bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒12‒20
fifty-eight papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Cell Metab. 2020 Dec 10. pii: S1550-4131(20)30657-4. [Epub ahead of print]
      Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.
    Keywords:  SHMT; cancer metabolism; folate cycle; in vivo; isotope tracing; metabolomics; mitochondria; one-carbon flux; physiologic medium; reduced folate carrier; serine hydroxymethyltransferase
  2. Dev Cell. 2020 Dec 07. pii: S1534-5807(20)30925-4. [Epub ahead of print]
      Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function, and neurodegeneration. The compositional and functional alterations in NPC lysosomes and nature of aberrant cholesterol-mTORC1 signaling contribution to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion, enhanced membrane damage, and defective mitophagy. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.
    Keywords:  ESCRT; NPC1; autophagy; cholesterol; lysosome; mTORC1; mitochondria; proteolysis; proteomics
  3. Cancers (Basel). 2020 Dec 15. pii: E3787. [Epub ahead of print]12(12):
      Patients with pancreatic ductal adenocarcinoma (PDAC) suffer debilitating and deadly weight loss, known as cachexia. Development of therapies requires biomarkers to diagnose, and monitor cachexia; however, no such markers are in use. Via Somascan, we measured ~1300 plasma proteins in 30 patients with PDAC vs. 11 controls. We found 60 proteins specific to local PDAC, 46 to metastatic, and 67 to presence of >5% cancer weight loss (FC ≥ |1.5|, p ≤ 0.05). Six were common for cancer stage (Up: GDF15, TIMP1, IL1RL1; Down: CCL22, APP, CLEC1B). Four were common for local/cachexia (C1R, PRKCG, ELANE, SOST: all oppositely regulated) and four for metastatic/cachexia (SERPINA6, PDGFRA, PRSS2, PRSS1: all consistently changed), suggesting that stage and cachexia status might be molecularly separable. We found 71 proteins that correlated with cachexia severity via weight loss grade, weight loss, skeletal muscle index and radiodensity (r ≥ |0.50|, p ≤ 0.05), including some known cachexia mediators/markers (LEP, MSTN, ALB) as well as novel proteins (e.g., LYVE1, C7, F2). Pathway, correlation, and upstream regulator analyses identified known (e.g., IL6, proteosome, mitochondrial dysfunction) and novel (e.g., Wnt signaling, NK cells) mechanisms. Overall, this study affords a basis for validation and provides insights into the processes underpinning cancer cachexia.
    Keywords:  biomarkers; cachexia; humans; neoplasms; pancreatic adenocarcinoma; paracrine communication; proteome; weight loss
  4. Cancer Cell. 2020 Nov 28. pii: S1535-6108(20)30598-5. [Epub ahead of print]
      Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-β in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.
    Keywords:  DNA sensing; MLH1; MSI; STING; T cell infiltration; cGAS; cancer; checkpoint blockade; cytosolic DNA; mismatch repair
  5. Nature. 2020 Dec 16.
      Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
  6. Mitochondrion. 2020 Dec 11. pii: S1567-7249(20)30224-5. [Epub ahead of print]
      Mitochondria play vital role in regulating the cellular energetics and metabolism. Further, it is a signaling hub for cell survival and apoptotic pathways. One of the key determinants that calibrate both cellular energetics and survival functions is mitochondrial calcium (Ca2+) dynamics. Mitochondrial Ca2+ regulates three Ca2+-sensitive dehydrogenase enzymes involved in tricarboxylic acid cycle (TCA) cycle thereby directly controlling ATP synthesis. On the other hand, excessive Ca2+ concentration within the mitochondrial matrix elevates mitochondrial reactive oxygen species (mROS) levels and causes mitochondrial membrane depolarization. This leads to opening of the mitochondrial permeability transition pore (mPTP) and release of cytochrome c into cytosol eventually triggering apoptosis. Therefore, it is critical for cell to maintain mitochondrial Ca2+ concentration. Since cells can neither synthesize nor metabolize Ca2+, it is the dynamic interplay of Ca2+ handling proteins involved in mitochondrial Ca2+ influx and efflux that take the center stage. In this review we would discuss the key molecular machinery regulating mitochondrial Ca2+ concentration. We would focus on the channel complex involved in bringing Ca2+ into mitochondrial matrix i.e. Mitochondrial Ca2+ Uniporter (MCU) and its key regulators Mitochondrial Ca2+ Uptake proteins (MICU1, 2 and 3), MCU regulatory subunit b (MCUb), Essential MCU Regulator (EMRE) and Mitochondrial Ca2+ Uniporter Regulator 1 (MCUR1). Further, we would deliberate on major mitochondrial Ca2+ efflux proteins i.e. Mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) and Leucine zipper EF hand-containing transmembrane1 (Letm1). Moreover, we would highlight the physiological functions of these proteins and discuss their relevance in human pathophysiology. Finally, we would highlight key outstanding questions in the field.
    Keywords:  EMRE; Letm1; MCU complex; MCUR1; MICU 1/2/3; Mitochondrial calcium dynamics; NCLX
  7. Cancer Cell. 2020 Dec 03. pii: S1535-6108(20)30596-1. [Epub ahead of print]
      Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.
    Keywords:  DNA breaks; MLH1; RPA exhaustion; Rad51; cGAS-STING; chromosome instability; cytosolic DNA; exonuclease 1; mismatch repair
  8. Cancer Discov. 2020 Dec 16. pii: CD-20-0652. [Epub ahead of print]
      KRAS-mutant colorectal cancers (CRC) are resistant to therapeutics, presenting a significant problem for ~40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant CRC. Using Kras-mutant mouse models and mouse- and patient-derived organoids we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of CRCs have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent co-targeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population who may benefit from its clinical application.
