bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2020‒11‒01
sixty papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. J Clin Invest. 2020 Oct 29. pii: 135124. [Epub ahead of print]
      The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We show the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyses phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol 4-phosphate (PI(4)P) and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report loss of INPP5K in muscle causes severe disease, autophagy inhibition and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppresses autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterised by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane-recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules which we propose interferes with ALR completion. Inhibition of PI(4,5)P2 synthesis, or expression of wild-type, but not INPP5K-disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes is integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.
    Keywords:  Autophagy; Cell Biology; Lysosomes; Muscle Biology; Skeletal muscle
  2. Cell Rep. 2020 Oct 27. pii: S2211-1247(20)31310-3. [Epub ahead of print]33(4): 108321
      The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, we show that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status.
    Keywords:  AMPK; Drosophila; Leptin; Upd2; fat body; insulin-producing cells; inter-organ communication; metabolism; p53; starvation
  3. Proc Natl Acad Sci U S A. 2020 Oct 30. pii: 202013644. [Epub ahead of print]
      Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.
    Keywords:  AMD3100; CXCR4; colorectal cancer; immunotherapy; pancreatic cancer
  4. JCSM Rapid Commun. 2020 Jul-Dec;3(2):3(2): 115-128
      Background: Cachexia, a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitory factor (LIF) which induces cachexia. We characterized how LIF promotes cachexia-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues.Methods: Cachexia was induced in vivo with the heterotopic allotransplanted administration of C26c20 colon adenocarcinoma cells or the intraperitoneal administration of recombinant LIF in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. Cachexia-associated lipolysis was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These adipocytes were processed for glycerol release into the media, immunoblot analysis, and RT-PCR.
    Results: Tumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing cachexia-associated adipose wasting and anorexia. We identified two JAK inhibitors that block IL-6 family-mediated adipocyte lipolysis and IL-6 induction using an in vitro cachexia lipolysis assay. JAK inhibitors administered to the in vivo C26c20 cancer cachexia mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the cachexia serum cyto/adipokine signature, 3) a delay in cancer cachexia-associated anorexia and adipose loss, and 4) an improvement in overall survival.
    Conclusions: JAK inhibitors suppress LIF-associated adipose loss and anorexia in both in vitro and in vivo models of cancer cachexia.
    Keywords:  IL-6; cachexia; cancer; janus kinase; leptin; leukemia inhibitory factor
  5. Cancer Discov. 2020 Oct 30. pii: CD-20-0775. [Epub ahead of print]
      Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAFs) support PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit NK cell mediated killing of tumor cells. These pro-tumor functions are controlled by a signaling circuit downstream to NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.
  6. J Biol Chem. 2020 Oct 29. pii: jbc.RA119.011060. [Epub ahead of print]
      RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.
    Keywords:  GTPase Kras (KRAS); PTPN2; cell proliferation; cell signaling; extracellular-signal-regulated kinase (ERK); plasma membrane; tyrosine-protein phosphatase (tyrosine phosphatase)
  7. Nat Metab. 2020 Oct 26.
      In addition to fatty acids, glucose and lactate are important myocardial substrates under physiologic and stress conditions. They are metabolized to pyruvate, which enters mitochondria via the mitochondrial pyruvate carrier (MPC) for citric acid cycle metabolism. In the present study, we show that MPC-mediated mitochondrial pyruvate utilization is essential for the partitioning of glucose-derived cytosolic metabolic intermediates, which modulate myocardial stress adaptation. Mice with cardiomyocyte-restricted deletion of subunit 1 of MPC (cMPC1-/-) developed age-dependent pathologic cardiac hypertrophy, transitioning to a dilated cardiomyopathy and premature death. Hypertrophied hearts accumulated lactate, pyruvate and glycogen, and displayed increased protein O-linked N-acetylglucosamine, which was prevented by increasing availability of non-glucose substrates in vivo by a ketogenic diet (KD) or a high-fat diet, which reversed the structural, metabolic and functional remodelling of non-stressed cMPC1-/- hearts. Although concurrent short-term KDs did not rescue cMPC1-/- hearts from rapid decompensation and early mortality after pressure overload, 3 weeks of a KD before transverse aortic constriction was sufficient to rescue this phenotype. Together, our results highlight the centrality of pyruvate metabolism to myocardial metabolism and function.
