model in which PQM-1 controls a trade-off between lipid metabolic activity in the mother and her progeny to promote the survival of the species under hypoxic conditions.
  52. J Clin Invest. 2020 Sep 29. pii: 140837. [Epub ahead of print]
      Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost the CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made towards harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.
    Keywords:  Antigen presentation; Breast cancer; Cancer immunotherapy; Immunology; Oncology
  53. Nat Commun. 2020 09 30. 11(1): 4909
      Effectively activating macrophages against cancer is promising but challenging. In particular, cancer cells express CD47, a 'don't eat me' signal that interacts with signal regulatory protein alpha (SIRPα) on macrophages to prevent phagocytosis. Also, cancer cells secrete stimulating factors, which polarize tumor-associated macrophages from an antitumor M1 phenotype to a tumorigenic M2 phenotype. Here, we report that hybrid cell membrane nanovesicles (known as hNVs) displaying SIRPα variants with significantly increased affinity to CD47 and containing M2-to-M1 repolarization signals can disable both mechanisms. The hNVs block CD47-SIRPα signaling axis while promoting M2-to-M1 repolarization within tumor microenvironment, significantly preventing both local recurrence and distant metastasis in malignant melanoma models. Furthermore, by loading a stimulator of interferon genes (STING) agonist, hNVs lead to potent tumor inhibition in a poorly immunogenic triple negative breast cancer model. hNVs are safe, stable, drug loadable, and suitable for genetic editing. These properties, combined with the capabilities inherited from source cells, make hNVs an attractive immunotherapy.
  54. J Gastroenterol Hepatol. 2020 Sep 30.
      INTRODUCTION: Even though ductal interventions in chronic pancreatitis (CP) is known to improve pain, its impact on diabetes is unclear. In this cohort study, we evaluated the impact of ductal interventions on diabetes in these patients.METHODS: Consecutive patients with CP visiting the Pancreas Clinic between 1st August 2011 and 31st July 2012 were enrolled and followed till December 2018. Detailed clinical, laboratory, imaging, and treatment data were recorded at enrollment and follow-up. Patients were followed 6-monthly through hospital visit and/or telephonic interview. Risk factors for diabetes were evaluated using logistic regression. The impact of ductal interventions on diabetes was evaluated using Kaplan Meier survival analyses and Cox Proportional Hazard.
    RESULTS: 644 patients were enrolled of which 137 were excluded. 326 (64.3%) patients had idiopathic CP. 283 (55.8%) patients underwent ductal intervention. The cumulative incidence of diabetes was 57.9%. Median duration between symptom onset and ductal intervention were similar irrespective of diabetes [2.6 (0.6-6.0) vs 3.0 (1.0-5.5) yrs; p=0.69]. Alcohol intake and pancreatic ductal calculi were independent risk factors for diabetes (OR [95% CI] of 2.05 (1.18-3.55), p=0.01 and 2.05 (1.28-3.28), p=0.003 respectively). Kaplan Meier analyses revealed that diabetes free interval- was significantly longer in patients undergoing ductal interventions, predominantly in those with idiopathic CP with obstructive ductal calculi [HR (95%CI) 0.39 (0.28-0.55); p< 0.0001]. There were no differences in glycemic status in patients with non-idiopathic CP and those with preexisting diabetes.
    DISCUSSION: Early ductal intervention could delay development of diabetes in patients with idiopathic CP with obstructive ductal calculi.
    Keywords:  ERCP; ESWL; chronic pancreatitis; diabetes; ductal intervention; lateral pancreaticojejunostomy; risk factors
  55. Aging Cell. 2020 Sep 29. e13248
      Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in-depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20-70 years of age that encode for all subunits comprising complexes I-V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. "Oxidative phosphorylation" was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I-V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt-Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells.
  56. Elife. 2020 Oct 01. pii: e60445. [Epub ahead of print]9
      Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/- zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/- myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.
    Keywords:  developmental biology; regenerative medicine; stem cells; zebrafish
  57. Nat Cancer. 2020 May;1(5): 533-545
      Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.
  58. Trends Pharmacol Sci. 2020 Sep 26. pii: S0165-6147(20)30204-2. [Epub ahead of print]
      Metformin can improve patients' hyperglycemia through significant suppression of hepatic glucose production. However, up to 300 times higher concentrations of metformin accumulate in the intestine than in the circulation, where it alters nutrient metabolism in intestinal epithelial cells and microbiome, leading to increased lactate production. Hepatocytes use lactate to make glucose at the cost of energy expenditure, creating a futile intestine-liver cycle. Furthermore, metformin reduces blood lipopolysaccharides and its initiated low-grade inflammation and increased oxidative phosphorylation in liver and adipose tissues. These metformin effects result in the improvement of insulin sensitivity and glucose utilization in extrahepatic tissues. In this review, I discuss the current understanding of the impact of metformin on systemic metabolism and its molecular mechanisms of action in various tissues.