  9. Trends Cancer. 2020 Dec 11. pii: S2405-8033(20)30289-2. [Epub ahead of print]
      Deeper and broader sequencing of circulating tumor DNA (ctDNA) has identified a wealth of cancer markers in the circulation, resulting in a paradigm shift towards data science-driven liquid biopsies in oncology. Although panel sequencing for actionable mutations in plasma is moving towards the clinic, the next generation of liquid biopsies is increasingly shifting from analyzing digital mutation signals towards analog signals, requiring a greater role for machine learning. Concomitantly, there is an increasing acceptance that these cancer signals do not have to arise from the tumor itself. In this Opinion, we discuss the opportunities and challenges arising from increasingly complex cancer liquid biopsy data.
    Keywords:  cancer; cell-free DNA; circulating tumor DNA; liquid biopsy; oncology
  10. Biol Open. 2020 Dec 16. pii: bio056747. [Epub ahead of print]9(12):
      Mechanisms that enhance energy expenditure are attractive therapeutic targets for obesity. Previously we have demonstrated that mice lacking cd47 are leaner, exhibit increased energy expenditure, and are protected against diet-induced obesity. In this study, we further defined the physiological role of cd47 deficiency in regulating mitochondrial function and energy expenditure in both white and brown adipose tissue. We observed that cd47 deficient mice (under normal chow diet) had comparable amount of white fat mass but reduced white adipocyte size as compared to wild-type mice. Subsequent ex vivo and in vitro studies suggest enhanced lipolysis, and not impaired lipogenesis or energy utilization, contributes to this phenotype. In contrast to white adipose tissue, there were no obvious morphological differences in brown adipose tissue between wild-type and knockout mice. However, mitochondria isolated from brown fat of cd47 deficient mice had significantly higher rates of free fatty acid-mediated uncoupling. This suggests that enhanced fuel availability via white adipose tissue lipolysis may perpetuate elevated brown adipose tissue energy expenditure and contributes to the lean phenotype observed in cd47 deficient mice.
    Keywords:  Brown fat; CD47; Lipolysis; Mitochondria; White fat
  11. Curr Biol. 2020 Dec 08. pii: S0960-9822(20)31748-6. [Epub ahead of print]
      Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. Although in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that, in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear-envelope-rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.
    Keywords:  DNA damage; cancer; cell compression; confined migration; metastasis; nuclear deformation; nuclear envelope rupture; nuclear mechanobiology; replication stress
  12. Nat Commun. 2020 12 11. 11(1): 6339
      Ferroptosis is a more recently recognized form of cell death that relies on iron-mediated oxidative damage. Here, we evaluate the impact of high-iron diets or depletion of Gpx4, an antioxidant enzyme reported as an important ferroptosis suppressor, in the pancreas of mice with cerulean- or L-arginine-induced pancreatitis, and in an oncogenic Kras murine model of spontaneous pancreatic ductal adenocarcinoma (PDAC). We find that either high-iron diets or Gpx4 depletion promotes 8-OHG release and thus activates the TMEM173/STING-dependent DNA sensor pathway, which results in macrophage infiltration and activation during Kras-driven PDAC in mice. Consequently, the administration of liproxstatin-1 (a ferroptosis inhibitor), clophosome-mediated macrophage depletion, or pharmacological and genetic inhibition of the 8-OHG-TMEM173 pathway suppresses Kras-driven pancreatic tumorigenesis in mice. GPX4 is also a prognostic marker in patients with PDAC. These findings provide pathological and mechanistic insights into ferroptotic damage in PDAC tumorigenesis in mice.
  13. Cancer Metab. 2020 Dec 11. 8(1): 29
      BACKGROUND: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown.METHODS: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma.
    RESULTS: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients.
    CONCLUSION: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM.
    TRIAL REGISTRATION: NCT03384108 and NCT03119883.
    Keywords:  Glutamine; Myeloma; Plasma cell malignancies; Stable isotope metabolomics
  14. Epigenetics. 2020 Dec 14.
      Unstructured Abstract DNA methylation (DNAm) age may reflect age-related variations in biological changes and abnormalities related to aging. DNAm age acceleration measures have been associated with a number of cancers, but to our knowledge, have not been examined in relation to pancreatic cancer risk or survival. DNAm levels in leukocytes of prediagnostic blood samples of 393 pancreatic cancer cases and 431 matched controls, pooled from three large prospective cohort studies, were used to estimate DNAm age, epigenetic age acceleration (AA) and intrinsic epigenetic age acceleration (IEAA) metrics. Logistic regression and Cox proportional hazard regression models were used to examine the relationship between the various AA and IEAA metrics and pancreatic cancer risk and survival, respectively. The results showed that pancreatic cancer risk was significantly increased across all IEAA metrics, ranging from 83% to 95% increased risk when comparing the third and highest quartiles to the lowest quartile of IEAA. Consistent with these findings, the results from multivariate spline regression analyses showed non-linear relationships between all three IEAA metrics and pancreatic cancer risk with apparent threshold effect including 2 turning points at minimal and at maximal risks, respectively. There is no evidence of significant association between pancreatic cancer survival and any of the epigenetic AA or IEAA metrics. Our results indicate DNAm age acceleration, measured in blood prior to cancer diagnosis, is associated with an increased risk of pancreatic cancer in a complex nonlinear, dose-response manner. Epigenetic IEAA metrics may be a useful addition to current methods for pancreatic cancer risk prediction.
    Keywords:  DNA methylation; Pancreatic cancer; epigenetic clock; prospective study; risk
  15. Nature. 2020 Dec 16.
      FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.
  16. Front Cell Dev Biol. 2020 ;8 594416
      Since the identification and definition of the hallmarks of aging, these aspects of molecular and cellular decline have been most often described as isolated or distinct mechanisms. However, there is significant evidence demonstrating interplay between most of these hallmarks and that they have the capacity to influence and regulate one another. These interactions are demonstrable across the tree of life, yet not all aspects are conserved. Here, we describe an integrative view on the hallmarks of aging by using the hallmark "mitochondrial dysfunction" as a focus point, and illustrate its capacity to both influence and be influenced by the other hallmarks of aging. We discuss the effects of mitochondrial pathways involved in aging, such as oxidative phosphorylation, mitochondrial dynamics, mitochondrial protein synthesis, mitophagy, reactive oxygen species and mitochondrial DNA damage in relation to each of the primary, antagonistic and integrative hallmarks. We discuss the similarities and differences in these interactions throughout the tree of life, and speculate how speciation may play a role in the variation in these mechanisms. We propose that the hallmarks are critically intertwined, and that mapping the full extent of these interactions would be of significant benefit to the aging research community.