  8. Cell Death Discov. 2020 ;6 107
      Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.
    Keywords:  Mitophagy; Stress signalling
  9. Oncogene. 2020 Oct 27.
      Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from KrasG12D; Pdx1-Cre (KC) and KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.
  10. Cancer Genomics Proteomics. 2020 Nov-Dec;17(6):17(6): 695-705
      BACKGROUND/AIM: The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model.MATERIALS AND METHODS: We established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice.
    RESULTS: G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes.
    CONCLUSION: Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.
    Keywords:  Carcinogenesis; PDAC; PanIN; chromatin accessibility; epigenetics
  11. J Exp Clin Cancer Res. 2020 Oct 28. 39(1): 227
      Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, whose main molecular trait is the MAPK pathway activation due to KRAS mutation, which is present in 90% of cases.The genetic landscape of KRAS wild type PDAC can be divided into three categories. The first is represented by tumors with an activated MAPK pathway due to BRAF mutation that occur in up to 4% of cases. The second includes tumors with microsatellite instability (MSI) due to defective DNA mismatch repair (dMMR), which occurs in about 2% of cases, also featuring a high tumor mutational burden. The third category is represented by tumors with kinase fusion genes, which marks about 4% of cases. While therapeutic molecular targeting of KRAS is an unresolved challenge, KRAS-wild type PDACs have potential options for tailored treatments, including BRAF antagonists and MAPK inhibitors for the first group, immunotherapy with anti-PD-1/PD-L1 agents for the MSI/dMMR group, and kinase inhibitors for the third group.This calls for a complementation of the histological diagnosis of PDAC with a routine determination of KRAS followed by a comprehensive molecular profiling of KRAS-negative cases.
    Keywords:  BRAF; KRAS; MSI; dMMR; fusion genes; pancreatic cancer
  12. Autophagy. 2020 Oct 28.
      Excessive inflammation may lead to irreparable injury and even death, but the key mediators and underlying mechanisms remain unclear. Our recent findings indicate that SQSTM1/p62 (sequestosome 1), a well-known macroautophagy/autophagy receptor, is a lethal inflammatory mediator of sepsis and septic shock. The release of SQSTM1 occurs during tissue damage or microbial invasion through two main ways: one is passive and the other is active. Passive release occurs in the context of GSDMD-mediated pyroptosis. Active SQSTM1 secretion requires two basic steps: the first step is the expression and phosphorylation of SQSTM1 mediated by STING1/STING/TMEM173, and then the unconventional secretion of SQSTM1 by secretory lysosomes. After release, the extracellular SQSTM1 binds to membrane receptor INSR to activate glycolysis, leading to subsequent production of pro-inflammatory cytokines in a transcription factor NFKB-dependent manner. Functionally, genetic deletion or pharmacological inhibition of the SQSTM1-INSR pathway limits tissue damage, systemic inflammation, organ failure, and death in experimental sepsis models in mice. Moreover, the activation of the SQSTM1-INSR pathway is related to the severity of sepsis in patients. These findings highlight a pathological role of extracellular SQSTM1 in infection, inflammation, and immunity.
    Keywords:  DAMP; INSR; SQSTM1; STING1; TLR4; autophagy; immunometabolism; inflammasome; sepsis
  13. J Biol Chem. 2020 Oct 27. pii: jbc.RA120.015238. [Epub ahead of print]
      Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. While much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is unknown. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable to hypoxia with activation of downstream target genes. Addition of fatty acids to culture media suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult fluorescent reporter mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.