    Keywords:  Metformin; insulin resistance; mitochondria; nutrient metabolism
  59. J Clin Med. 2020 Sep 25. pii: E3102. [Epub ahead of print]9(10):
      Pancreatic intraepithelial neoplasms (PanINs) and intraductal papillary mucinous neoplasms (IPMNs) are common pancreatic adenocarcinoma precursor lesions. However, data regarding their respective associations with survival rate and prognosis are lacking. We retrospectively evaluated 72 pancreatic adenocarcinoma tumor resection patients at the University of Kansas Hospital between August 2009 and March 2019. Patients were divided into one of two groups, PanIN or IPMN, based on the results of the surgical pathology report. We compared baseline characteristics, overall survival (OS), and progression free survival (PFS) between the two groups, as well as OS and PFS based on local or distant tumor recurrence for both groups combined. 52 patients had PanINs and 20 patients had IPMNs. Patients who had an IPMN precursor lesion had better median PFS and OS when compared to patients with PanIN precursor lesions. However, the location of tumor recurrence (local or distant) did not show a statistically significant difference in OS.
    Keywords:  IPMN; PanIN; pancreatic adenocarcinoma; pancreatic cancer; precursor lesion
  60. JCI Insight. 2020 Sep 29. pii: 137474. [Epub ahead of print]
      With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat Type 2 diabetes (T2DM) can extend mouse lifespan, with greater effect in males than in females. Utilizing a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological and biochemical changes that occur during aging, and determined which of the effects of age and drug were sex-dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids - changes which have previously been associated with aging, cardiac dysfunction and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging anti-aging effects in multiple tissues, and may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.
    Keywords:  Aging; Cellular senescence
  61. Mol Cell Biochem. 2020 Oct 01.
      Telomeres are protective structures that are shortened during the lifetime, resulting in aging and degenerative diseases. Subjects experiencing aging and degenerative disorders present smaller telomeres than young and healthy ones. The size of these structures can be stabilized by telomerase, an enzyme which is inactive in adult tissues but functional in fetal and newborn tissues and adult testes and ovaries. The aim of this study was to perform a systematic review to evaluate the effect of telomerase activation in the treatment of degenerative and aging disorders. We accomplished the search using the Pubmed interface for papers published from September 1985 to April 16th, 2020. We found twenty one studies that matched our eligibility criteria. I concluded that telomerase is probably a potential and safe treatment for aging and degenerative diseases, demonstrating neither side effects nor risk of cancer in the selected studies. Further studies in humans are needed to confirm safety and efficiency of this treatment.
    Keywords:  Aging; Biological therapy; Enzyme activation; Systematic review; Telomerase; Telomere
  62. J Cell Physiol. 2020 Sep 30.
      Fibrosis is a common pathological condition associated with abnormal repair after tissue injury. However, the etiology and molecular mechanisms of fibrosis are still not well-understood. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) belongs to the TNF superfamily and acts by binding to its receptor, fibroblast growth factor-inducible 14 (Fn14), thereby activating a variety of intracellular signal transduction pathways in various types of cells. Besides promoting the expression of growth factors, activation of TWEAK/Fn14 signaling after tissue injury can promote the expression of pro-inflammatory cytokines, which trigger the immune response, thereby exacerbating the injury. Severe or repetitive injury leads to a dysregulated tissue repair process, in which the TWEAK/Fn14 axis promotes the activation and proliferation of myofibroblasts, induces the secretion of the extracellular matrix, and regulates profibrotic mediators to further perpetuate and sustain the fibrotic process. In this review, we summarize the available experimental evidence on the underlying molecular mechanisms by which the TWEAK/Fn14 pathway mediates the development and progression of fibrosis. In addition, we discuss the therapeutic potential of the TWEAK/Fn14 pathway in fibrosis-associated diseases based on evidence derived from multiple models and cells from injured tissue and fibrotic tissue.
    Keywords:  Fn14; TWEAK; fibrosis; injury; tumor necrosis factor
  63. Nat Cell Biol. 2020 Oct;22(10): 1180-1186
      Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs)1-3. Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates4. Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.
  64. Nat Commun. 2020 09 29. 11(1): 4903
      The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.