    Keywords:  aging; hallmarks of aging; interplay; mitochondria; tree of life
  17. Nat Metab. 2020 12;2(12): 1482-1497
      White and beige adipocytes in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) are maintained by proliferation and differentiation of adipose progenitor cells (APCs). Here we use mice with tissue-specific telomerase reverse transcriptase (TERT) gene knockout (KO), which undergo premature telomere shortening and proliferative senescence in APCs, to investigate the effect of over-nutrition on APC exhaustion and metabolic dysfunction. We find that TERT KO in the Pdgfra+ cell lineage results in adipocyte hypertrophy, inflammation and fibrosis in SAT, while TERT KO in the Pdgfrb+ lineage leads to adipocyte hypertrophy in both SAT and VAT. Systemic insulin resistance is observed in both KO models and is aggravated by a high-fat diet. Analysis of human biopsies demonstrates that telomere shortening in SAT is associated with metabolic disease progression after bariatric surgery. Our data indicate that over-nutrition can promote APC senescence and provide a mechanistic link between ageing, obesity and diabetes.
  18. Sci Adv. 2020 Dec;pii: eabc5629. [Epub ahead of print]6(51):
      Circadian gene expression driven by transcription activators CLOCK and BMAL1 is intimately associated with dynamic chromatin remodeling. However, how cellular metabolism directs circadian chromatin remodeling is virtually unexplored. We report that the S-adenosylhomocysteine (SAH) hydrolyzing enzyme adenosylhomocysteinase (AHCY) cyclically associates to CLOCK-BMAL1 at chromatin sites and promotes circadian transcriptional activity. SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases, and timely hydrolysis of SAH by AHCY is critical to sustain methylation reactions. We show that AHCY is essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin, and subsequent circadian transcription. Depletion or targeted pharmacological inhibition of AHCY in mammalian cells markedly decreases the amplitude of circadian gene expression. In mice, pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription within the suprachiasmatic nucleus. These results reveal a previously unappreciated connection between cellular metabolism, chromatin dynamics, and circadian regulation.
  19. Cell Metab. 2020 Dec 08. pii: S1550-4131(20)30653-7. [Epub ahead of print]
      Caspase-4 is an intracellular sensor for cytosolic bacterial lipopolysaccharide (LPS) and underlies infection-elicited pyroptosis. It is unclear whether and how caspase-4 detects host-derived factors to trigger pyroptosis. Here we show that mitochondrial permeability transition (MPT) activates caspase-4 by promoting the assembly of a protein complex, which we term the Apaf-1 pyroptosome, for the execution of facilitated pyroptosis. MPT, when induced by bile acids, calcium overload, or an adenine nucleotide translocator 1 (ANT1) activator, triggers assembly of the pyroptosome comprised of Apaf-1 and caspase-4 with a stoichiometry ratio of 7:2. Unlike the direct cleavage of gasdermin D (GSDMD) by caspase-4 upon LPS ligation, caspase-4 activated in the Apaf-1 pyroptosome proceeds to cleave caspase-3 and thereby GSDME to induce pyroptosis. Caspase-4-initiated and GSDME-executed pyroptosis underlies cholestatic liver failure. These findings identify Apaf-1 pyroptosome as a pivotal machinery for cells sensing MPT signals and may shed light on understanding how cells execute intrinsic pyroptosis under sterile conditions.
    Keywords:  Apaf-1; Caspase-4; bile acid; gasdermin E; mitochondrial permeability transition; pyroptosis
  20. Gastroenterology. 2020 Dec 10. pii: S0016-5085(20)35546-3. [Epub ahead of print]
      BACKGROUND & AIMS: There is substantial interest in liquid biopsy approaches for cancer early detection, among subjects at risk, using multi-marker panels. CA19-9 is an established circulating biomarker for pancreatic cancer. However, its relevance for pancreatic cancer early detection or for monitoring subjects at risk has not been established.METHODS: CA19-9 levels were assessed in blinded sera from 175 subjects collected up to 5 years prior to diagnosis of pancreatic cancer and from 875 matched controls from the PLCO Cancer Screening Trial. For comparison of performance, CA19-9 was assayed in blinded independent sets of samples collected at diagnosis from 129 subjects with resectable pancreatic cancer and 275 controls (100 healthy subjects; 50 with chronic pancreatitis; and 125 with non-cancerous pancreatic cysts). The complementary value of two additional protein markers, TIMP1 and LRG1, was determined.
    RESULTS: In the PLCO cohort, levels of CA19-9 increased exponentially starting at two years prior to diagnosis with sensitivities reaching 60% at 99% specificity within 0-6 months prior to diagnosis for all cases and 50% at 99% specificity for cases diagnosed with early-stage disease. Performance was comparable for distinguishing newly diagnosed cases with resectable pancreatic cancer from healthy controls (64% sensitivity at 99% specificity). Comparison of resectable pancreatic cancer cases to subjects with chronic pancreatitis yielded 46% sensitivity at 99% specificity and for subjects with non-cancerous cysts 30% sensitivity at 99% specificity. For pre-diagnostic cases below cut-off value for CA19-9, the combination with LRG1 and TIMP1 yielded an increment of 13.2% in sensitivity at 99% specificity (p=0.031) in identifying cases diagnosed within 1 year of blood collection.
    CONCLUSION: CA19-9 can serve as an anchor marker for pancreatic cancer early detection applications.