    Keywords:  fatty acid; hypoxia-inducible factor (HIF); lipoprotein; low-density lipoprotein (LDL); lysosomal acid lipase; mitochondria
  14. Cancer Med. 2020 Oct 27.
      It is unclear to what extent patients with pancreatic cancer have cachexia and had a dietetic consult for nutritional support. The aim was to assess the prevalence of cachexia, dietitian consultation, and overall survival in these patients. This prospective multicenter cohort study included patients with pancreatic cancer, who participated in the Dutch Pancreatic Cancer Project and completed patient reported outcome measures (2015-2018). Additional data were obtained from the Netherlands Cancer Registry. Cachexia was defined as self-reported >5% body weight loss, or >2% in patients with a BMI <20 kg/m2 over the past half year. The Kaplan-Meier method was used to analyze overall survival. In total, 202 patients were included from 18 centers. Cachexia was present in 144 patients (71%) and 81 of those patients (56%) had dietetic consultation. Cachexia was present in 63% of 94 patients who underwent surgery, 77% of 70 patients who received palliative chemotherapy and 82% of 38 patients who had best supportive care. Dietitian consultation was reported in 53%, 52%, and 71%, respectively. Median overall survival did not differ between patients with and without cachexia, but decreased in those with severe weight loss (12 months (IQR 7-20) vs. 16 months (IQR 8-31), p = 0.02), as compared to those with <10% weight loss during the past half year. Two-thirds of patients with pancreatic cancer present with cachexia of which nearly half had no dietetic consultation. Survival was comparable in patients with and without cachexia, but decreased in patients with more severe weight loss.
  15. Cell. 2020 Oct 20. pii: S0092-8674(20)31309-X. [Epub ahead of print]
      Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.
    Keywords:  angiogenesis; immune heterogeneity; immune niche; innate immunity; neutrophil lifespan; neutrophils; single-cell analysis; tissue-resident cells
  16. Nat Genet. 2020 Nov;52(11): 1208-1218
      Cultured cell lines are the workhorse of cancer research, but the extent to which they recapitulate the heterogeneity observed among malignant cells in tumors is unclear. Here we used multiplexed single-cell RNA-seq to profile 198 cancer cell lines from 22 cancer types. We identified 12 expression programs that are recurrently heterogeneous within multiple cancer cell lines. These programs are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-mesenchymal transition and protein metabolism. Most of these programs recapitulate those recently identified as heterogeneous within human tumors. We prioritized specific cell lines as models of cellular heterogeneity and used them to study subpopulations of senescence-related cells, demonstrating their dynamics, regulation and unique drug sensitivities, which were predictive of clinical response. Our work describes the landscape of heterogeneity within diverse cancer cell lines and identifies recurrent patterns of heterogeneity that are shared between tumors and specific cell lines.
  17. Nat Struct Mol Biol. 2020 Oct 26.
      De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
  18. STAR Protoc. 2020 Sep 18. 1(2): 100048
      The metabolic activity of cells is interrelated with cell signaling, functions, and fate. Uncontrolled cancer cell proliferation requires metabolic adaptations. Research focusing on understanding the characteristics of cell metabolism is crucial for the development of novel diagnostic and therapeutic strategies. Here, we describe protocols for the ATP profiling of single cancer cells by fluorescence live-cell imaging. In response to distinct metabolic inhibitions, we record individual mitochondrial ATP dynamics using established Förster resonance energy transfer-based genetically encoded fluorescent ATP probes. For complete details on the use and execution of this protocol, please refer to Depaoli et al. (2018).