  65. Mol Oncol. 2020 Sep 27.
      Senescence refers to a cellular state featuring a stable cell-cycle arrest triggered in response to stress. This response also involves other distinct morphological and intracellular changes including alterations in gene expression and epigenetic modifications, elevated macromolecular damage, metabolism deregulation, and a complex proinflammatory secretory phenotype. The initial demonstration of oncogene-induced senescence in vitro established senescence as an important tumour suppressive mechanism, in addition to apoptosis. Senescence not only halts the proliferation of premalignant cells but also facilitates the clearance of affected cells through immunosurveillance. Failure to clear senescent cells owing to deficient immunosurveillance may, however, lead to a state of chronic inflammation that nurtures a pro-tumorigenic microenvironment favouring cancer initiation, migration, and metastasis. In addition, senescence is a response to post-therapy genotoxic stress. Therefore, tracking the emergence of senescent cells becomes pivotal to detect potential pro-tumorigenic events. Current protocols for the in vivo detection of senescence require the analysis of fixed or deep-frozen tissues, despite a significant clinical need for real-time bioimaging methods. Accuracy and efficiency of senescence detection is further hampered by a lack of universal and more specific senescence biomarkers. Recently, in an attempt to overcome these hurdles, an assortment of detection tools have been developed. These strategies all have significant potential for clinical utilization, and include flow cytometry combined with histo- or cytochemical approaches, nanoparticle-based targeted delivery of imaging contrast agents, OFF-ON fluorescent senoprobes, positron emission tomography (PET) senoprobes, and analysis of circulating SASP factors, extracellular vesicles, and cell-free nucleic acids isolated from plasma. Here we highlight the occurrence of senescence in neoplasia and advanced tumours, assess the impact of senescence on tumorigenesis, and discuss how the ongoing development of senescence detection tools might improve early detection of multiple cancers and response to therapy in the near future.
    Keywords:  Cellular senescence; cancer; detection; senoprobes; tumour microenvironment
  66. Oncogene. 2020 Sep 28.
      Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.
  67. Nat Commun. 2020 10 01. 11(1): 4913
      Reprograming of proline metabolism is critical for tumor growth. Here we show that PINCH-1 is highly expressed in lung adenocarcinoma and promotes proline synthesis through regulation of mitochondrial dynamics. Knockout (KO) of PINCH-1 increases dynamin-related protein 1 (DRP1) expression and mitochondrial fragmentation, which suppresses kindlin-2 mitochondrial translocation and interaction with pyrroline-5-carboxylate reductase 1 (PYCR1), resulting in inhibition of proline synthesis and cell proliferation. Depletion of DRP1 reverses PINCH-1 deficiency-induced defects on mitochondrial dynamics, proline synthesis and cell proliferation. Furthermore, overexpression of PYCR1 in PINCH-1 KO cells restores proline synthesis and cell proliferation, and suppresses DRP1 expression and mitochondrial fragmentation. Finally, ablation of PINCH-1 from lung adenocarcinoma in mouse increases DRP1 expression and inhibits PYCR1 expression, proline synthesis, fibrosis and tumor growth. Our results identify a signaling axis consisting of PINCH-1, DRP1 and PYCR1 that regulates mitochondrial dynamics and proline synthesis, and suggest an attractive strategy for alleviation of tumor growth.
  68. Nat Immunol. 2020 Sep 28.
      Foxp3+ regulatory T (Treg) cells expressing the interleukin (IL)-33 receptor ST2 mediate tissue repair in response to IL-33. Whether Treg cells also respond to the alarmin IL-33 to regulate specific aspects of the immune response is not known. Here we describe an unexpected function of ST2+ Treg cells in suppressing the innate immune response in the lung to environmental allergens without altering the adaptive immune response. Following allergen exposure, ST2+ Treg cells were activated by IL-33 to suppress IL-17-producing γδ T cells. ST2 signaling in Treg cells induced Ebi3, a component of the heterodimeric cytokine IL-35 that was required for Treg cell-mediated suppression of γδ T cells. This response resulted in fewer eosinophil-attracting chemokines and reduced eosinophil recruitment into the lung, which was beneficial to the host in reducing allergen-induced inflammation. Thus, we define a fundamental role for ST2+ Treg cells in the lung as a negative regulator of the early innate γδ T cell response to mucosal injury.