    Keywords:  Biomarker; Detection; Pancreatic Cancer
  21. Front Physiol. 2020 ;11 543564
      A metabolic hallmark of many cancers is the increase in glucose consumption coupled to excessive lactate production. Mindful that L-lactate originates only from pyruvate, the question arises as to how can this be sustained in those tissues where pyruvate kinase activity is reduced due to dimerization of PKM2 isoform or inhibited by oxidative/nitrosative stress, posttranslational modifications or mutations, all widely reported findings in the very same cells. Hereby 17 pathways connecting glucose to lactate bypassing pyruvate kinase are reviewed, some of which transit through the mitochondrial matrix. An additional 69 converging pathways leading to pyruvate and lactate, but not commencing from glucose, are also examined. The minor production of pyruvate and lactate by glutaminolysis is scrutinized separately. The present review aims to highlight the ways through which L-lactate can still be produced from pyruvate using carbon atoms originating from glucose or other substrates in cells with kinetically impaired pyruvate kinase and underscore the importance of mitochondria in cancer metabolism irrespective of oxidative phosphorylation.
    Keywords:  Warburg effect; cancer; glycolysis; lactate dehydrogenase; metabolomics; mitochondria; oncometabolism
  22. FEBS Lett. 2020 Dec 12.
      Most of the genetic information has been lost or transferred to the nucleus during the evolution of mitochondria. Neverthelss, mitochondria have retained their own genome that is essential for oxidative phosphorylation (OXPHOS). In mammals, a gene-dense circular mitochondrial DNA (mtDNA) of about 16.5kb encodes 13 proteins, which constitute only 1% of the mitochondrial proteome. Mammalian mtDNA is present in thousands of copies per cell and mutations often affect only a fraction of them. Most pathogenic human mtDNA mutations are recessive and only cause OXPHOS defects if present above a certain critical threshold. However, emerging evidence strongly suggests that the proportion of mutated mtDNA copies is not the only determinant of disease but that also the absolute copy number matters. In this review, we critically discuss current knowledge of the role of mtDNA copy number regulation in various types of human diseases, including mitochondrial disorders, neurodegenerative disorders, and cancer, and during ageing. We also provide an overview of new exciting therapeutic strategies to directly manipulate mtDNA to restore OXPHOS in mitochondrial diseases.
    Keywords:  Alzheimer; Parkinson; TFAM; ageing; cancer; mitochondria; mitochondrial diseases; mtDNA; mtDNA copy number; neurodegenerative disorders; s disease
  23. FASEB J. 2021 Jan;35(1): e21218
      Growth differentiating factor-15 (GDF15) is an emerging target for the treatment of obesity and metabolic disease partly due to its ability to suppress food intake. GDF15 expression and secretion are thought to be regulated by a cellular integrated stress response, which involves endoplasmic reticulum (ER) stress. AMPK is another cellular stress sensor, but the relationship between AMPK, ER stress, and GDF15 has not been assessed in vivo. Wildtype (WT), AMPK β1 deficient (AMPKβ1-/- ), and CHOP-/- mice were treated with three distinct AMPK activators; AICAR, which is converted to ZMP mimicking the effects of AMP on the AMPKγ isoform, R419, which indirectly activates AMPK through inhibition of mitochondrial respiration, or A769662, a direct AMPK activator which binds the AMPKβ1 isoform ADaM site causing allosteric activation. Following treatments, liver Gdf15, markers of ER-stress, AMPK activity, adenine nucleotides, circulating GDF15, and food intake were assessed. AICAR and R419 caused ER and energetic stress, increased GDF15 expression and secretion, and suppressed food intake. Direct activation of AMPK β1 containing complexes by A769662 increased hepatic Gdf15 expression, circulating GDF15, and suppressed food intake, independent of ER stress. The effects of AICAR, R419, and A769662 on GDF15 were attenuated in AMPKβ1-/- mice. AICAR and A769662 increased GDF15 to a similar extent in WT and CHOP-/- mice. Herein, we provide evidence that AMPK plays a role in mediating the induction of GDF15 under conditions of energetic stress in mouse liver in vivo.
    Keywords:  AMPK; CHOP; ER stress; GDF15; energetic stress; liver
  24. Carcinogenesis. 2020 Dec 15. pii: bgaa135. [Epub ahead of print]
      In the past two decades, a ponderous epidemiological literature has causally linked tumor onset to environmental exposure to carcinogens. As consequence, risk assessment studies have been carried out with the aim to identify both predictive models of estimating cancer risks within exposed populations and establishing rules for minimizing hazard when handling carcinogenic compounds. The central assumption of these works is that neoplastic transformation is directly related to the mutational burden of the cell without providing further mechanistic clues to explain increased cancer onset after carcinogen exposure. Nevertheless, in the last few years, a growing number of studies have implemented the traditional models of cancer aetiology, proposing that neoplastic transformation is a complex process in which several parameters and crosstalk between tumor and microenvironmental cells must be taken into account and integrated with mutagenesis. In this conceptual framework, the current strategies of risk assessment that are solely based on the "mutator model" require an urgent update and revision to keep pace with advances in our understanding of cancer biology. We will approach this topic revising the most recent theories on the biological mechanisms involved in tumor formation in order to envision a roadmap leading to a future regulatory framework for a new, protective policy of risk assessment.
  25. Nat Metab. 2020 12;2(12): 1472-1481
      Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.
  26. Elife. 2020 Dec 15. pii: e63614. [Epub ahead of print]9
      The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.
    Keywords:  cell biology; human
  27. Cancers (Basel). 2020 Dec 15. pii: E3774. [Epub ahead of print]12(12):
      Pancreatic cancer is a malignant disease with high mortality and a dismal prognosis. Circulating tumor cell (CTC) detection and characterization have emerged as essential techniques for early detection, prognostication, and liquid biopsy in many solid malignancies. Unfortunately, due to the low EPCAM expression in pancreatic cancer CTCs, no specific marker is available to identify and isolate this rare cell population. This study analyzed single-cell RNA sequencing profiles of pancreatic CTCs from a genetically engineered mouse model (GEMM) and pancreatic cancer patients. Through dimensionality reduction analysis, murine pancreatic CTCs were grouped into three clusters with different biological functions. CLIC4 and GAS2L1 were shown to be overexpressed in pancreatic CTCs in comparison with peripheral blood mononuclear cells (PBMCs). Further analyses of PBMCs and RNA-sequencing datasets of enriched pancreatic CTCs were used to validate the overexpression of GAS2L1 in pancreatic CTCs. A combinatorial approach using both GAS2L1 and EPCAM expression leads to an increased detection rate of CTCs in PDAC in both GEMM and patient samples. GAS2L1 is thus proposed as a novel biomarker of pancreatic cancer CTCs.