  69. Aging (Albany NY). 2020 Sep 29. 12
      DNA methylation (DNAm) age estimators are widely used to study aging-related conditions. It is not yet known whether DNAm age is associated with the accumulation of stochastic epigenetic mutations (SEMs), which reflect dysfunctions of the epigenetic maintenance system. Here, we defined epigenetic mutation load (EML) as the total number of SEMs per individual. We assessed associations between EML and DNAm age acceleration estimators using biweight midcorrelations in four population-based studies (total n = 6,388). EML was not only positively associated with chronological age (meta r = 0.171), but also with four measures of epigenetic age acceleration: the Horvath pan tissue clock, intrinsic epigenetic age acceleration, the Hannum clock, and the GrimAge clock (meta-analysis correlation ranging from r = 0.109 to 0.179). We further conducted pathway enrichment analyses for each participant's SEMs. The enrichment result demonstrated the stochasticity of epigenetic mutations, meanwhile implicated several pathways: signaling, neurogenesis, neurotransmitter, glucocorticoid, and circadian rhythm pathways may contribute to faster DNAm age acceleration. Finally, investigating genomic-region specific EML, we found that EMLs located within regions of transcriptional repression (TSS1500, TSS200, and 1stExon) were associated with faster age acceleration. Overall, our findings suggest a role for the accumulation of epigenetic mutations in the aging process.
    Keywords:  DNA methylation; aging; epigenetic clock; epigenetic mutation load; stochastic epigenetic mutation
  70. Annu Rev Pharmacol Toxicol. 2020 Sep 30.
      Senescence is the consequence of a signaling mechanism activated in stressed cells to prevent proliferation of cells with damage. Senescent cells (Sncs) often develop a senescence-associated secretory phenotype to prompt immune clearance, which drives chronic sterile inflammation and plays a causal role in aging and age-related diseases. Sncs accumulate with age and at anatomical sites of disease. Thus, they are regarded as a logical therapeutic target. Senotherapeutics are a new class of drugs that selectively kill Sncs (senolytics) or suppress their disease-causing phenotypes (senomorphics/senostatics). Since 2015, several senolytics went from identification to clinical trial. Preclinical data indicate that senolytics alleviate disease in numerous organs, improve physical function and resilience, and suppress all causes of mortality, even if administered to the aged. Here, we review the evidence that Sncs drive aging and disease, the approaches to identify and optimize senotherapeutics, and the current status of preclinical and clinical testing of senolytics. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 61 is January 7, 2021. Please see for revised estimates.
  71. Cancer Med. 2020 Sep 30.
      BACKGROUND: Real-world practice patterns, treatment sequencing, and outcomes in patients with metastatic pancreatic cancer remain unclear. Previous research indicates that the likelihood of patients with metastatic pancreatic cancer receiving or continuing cancer-directed therapy is low-a phenomenon called nihilism. This retrospective, descriptive analysis examined clinical characteristics, treatment patterns, and outcomes for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC).METHODS: Treatment patterns were examined using electronic health records from the Flatiron Health database covering the period from January 1, 2014, to June 30, 2019. Real-world overall survival [rwOS]) was compared for a subgroup of patients receiving treatment and a matched subgroup not receiving treatment.
    RESULTS: Of 7666 patients, 5687 (74.2%) received at least one line of systemic therapy. A greater proportion of patients receiving treatment than not receiving treatment had an initial diagnosis of stage IV disease (68.8% vs 61.2%, respectively). Among patients receiving an initial therapy, fewer than half (38.2%; 2174/5687) received second-line treatment, mostly because they died, and only 34.3% (745/2174) of those receiving second-line treatment advanced to third-line treatment. The rwOS for patients receiving at least one line of systemic therapy was 8.1 months versus 2.6 months for matched patients not receiving treatment (hazard ratio, 0.41; 95% confidence interval, 0.38-0.45; 1470 patients per group).
    CONCLUSIONS: Systemic therapy provided significant clinical benefit for patients who were eligible and chose to receive it, particularly when treatment was consistent with guideline recommendations. The large proportion of patients initiating treatment suggests that nihilism with mPDAC is diminishing.
    Keywords:  antineoplastic combined chemotherapy protocols; carcinoma; electronic health records; pancreatic ductal; retrospective studies; survival analysis
  72. J Clin Invest. 2020 Sep 28. pii: 142237. [Epub ahead of print]
      Growth hormone-secreting (GH-secreting) pituitary tumors are driven by oncogenes that induce cAMP signaling. In this issue of the JCI, Ben-Shlomo et al. performed a whole-exome study of pituitary adenomas. GH-secreting tumors had a high frequency of whole chromosome or chromosome arm copy number alterations and were associated with an increase in the tumor protein p53 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1, which are findings consistent with induction of a response to DNA damage. Further, treatment of mouse pituitary cells with cAMP pathway agonists in vitro and in vivo elicited biomarkers of DNA replication stress or double-strand breaks. The findings of Ben Shlomo et al. indicate that oncoproteins that drive constitutively high cAMP signaling pathway output in susceptible cell types can elicit DNA replication stress and may promote genomic instability.
  73. Science. 2020 Oct 02. 370(6512): 50-56
      Sleep is evolutionarily conserved across all species, and impaired sleep is a common trait of the diseased brain. Sleep quality decreases as we age, and disruption of the regular sleep architecture is a frequent antecedent to the onset of dementia in neurodegenerative diseases. The glymphatic system, which clears the brain of protein waste products, is mostly active during sleep. Yet the glymphatic system degrades with age, suggesting a causal relationship between sleep disturbance and symptomatic progression in the neurodegenerative dementias. The ties that bind sleep, aging, glymphatic clearance, and protein aggregation have shed new light on the pathogenesis of a broad range of neurodegenerative diseases, for which glymphatic failure may constitute a therapeutically targetable final common pathway.
  74. J Physiol. 2020 Sep 29.
      KEY POINTS: In humans, hypohydration attenuates sweat secretion and attenuates whole-body heat loss, probably to mitigate further fluid losses and thereby support blood pressure regulation. Recently, however, we demonstrated that the hypohydration-mediated reduction in net whole-body heat exchange (evaporative heat loss - dry heat gain) was blunted in middle-aged compared to younger men during moderate exercise in dry heat; albeit, the underpinning mechanisms could not be determined. Here we evaluated the hypothesis that those findings stemmed from a diminished influence of extracellular hyperosmolality on net whole-body heat exchange in middle-aged-to-older compared to young men. Consistent with that hypothesis, extracellular hyperosmolality induced by an intravenous infusion of hypertonic saline (3% NaCl) reduced net heat exchange and augmented rectal temperature to a greater extent in the young compared to middle-aged-to-older men. Thus, age-related differences in the influence of hypohydration on thermoregulatory function appear to be due to blunted sensitivity to hyperosmolality with ageing.ABSTRACT: We recently demonstrated that sweating-induced hypohydration attenuated whole-body heat dissipation to a greater extent in young compared to middle-aged men during exercise-heat stress. Here, we evaluated whether this divergent response stemmed from an attenuated influence of extracellular hyperosmolality on heat exchange with ageing. To achieve this, ten young (mean (SD): 25 (5) years) and ten middle-aged-to-older (61 (5) years) men completed two trials involving a 90-min intravenous infusion of isosmotic saline (0.9% NaCl; ISO) or hyperosmotic saline (3.0% NaCl; HYP) followed by 60 min of cycling at a fixed metabolic heat production of 250 W/m2 (∼50% peak aerobic power) in dry heat (40°C, ∼17% relative humidity). Whole-body net heat exchange (evaporative heat loss - dry heat gain) was measured via direct calorimetry. Rectal temperature was monitored continuously. Heat exchange was attenuated in HYP compared to ISO in the young (233 (20) vs. 251 (17) W/m2 ; P = 0.002) but not older group (229 (16) vs. 227 (20) W/m2 ; P = 0.621). Further, heat exchange was lower in the middle-aged-to-older vs. young men in ISO (P = 0.034) but not in HYP (P = 0.623). Similarly, end-exercise rectal temperature was greater in HYP relative to ISO in the young (38.3 (0.4)°C vs. 37.9 (0.3)°C; P = 0.015) but not the middle-aged-to-older men (38.3 (0.3)°C vs. 38.2 (0.2)°C; P = 0.652). Compared to the young, rectal temperature was greater in the middle-aged-to-older during ISO (P = 0.035) whereas no between-group difference was observed in HYP (P = 0.746). Our findings indicate that ageing blunts the effect of extracellular hyperosmolality on thermoregulatory function during exercise-heat stress.
    Keywords:  ageing; exercise; fluid regulation; heat stress; hyperosmolality; thermoregulation
  75. Target Oncol. 2020 Sep 29.
      Pancreatic cancer is expected to become the second leading cause of cancer-related death within the next few years. Current therapeutic strategies have limited effectiveness and therefore there is an urgency to develop novel effective therapies. The receptor tyrosine kinase subfamily TAM (Tyro3, Axl, MerTK) is directly implicated in the pathogenesis of the metastatic, chemoresistant, and immunosuppressive phenotype in pancreatic cancer. TAM inhibitors are promising investigational therapies for pancreatic cancer due to their potential to target multiple aspects of pancreatic cancer biology. Specifically, recent mechanistic investigations and therapeutic combinations in the preclinical setting suggest that TAM inhibition with chemotherapy, targeted therapy, and immunotherapy should be evaluated clinically.
  76. Proc Natl Acad Sci U S A. 2020 Sep 28. pii: 202005330. [Epub ahead of print]
      The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα -/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.