    Keywords:  biomarkers; circulating tumor cells; computational biology; genetically engineered mouse model; liquid biopsy; mice; pancreatic ductal adenocarcinoma; pancreatic neoplasms; single-cell RNA sequencing
  28. Nat Protoc. 2020 Dec 11.
      Understanding cell-cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell-cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2-3 weeks to generate the labeling cells and 1-2 weeks to test their labeling ability.
  29. Cancers (Basel). 2020 Dec 15. pii: E3770. [Epub ahead of print]12(12):
      Pancreatic cancer is projected to become the second deadliest cancer by 2030 in the United States, and the overall five-year survival rate stands still at around 9%. The stroma compartment can make up more than 90% of the pancreatic tumor mass, contributing to the hypoxic tumor microenvironment. The dense stroma with extracellular matrix proteins can be a physical and metabolic barrier reducing therapeutic efficacy. Cancer-associated fibroblasts are a source of extracellular matrix proteins. Therefore, targeting these cells, or extracellular matrix proteins, have been considered as therapeutic strategies. However, several studies show that deletion of cancer-associated fibroblasts may have tumor-promoting effects. Cancer-associated fibroblasts are derived from a variety of different cell types, such as pancreatic stellate cells and mesenchymal stem cells, and constitute a diverse cell population consisting of several functionally heterogeneous subtypes. Several subtypes of cancer-associated fibroblasts exhibit a tumor-restraining function. This review article summarizes recent findings regarding origin and functional heterogeneity of tumor-promoting as well as tumor-restraining cancer-associated fibroblasts. A better understanding of cancer-associated fibroblast heterogeneity could provide more specific and personalized therapies for pancreatic cancer patients in the future.
    Keywords:  cancer-associated fibroblasts; cancer-restraining cancer-associated fibroblast; cellular heterogeneity; mesenchymal stem cells; pancreatic cancer; pancreatic stellate cells
  30. Elife. 2020 Dec 15. pii: e63665. [Epub ahead of print]9
      It has been known adipocytes increase p53 expression and activity in obesity, however, only canonical p53 functions (i.e., senescence and apoptosis) are attributed to inflammation-associated metabolic phenotypes. Whether or not p53 is directly involved in mature adipocyte metabolic regulation remains unclear. Here we show p53 protein expression can be up-regulated in adipocytes by nutrient starvation without activating cell senescence, apoptosis, or a death-related p53 canonical pathway. Inducing the loss of p53 in mature adipocytes significantly reprograms energy metabolism and this effect is primarily mediated through a AMP-activated protein kinase (AMPK) pathway and a novel downstream transcriptional target, lysosomal acid lipase (LAL). The pathophysiological relevance is further demonstrated in a conditional and adipocyte-specific p53 knockout mouse model. Overall, these data support a non-canonical p53 function in the regulation of adipocyte energy homeostasis and indicate that the dysregulation of this pathway may be involved in developing metabolic dysfunction in obesity.
    Keywords:  cell biology; medicine; mouse
  31. Mol Cell. 2020 Dec 08. pii: S1097-2765(20)30832-7. [Epub ahead of print]
      Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.
    Keywords:  assembly; dynamics; exchange; nuclear pore complex; nucleoporin; quantitative proteomics; turnover
  32. Lancet Gastroenterol Hepatol. 2020 Dec 15. pii: S2468-1253(20)30330-7. [Epub ahead of print]
      BACKGROUND: The optimal preoperative treatment for locally advanced pancreatic cancer is unknown. We aimed to compare the efficacy and safety of nab-paclitaxel plus gemcitabine with nab-paclitaxel plus gemcitabine followed by fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) as multidrug induction chemotherapy regimens in locally advanced pancreatic cancer.METHODS: In this open-label, multicentre, randomised phase 2 study, done at 28 centres in Germany, eligible patients were adults (aged 18-75 years) with an Eastern Cooperative Oncology Group performance status of 0 or 1 and histologically or cytologically confirmed, treatment-naive locally advanced pancreatic adenocarcinoma, as determined by local multidisciplinary team review. After two cycles of nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 (administered intravenously on days 1, 8, and 15 of each 28-day cycle), patients without progressive disease or unacceptable adverse events were randomly assigned (1:1) to receive either two additional cycles of nab-paclitaxel plus gemcitabine (nab-paclitaxel plus gemcitabine group) or four cycles of sequential FOLFIRINOX (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, irinotecan 180 mg/m2, fluorouracil 400 mg/m2 by intravenous bolus followed by a continuous intravenous infusion of 2400 mg/m2 for 46 h on day 1 of each 14-day cycle; sequential FOLFIRINOX group). Randomisation was done by the clinical research organisation on request of the trial centre using a permuted block design (block size 2 and 4). Patients, investigators, and study team members were not masked to treatment allocation. The primary endpoint was surgical conversion rate (complete macroscopic tumour resection) in the randomised population by intention-to-treat analysis, which was assessed by surgical exploration in all patients with at least stable disease after completion of induction chemotherapy. This trial is registered with, NCT02125136.
    FINDINGS: Between Nov 18, 2014, and April 27, 2018, 168 patients were registered and 130 were randomly assigned to either the nab-paclitaxel plus gemcitabine group (64 patients) or the sequential FOLFIRINOX group (66 patients). Surgical exploration after completed induction chemotherapy was done in 40 (63%) of 64 patients in the nab-paclitaxel plus gemcitabine group and 42 (64%) of 66 patients in the sequential FOLFIRINOX group. 23 patients in the nab-paclitaxel plus gemcitabine group and 29 in the sequential FOLFIRINOX group had complete macroscopic tumour resection, yielding a surgical conversion rate of 35·9% (95% CI 24·3-48·9) in the nab-paclitaxel plus gemcitabine group and 43·9% (31·7-56·7) in the sequential FOLFIRINOX group (odds ratio 0·72 [95% CI 0·35-1·45]; p=0·38). At a median follow-up of 24·9 months (95% CI 21·8-27·6), median overall survival was 18·5 months (95% CI 14·4-21·5) in the nab-paclitaxel plus gemcitabine group and 20·7 months (13·9-28·7) in the sequential FOLFIRINOX group (hazard ratio 0·86 [95% CI 0·55-1·36]; p=0·53). All other secondary efficacy endpoints, such as investigator-assessed progression-free survival, radiographic response rate, CA 19-9 response rate, and R0 resection rate, were not significantly different between the two treatment groups except for improved histopathological downstaging in evaluable resection specimens from the sequential FOLFIRINOX group (ypT1/2 stage: 20 [69%] of 29 patients in the sequential FOLFIRINOX group vs four [17%] of 23 patients in the nab-paclitaxel plus gemcitabine group, p=0·0003; ypN0 stage: 15 [52%] of 29 patients in the sequential FOLFIRINOX group vs four [17%] of 23 patients in the nab-paclitaxel plus gemcitabine group, p=0·02). Grade 3 or higher treatment-emergent adverse events during induction chemotherapy occurred in 35 (55%) of 64 patients in nab-paclitaxel plus gemcitabine group and in 35 (53%) of 66 patients in the sequential FOLFIRINOX group. The most common of which were neutropenia (18 [28%] in nab-paclitaxel plus gemcitabine group, 16 [24%] in the sequential FOLFIRINOX group), nausea and vomiting (two [3%] in nab-paclitaxel plus gemcitabine group, eight [12%] in the sequential FOLFIRINOX group), and bile duct obstruction with cholangitis (six [9%] in nab-paclitaxel plus gemcitabine group, seven [11%] in the sequential FOLFIRINOX group). No deaths were caused by treatment-related adverse events during the induction chemotherapy phase.
    INTERPRETATION: Our findings suggest that nab-paclitaxel plus gemcitabine is similarly active and safe as nab-paclitaxel plus gemcitabine followed by FOLFIRINOX as multidrug induction chemotherapy regimens for locally advanced pancreatic cancer. Although conversion to resectability was achieved in about a third of patients, additional evidence is required to determine whether this translates into improved overall survival.
    FUNDING: Celgene.
  33. J Cell Sci. 2020 Dec 11. pii: jcs.243394. [Epub ahead of print]
      Silicosis is characterized by silica exposure-induced lung interstitial fibrosis and formation of silicotic nodules, resulting in lung stiffening. The acetylation of microtubules mediated by α-tubulin N-acetyltransferase 1 (α-TAT1) is a posttranslational modification that promotes microtubule stability in response to mechanical stimulation. α-TAT1 and downstream-acetylated α-tubulin (Ac-α-Tub) are decreased in silicosis, promoting the epithelial-mesenchymal transition (EMT); however, the underlying mechanisms are unknown. We found that silica, matrix stiffening, or their combination triggered Ac-α-Tub downregulation in alveolar epithelial cells, followed by DNA damage and replication stress. α-TAT1 elevated Ac-α-Tub to limit replication stress and the EMT via trafficking of p53-binding protein 1 (53BP1). The results provide evidence that α-TAT1/Ac-α-Tub inhibits the EMT and silicosis fibrosis by preventing 53BP1 mislocalization and relieving DNA damage. This study provides insight into how the cell cycle is regulated during the EMT, and why the decrease in α-TAT1/Ac-α-Tub promotes silicosis fibrosis.
    Keywords:  Acetylated α-tubulin; DNA damage; Epithelial–mesenchymal transition; Silicosis; α-tubulin N-acetyltransferase 1
  34. Elife. 2020 Dec 16. pii: e56849. [Epub ahead of print]9
      Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.
    Keywords:  C. elegans; E. coli; aging; bacteria-host interaction; developmental biology; longevity; mitochondria; optogenetics
  35. Mol Cell. 2020 Dec 08. pii: S1097-2765(20)30833-9. [Epub ahead of print]
      Stalling during translation triggers ribosome quality control (RQC) to maintain proteostasis. Recently, stalling has also been linked to the activation of integrated stress response (ISR) by Gcn2. How the two processes are coordinated is unclear. Here, we show that activation of RQC by Hel2 suppresses that of Gcn2. We further show that Hel2 and Gcn2 are activated by a similar set of agents that cause ribosome stalling, with maximal activation of Hel2 observed at a lower frequency of stalling. Interestingly, inactivation of one pathway was found to result in the overactivation of the other, suggesting that both are activated by the same signal of ribosome collisions. Notably, the processes do not appear to be in direct competition with each other; ISR prefers a vacant A site, whereas RQC displays no preference. Collectively, our findings provide important details about how multiple pathways that recognize stalled ribosomes coordinate to mount the appropriate response.
    Keywords:  Gcn2; Hel2; RNA damage; alkylation; integrated stress response; ribosome quality control; ribosome rescue
  36. Sci Adv. 2020 Dec;pii: eabc7209. [Epub ahead of print]6(51):
      The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains: sheets and interconnected tubules. These domains undergo dynamic reshaping in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status: The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven repositioning of lysosomes, which leads to both a redistribution of the ER tubules and a change of its global morphology. Therefore, lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.
  37. Sci Rep. 2020 Dec 17. 10(1): 22111
      The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and D-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.
  38. Elife. 2020 Dec 15. pii: e58764. [Epub ahead of print]9
      To maintain energy homeostasis during cold exposure, the increased energy demands of thermogenesis must be counterbalanced by increased energy intake. To investigate the neurobiological mechanisms underlying this cold-induced hyperphagia, we asked whether agouti-related peptide (AgRP) neurons are activated when animals are placed in a cold environment and, if so, whether this response is required for the associated hyperphagia. We report that AgRP-neuron activation occurs rapidly upon acute cold exposure, as do increases of both energy expenditure and energy intake, suggesting the mere perception of cold is sufficient to engage each of these responses. We further report that silencing of AgRP neurons selectively blocks the effect of cold exposure to increase food intake but has no effect on energy expenditure. Together, these findings establish a physiologically important role for AgRP neurons in the hyperphagic response to cold exposure.