    Keywords:  NR1D1; circadian clock; energy metabolism; liver; nuclear hormone receptor
  77. Lab Invest. 2020 Sep 29.
      Metabolic flux analysis (MFA) aims at revealing the metabolic reaction rates in a complex biochemical network. To do so, MFA uses the input of stable isotope labeling patterns of the intracellular metabolites. Elementary metabolic unit (EMU) is the computational framework to simulate the metabolite labeling patterns in a network, which was originally designed for simulating mass isotopomer distributions (MIDs) at the MS1 level. Recently, the EMU framework is expanded to simulate tandem mass spectrometry data. Tandem mass spectrometry has emerged as a new experimental approach to provide information on the positional isotope labeling of metabolites and therefore greatly improves the precision of MFA. In this review, we will discuss the new EMU framework that can accommodate the tandem mass isotopomer distributions (TMIDs) data. We will also analyze the improvement on the MFA precision by using TMID. Our analysis shows that combining the MIDs of the parent and daughter ions and the TMID for the MFA is more powerful than using TMID alone.
  78. Cancers (Basel). 2020 Sep 24. pii: E2750. [Epub ahead of print]12(10):
      Pancreatic ductal adenocarcinoma (PDAC) represents 90% of pancreatic malignancies. In contrast to many other tumor entities, the prognosis of PDAC has not significantly improved during the past thirty years. Patients are often diagnosed too late, leading to an overall five-year survival rate below 10%. More dramatically, PDAC cases are on the rise and it is expected to become the second leading cause of death by cancer in western countries by 2030. Currently, the use of gemcitabine/nab-paclitaxel or FOLFIRINOX remains the standard chemotherapy treatment but still with limited efficiency. There is an urgent need for the development of early diagnostic and therapeutic tools. To this point, in the past 5 years, organoid technology has emerged as a revolution in the field of PDAC personalized medicine. Here, we are reviewing and discussing the current technical and scientific knowledge on PDAC organoids, their future perspectives, and how they can represent a game change in the fight against PDAC by improving both diagnosis and treatment options.
    Keywords:  3D cell culture; PDAC; chemotherapy; drug response; organoids; personalized medicine
  79. Cells. 2020 Sep 29. pii: E2197. [Epub ahead of print]9(10):
      Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or Cox10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1-5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1α during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1α regulate the assembly of respiratory complexes.
    Keywords:  COX10; HIF-1α; Rieske iron sulfur protein; complex I; complex III; complex IV; hypoxia; mitochondrial respiratory supercomplexes; oxidative phosphorylation
  80. Eur Radiol. 2020 Sep 30.
      OBJECTIVES: To retrospectively examine US, CT, and MR imaging examinations of missed or misinterpreted pancreatic ductal adenocarcinoma (PDAC), and identify factors which may have confounded detection or interpretation.METHODS: We reviewed 107 examinations in 66/257 patients (26%, mean age 73.7 years) diagnosed with PDAC in 2014 and 2015, with missed or misinterpreted imaging findings as determined by a prior study. For each patient, images and reports were independently reviewed by two radiologists, and in consensus, the following factors which may have confounded assessment were recorded: inherent tumor factors, concurrent pancreatic pathology, technical limitations, and cognitive biases. Secondary signs of PDAC associated with each examination were recorded and compared with the original report to determine which findings were missed.
    RESULTS: There were 66/107 (62%) and 49/107 (46%) cases with missed and misinterpreted imaging findings, respectively. A significant number of missed tumors were < 2 cm (45/107, 42%), isoattenuating on CT (32/72, 44%) or non-contour deforming (44/107, 41%). Most (29/49, 59%) misinterpreted examinations were reported as uncomplicated pancreatitis. Almost all examinations (94/107, 88%) demonstrated secondary signs; pancreatic duct dilation was the most common (65/107, 61%) and vascular invasion was the most commonly missed 35/39 (90%). Of the CT and MRIs, 28 of 88 (32%) had suboptimal contrast dosing. Inattentional blindness was the most common cognitive bias, identified in 55/107 (51%) of the exams.
    CONCLUSION: Recognizing pitfalls of PDAC detection and interpretation, including intrinsic tumor features, secondary signs, technical factors, and cognitive biases, can assist radiologists in making an early and accurate diagnosis.
    KEY POINTS: • There were 66/107 (62%) and 49/107 (46%) cases with missed and misinterpreted imaging findings, respectively, with tumoral, technical, and cognitive factors leading to the misdiagnosis of pancreatic ductal adenocarcinoma. • The majority (29/49, 59%) of misinterpreted cases of pancreatic ductal adenocarcinoma were mistaken for pancreatitis, where an underlying mass or secondary signs were not appreciated due to inflammatory changes. • The most common missed secondary sign of pancreatic ductal adenocarcinoma was vascular encasement, missed in 35/39 (90%) of cases, indicating the importance of evaluating the peri-pancreatic vasculature.