    Keywords:  mouse; neuroscience
  39. Nat Rev Mol Cell Biol. 2020 Dec 16.
      Cellular senescence, first described in vitro in 1961, has become a focus for biotech companies that target it to ameliorate a variety of human conditions. Eminently characterized by a permanent proliferation arrest, cellular senescence occurs in response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation and persistent DNA damage. Cellular senescence can also be a controlled programme occurring in diverse biological processes, including embryonic development. Senescent cell extrinsic activities, broadly related to the activation of a senescence-associated secretory phenotype, amplify the impact of cell-intrinsic proliferative arrest and contribute to impaired tissue regeneration, chronic age-associated diseases and organismal ageing. This Review discusses the mechanisms and modulators of cellular senescence establishment and induction of a senescence-associated secretory phenotype, and provides an overview of cellular senescence as an emerging opportunity to intervene through senolytic and senomorphic therapies in ageing and ageing-associated diseases.
  40. Cell Stress. 2020 Nov 25. 4(12): 273-277
      Cancer represents the leading public health problem throughout the world. Globally, about one out of six deaths is related to cancer, which is largely due to the metastatic lesions. However, there are no effective strategies for targeting cancer metastasis. Identification of the key druggable targets maintaining metastasis is crucial for cancer treatment. In our recent study (Cai et al. (2020), Mol Cell, doi: 10.1016/j.molcel.2020.09.018), we found that activity of AMPK was enriched in metastatic tumors compared to primary tumors. Depletion of AMPK rendered cancer cells more sensitive to metabolic and oxidative stress, leading to the impairment of breast cancer lung metastasis. Activation of AMPK rewired cancer metabolism towards TCA cycle, which protects disseminated cancer cells from both metabolic and oxidative stress-induced cell death, and facilitates cancer metastasis. Further, AMPK critically maintained the activity of pyruvate dehydrogenase complex (PDH), the rate limiting enzyme involved in TCA cycle, thus favoring the pyruvate metabolism towards TCA cycle rather than converting it to lactate. Mechanistically, AMPK was shown to co-localize with PDHA, the catalytic subunit of PDH, in the mitochondrial matrix and directly triggered the phosphorylation of PDHA on Ser295 and Ser314. Hyper-phosphorylation of Ser295 and Ser314 of PDHA promotes lung metastasis through elevating activity of PDH. Of note, PDHA Ser314 phosphorylation abrogated the interaction between PDHA and PDHKs leading to the dephosphorylation on previously reported S293 site, whose phosphorylation serves as a negative signal for PDH activation, while S295 phosphorylation serves as an intrinsic catalytic site required for pyruvate metabolism. Our study presented the first evidence for the pro-metastatic property of the AMPK-PDH axis and advance our current understanding of how PDH is activated under physiological and pathological conditions.
    Keywords:  AMPK; PDHA; TCA cycle; cancer metastasis; metabolic stress; oxidative stress
  41. Front Cell Dev Biol. 2020 ;8 569377
      Proteoglycans are a diverse group of molecules which are characterized by a central protein backbone that is decorated with a variety of linear sulfated glycosaminoglycan side chains. Proteoglycans contribute significantly to the biochemical and mechanical properties of the interstitial extracellular matrix where they modulate cellular behavior by engaging transmembrane receptors. Proteoglycans also comprise a major component of the cellular glycocalyx to influence transmembrane receptor structure/function and mechanosignaling. Through their ability to initiate biochemical and mechanosignaling in cells, proteoglycans elicit profound effects on proliferation, adhesion and migration. Pathologies including cancer and cardiovascular disease are characterized by perturbed expression of proteoglycans where they compromise cell and tissue behavior by stiffening the extracellular matrix and increasing the bulkiness of the glycocalyx. Increasing evidence indicates that a bulky glycocalyx and proteoglycan-enriched extracellular matrix promote malignant transformation, increase cancer aggression and alter anti-tumor therapy response. In this review, we focus on the contribution of proteoglycans to mechanobiology in the context of normal and transformed tissues. We discuss the significance of proteoglycans for therapy response, and the current experimental strategies that target proteoglycans to sensitize cancer cells to treatment.
    Keywords:  GAG; cancer; glycocalyx; mechanosignaling; proteoglycans
  42. Cancer Sci. 2020 Dec 19.
      Cancer metabolism is influenced by availability of nutrients in the microenvironment and can to some extent be manipulated by dietary modifications that target oncogenic metabolism. As yet, few dietary interventions have been scientifically proven to mitigate disease progression or enhance any other kind of therapy in human cancer. But recent advances in understanding of cancer metabolism enable us to predict or devise effective dietary interventions that might antagonize tumor growth. In fact, evidence emerging from preclinical models suggests that appropriate combinations of specific cancer therapies with dietary interventions could critically impact therapeutic efficacy. Here, we review the potential benefits of precision nutrition approaches in augmenting efficacy of cancer treatment.
  43. Nature. 2019 Dec 16.
    Keywords:  Arts; Culture
  44. Cell. 2020 Dec 09. pii: S0092-8674(20)31544-0. [Epub ahead of print]
      The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.
    Keywords:  TAD; chromatin; condensate; condensation; euchromatin; heterochromatin; histone acetylation; nucleosomal array; nucleus; phase separation
  45. Sci Rep. 2020 Dec 18. 10(1): 22334
      Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
  46. Sci Adv. 2020 Dec;pii: eabc4308. [Epub ahead of print]6(50):
      Early cancer detection aims to find tumors before they progress to an incurable stage. To determine the potential of circulating tumor DNA (ctDNA) for cancer detection, we developed a mathematical model of tumor evolution and ctDNA shedding to predict the size at which tumors become detectable. From 176 patients with stage I to III lung cancer, we inferred that, on average, 0.014% of a tumor cell's DNA is shed into the bloodstream per cell death. For annual screening, the model predicts median detection sizes of 2.0 to 2.3 cm representing a ~40% decrease from the current median detection size of 3.5 cm. For informed monthly cancer relapse testing, the model predicts a median detection size of 0.83 cm and suggests that treatment failure can be detected 140 days earlier than with imaging-based approaches. This mechanistic framework can help accelerate clinical trials by precomputing the most promising cancer early detection strategies.
  47. Nucleic Acids Res. 2020 Dec 16. pii: gkaa1202. [Epub ahead of print]
      DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.
  48. Proc Natl Acad Sci U S A. 2020 Dec 14. pii: 202013724. [Epub ahead of print]
      The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10-3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10-7 Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.
    Keywords:  DNA repair; error-corrected sequencing; genetic toxicology; genotoxicity; preclinical cancer risk assessment
  49. Br J Cancer. 2020 Dec 18.
      BACKGROUND: Schlafen 11 (SLFN11) has been linked with response to DNA-damaging agents (DDA) and PARP inhibitors. An in-depth understanding of several aspects of its role as a biomarker in cancer is missing, as is a comprehensive analysis of the clinical significance of SLFN11 as a predictive biomarker to DDA and/or DNA damage-response inhibitor (DDRi) therapies.METHODS: We used a multidisciplinary effort combining specific immunohistochemistry, pharmacology tests, anticancer combination therapies and mechanistic studies to assess SLFN11 as a potential biomarker for stratification of patients treated with several DDA and/or DDRi in the preclinical and clinical setting.
    RESULTS: SLFN11 protein associated with both preclinical and patient treatment response to DDA, but not to non-DDA or DDRi therapies, such as WEE1 inhibitor or olaparib in breast cancer. SLFN11-low/absent cancers were identified across different tumour types tested. Combinations of DDA with DDRi targeting the replication-stress response (ATR, CHK1 and WEE1) could re-sensitise SLFN11-absent/low cancer models to the DDA treatment and were effective in upper gastrointestinal and genitourinary malignancies.
    CONCLUSION: SLFN11 informs on the standard of care chemotherapy based on DDA and the effect of selected combinations with ATR, WEE1 or CHK1 inhibitor in a wide range of cancer types and models.
  50. Elife. 2020 Dec 15. pii: e55793. [Epub ahead of print]9
      Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.
    Keywords:  D. melanogaster; developmental biology; genetics; genomics
  51. Genomics Proteomics Bioinformatics. 2020 Dec 10. pii: S1672-0229(20)30133-9. [Epub ahead of print]
      Aging is a complex process that can be characterized by functional and cognitive decline in an individual. Aging can be assessed based on the functional capacity of vital organs and their intricate interactions with one another. Thus, the nature of aging can be described by focusing on a specific organ and an individual itself. However, to fully understand the complexity of aging, one must investigate not only a single tissue or biological process but also its complex interplay and interdependencies with other biological processes. Here, using RNA-seq, we monitored changes in the transcriptome during aging in four tissues (including brain, blood, skin and liver) in mice at 9 months, 15 months, and 24 months, with a final evaluation at the very old age of 30 months. We identified several genes and processes that were differentially regulated during aging in both tissue-dependent and tissue-independent manners. Most importantly, we found that the electron transport chain (ETC) of mitochondria was similarly affected at the transcriptome level in the four tissues during the aging process. We also identified the liver as the tissue showing the largest variety of differentially expressed genes (DEGs) over time. Lcn2 (Lipocalin-2) was found to be similarly regulated among all tissues, and its effect on longevity and survival was validated using its orthologue in Caenorhabditis elegans. Our study demonstrated that the molecular processes of aging are relatively subtle in their progress, and the aging process of every tissue depends on the tissue's specialized function and environment. Hence, individual gene or process alone cannot be described as the key of aging in the whole organism.
    Keywords:  Aging; Electron transport chain; Inflammaging; RNA-seq analysis; Tissue aging
  52. Cell. 2020 Dec 10. pii: S0092-8674(20)31564-6. [Epub ahead of print]
      Barrier tissue immune responses are regulated in part by nociceptors. Nociceptor ablation alters local immune responses at peripheral sites and within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Using high-resolution imaging, viral tracing, single-cell transcriptomics, and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is responsive to lymph-borne inflammatory signals. Transcriptomics profiling revealed that multiple sensory neuron subsets, predominantly peptidergic nociceptors, innervate LNs, distinct from those innervating surrounding skin. To uncover LN-resident cells that may interact with LN-innervating sensory neurons, we generated a LN single-cell transcriptomics atlas and nominated nociceptor target populations and interaction modalities. Optogenetic stimulation of LN-innervating sensory fibers triggered rapid transcriptional changes in the predicted interacting cell types, particularly endothelium, stromal cells, and innate leukocytes. Thus, a unique population of sensory neurons monitors peripheral LNs and may locally regulate gene expression.
    Keywords:  lymph node; neuro-immunology; nociceptor; optogenetics; sensory neuron; single-cell RNA-seq
  53. Sci Adv. 2020 Dec;pii: eabd6798. [Epub ahead of print]6(50):
      Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac I Ks current. Although cryo-electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP2 to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.
  54. Sci Adv. 2020 Dec;pii: eabc2697. [Epub ahead of print]6(50):
      A functional lymphatic vasculature is essential for tissue fluid homeostasis, immunity, and lipid clearance. Although atherosclerosis has been linked to adventitial lymphangiogenesis, the functionality of aortic lymphatic vessels draining the diseased aorta has never been assessed and the role of lymphatic drainage in atherogenesis is not well understood. We develop a method to measure aortic lymphatic transport of macromolecules and show that it is impaired during atherosclerosis progression, whereas it is ameliorated during lesion regression induced by ezetimibe. Disruption of aortic lymph flow by lymphatic ligation promotes adventitial inflammation and development of atherosclerotic plaque in hypercholesterolemic mice and inhibits ezetimibe-induced atherosclerosis regression. Thus, progression of atherosclerotic plaques may result not only from increased entry of atherogenic factors into the arterial wall but also from reduced lymphatic clearance of these factors as a result of aortic lymph stasis. Our findings suggest that promoting lymphatic drainage might be effective for treating atherosclerosis.