    Keywords:  Pancreatic ductal carcinoma; Pancreatic neoplasms; Pancreatitis; Retrospective studies
  81. Nat Commun. 2020 Oct 02. 11(1): 4951
      Immunogenic cell death (ICD) and tumour-infiltrating T lymphocytes are severely weakened by elevated reactive oxygen species (ROS) in the tumour microenvironment. It is therefore of critical importance to modulate the level of extracellular ROS for the reversal of immunosuppressive environment. Here, we present a tumour extracellular matrix (ECM) targeting ROS nanoscavenger masked by pH sensitive covalently crosslinked polyethylene glycol. The nanoscavenger anchors on the ECM to sweep away the ROS from tumour microenvironment to relieve the immunosuppressive ICD elicited by specific chemotherapy and prolong the survival of T cells for personalized cancer immunotherapy. In a breast cancer model, elimination of the ROS in tumour microenvironment elicited antitumour immunity and increased infiltration of T lymphocytes, resulting in highly potent antitumour effect. The study highlights a strategy to enhance the efficacy of cancer immunotherapy by scavenging extracellular ROS using advanced nanomaterials.
  82. Elife. 2020 Sep 29. pii: e60066. [Epub ahead of print]9
      Scars are a serious health concern for burn victims and individuals with skin conditions associated with wound healing. Here, we identify regenerative factors in neonatal murine skin that transforms adult skin to regenerate instead of only repairing wounds with a scar, without perturbing development and homeostasis. Using scRNA-seq to probe unsorted cells from regenerating, scarring, homeostatic, and developing skin, we identified neonatal papillary fibroblasts that form a transient regenerative cell type that promotes healthy skin regeneration in young skin. These fibroblasts are defined by the expression of a canonical Wnt transcription factor Lef1 and using gain- and loss of function genetic mouse models, we demonstrate that Lef1 expression in fibroblasts primes the adult skin macroenvironment to enhance skin repair, including regeneration of hair follicles with arrector pili muscles in healed wounds. Finally, we share our genomic data in an interactive, searchable companion website ( Together, these data and resources provide a platform to leverage the regenerative abilities of neonatal skin to develop clinically tractable solutions that promote the regeneration of adult tissue.
    Keywords:  Lef1; dermal papilla; fibroblast heterogeneity; mouse; regeneration; regenerative medicine; stem cells; wound healing
  83. Cancers (Basel). 2020 Sep 28. pii: E2780. [Epub ahead of print]12(10):
      Numerous findings have indicated that CSCs, which are present at a low frequency inside primary tumors, are the main cause of therapy resistance and cancer recurrence. Although various therapeutic methods targeting CSCs have been attempted for eliminating cancer cells completely, the complicated characteristics of CSCs have hampered such attempts. In analyzing the biological properties of CSCs, it was revealed that CSCs have a peculiar metabolism that is distinct from non-CSCs to maintain their stemness properties. The CSC metabolism involves not only the catabolic and anabolic pathways, but also intracellular signaling, gene expression, and redox balance. In addition, CSCs can reprogram their metabolism to flexibly respond to environmental changes. In this review, we focus on the flexible metabolic mechanisms of CSCs, and highlight the new therapeutics that target CSC metabolism.
    Keywords:  ROS; cancer stem cells; glucose metabolism; metastasis; mitochondrial metabolism; redox homeostasis
  84. Cancer Discov. 2020 Oct 01. pii: CD-20-0571. [Epub ahead of print]
      On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKA G667 and TRKC G696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations.
  85. Cell Stem Cell. 2020 Sep 24. pii: S1934-5909(20)30450-1. [Epub ahead of print]
      Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-β pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.
    Keywords:  CXCR4; DNA damage; Fanconi anemia; MYC; bone marrow failure; genotoxic stress; hematopoietic stem cells; p53; physiological stress; single-cell RNA sequencing
  86. Lancet Oncol. 2020 Oct;pii: S1470-2045(20)30328-4. [Epub ahead of print]21(10): e463-e476
      Immunotherapy represents a paradigm shift in oncology treatment. The goal of immunotherapy is to overcome immunosuppression induced by a tumour and its microenvironment, thereby allowing the immune system to target and kill cancer cells. The immunotherapy era began when the first immune checkpoint inhibitor, ipilimumab, was approved for use almost a decade ago. This therapeutic approach is associated with distinct types of response, including processes such as pseudoprogression (ie, increased tumour burden via radiology, which is not accompanied by clinical deterioration) and hyperprogression (ie, rapid progression of the disease as a result of immunotherapy). In this Review, we focus on therapeutic approaches for patients who progress on immunotherapy. We review the different types of clinical responses associated with immunotherapy and describe treatment options for this population.
  87. J Clin Invest. 2020 Sep 29. pii: 140966. [Epub ahead of print]
      Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer's disease; however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial-specific ablation of C3ar1. Using an in vitro model of the BBB, we identify intracellular Ca2+ as a downstream effector of C3a-C3aR signaling and a functional mediator of VE-cadherins junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation is also observed in a tau transgenic mouse model. Our studies suggest that heightened C3a-C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contribute to overall neuroinflammation in aging and neurodegenerative disease.
    Keywords:  Aging; Alzheimer's disease
  88. J Proteome Res. 2020 Sep 28.
      KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as non-receptor tyrosine kinases, MAPK kinases and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect the subtle differences between related oncogenic mutants and potentially lead to individualized treatments.
  89. Cancer Metab. 2020 ;8 21
      Background: Overweight or obesity has been evidenced as an important risk factor involved in the incidence, mortality, and therapy response of multiple malignancies. However, the differences between healthy and obesity tumor patients at the molecular and multi-omics levels remain unclear.Methods: Our study performed a comprehensive and multidimensional analysis in fourteen tumor types of The Cancer Genome Atlas (TCGA) and found body mass index (BMI)-related genes in multiple tumor types. Furthermore, we compared composite expression between normal, overweight, and obese patients of each immune cell subpopulation and metabolism gene subset. Statistical significance was calculated via the Kruskal-Wallis rank-sum test.
    Results: Our analysis revealed that BMI-related genes are enriched in multiple tumor-related biological pathways involved in intracellular signaling, immune response, and metabolism. We also found the different relationships between BMI and different immune cell infiltration and metabolic pathway activity. Importantly, we found that many clinically actionable genes were BMI-affect genes.
    Conclusion: Overall, our data indicated that BMI-associated molecular characteristics involved in tumor immune and metabolic pathways, which may highlight the clinical importance of considering BMI-associated molecular signatures in cancer precision medicine.
    Keywords:  Body mass index; Metabolic pathways; Molecular characteristics; Pan-cancer analysis; Tumor immune microenvironment
  90. Cancer Cell. 2020 Sep 15. pii: S1535-6108(20)30432-3. [Epub ahead of print]
      PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1+ T cells which closely associate with PD-L1+ cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.
    Keywords:  PD-1; PD-L1; T cells; checkpoint immunotherapy; dendritic cells; lymph node; tumor immunology
  91. Clin Nutr. 2020 Sep 18. pii: S0261-5614(20)30477-5. [Epub ahead of print]
      BACKGROUND & AIMS: A growing number of studies have shown that body fat and inflammation are associated with age-related changes in body muscle composition. However, most of these studies did not control for potential confounders. The aim was to determine whether there is an association between body fat and inflammatory cytokines with muscle mass/strength decline in community-dwelling older adults.METHODS: Anthropometric, physical and functionality variables were collected. Nutritional status was assessed by the MNA form. Dynapenia was assessed with handgrip strength on the dominant hand using a dynamometer. Sarcopenia was determined using adapted criteria from the European Working Group on Sarcopenia in Older People 2 (EWGSOP2). Inflammatory cytokines were evaluated in plasma using a multiplex assay. Associations to muscle mass/strength decline were analyzed using a multinominal logistic regression, adjusted for potential confounders.
    RESULTS: We recruited a convenience sample of 311 adults aged 60 years or older. Most of subjects were sufficiently active females with a median age of 68 years (interquartile range [IQR], 64-74 years), whereas about a half (46.3%) were at risk of malnutrition. The prevalence of dynapenia was 38.3%, whereas sarcopenia was 13.2%. After controlling for potential confounders, we found that relative fat mass index is independently associated with sarcopenia. Loss of strength was independently associated only with female sex, lower physical activity, worse nutrition and IL-10/TNF-α ratio, whereas female sex, an insufficiently active lifestyle and relative fat mass index were the key determinants of sarcopenia.
    CONCLUSIONS: These findings highlight the importance of physical activity and healthy diet as effective interventions to prevent muscle mass/strength decline, and points to IL-10/TNF-α ratio and body fat as independently associated factors for dynapenia and sarcopenia, respectively.
    Keywords:  Body fat; Dynapenia; Inflammation; Sarcopenia