  19. iScience. 2020 Oct 23. 23(10): 101630
      Understanding the mechanisms for cellular aging is a fundamental question in biology. Normal red blood cells (RBCs) survive for approximately 100 days, and their survival is likely limited by functional decline secondary to cumulative damage to cell constituents, which may be reflected in altered metabolic capabilities. To investigate metabolic changes during in vivo RBC aging, labeled cell populations were purified at intervals and assessed for abundance of metabolic intermediates using mass spectrometry. A total of 167 metabolites were profiled and quantified from cell populations of defined ages. Older RBCs maintained ATP and redox charge states at the cost of altered activity of enzymatic pathways. Time-dependent changes were identified in metabolites related to maintenance of the redox state and membrane structure. These findings illuminate the differential metabolic pathway usage associated with normal cellular aging and identify potential biomarkers to determine average RBC age and rates of RBC turnover from a single blood sample.
    Keywords:  Medical Biochemistry; Metabolomics
  20. Science. 2020 Oct 29. pii: eabb5390. [Epub ahead of print]
      Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional (2D) cell culture hindering testing of gene functions requiring tissue context. Here we present CRISPR-LIneage tracing at Cellular resolution in Heterogenous Tissue (CRISPR-LICHT), enabling parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized Immediate Early Response 3 Interacting Protein 1 (IER3IP1) regulating the unfolded protein response (UPR) and extracellular matrix (ECM) protein secretion crucial for tissue integrity, with dysregulation resulting in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain size control.
  21. Elife. 2020 Oct 27. pii: e56969. [Epub ahead of print]9
      PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine and endocrine mechanisms. We investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells.
    Keywords:  D. melanogaster; genetics; genomics
  22. Autophagy. 2020 Oct 30.
      Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1.
    Keywords:  AMPK; DARTS; MAPT; MTOR; Sert; VDAC1; antidepressant; tauopathy
  23. Pancreas. 2020 Nov/Dec;49(10):49(10): 1348-1354
      OBJECTIVES: The association of Lewis antigen phenotype with survival of patients with pancreatic ductal adenocarcinoma was investigated.METHODS: A total of 1187 patients diagnosed with pancreatic ductal adenocarcinoma were evaluated in a prospective cohort. Patients were classified into 3 different groups according to Lewis antigen phenotype: Lewis antigen (1) A positive [Le(a+b-)], (2) B positive [Le(a-b+)], and (3) negative [Le(a-b-)]. Risk of mortality was analyzed with Cox regression after adjusting for other predictors.
    RESULTS: The risk of mortality increased in the order of Le(a+b-), Le(a-b+), and Le(a-b-) [reference; hazard ratio (HR), 1.27; 95% confidence interval (CI)], 1.03-1.57; P = 0.02; and HR, 1.65; 95% CI, 1.31-2.09; P < 0.001] after adjusting for other predictors. Among patients with serum carbohydrate antigen (CA) 19-9 lower than 37 U/mL, the association seemed more apparent (reference; HR, 1.50; 95% CI, 0.77-2.29; P = 0.22; and HR, 2.10; 95% CI, 1.10-4.02; P < 0.02).
    CONCLUSIONS: The risk of mortality increased in the order of Le(a+b-), Le(a-b+), and Le(a-b-). The difference in prognosis according to the Lewis antigen phenotype was more pronounced in the low CA 19-9 group, which suggests that the Lewis antigen phenotype works as a biomarker predicting the prognosis of patients with pancreatic cancer with undetectable CA 19-9 level.
  24. Nature. 2020 Oct 28.
      Adipose tissue is usually classified on the basis of its function as white, brown or beige (brite)1. It is an important regulator of systemic metabolism, as shown by the fact that dysfunctional adipose tissue in obesity leads to a variety of secondary metabolic complications2,3. In addition, adipose tissue functions as a signalling hub that regulates systemic metabolism through paracrine and endocrine signals4. Here we use single-nucleus RNA-sequencing (snRNA-seq) analysis in mice and humans to characterize adipocyte heterogeneity. We identify a rare subpopulation of adipocytes in mice that increases in abundance at higher temperatures, and we show that this subpopulation regulates the activity of neighbouring adipocytes through acetate-mediated modulation of their thermogenic capacity. Human adipose tissue contains higher numbers of cells of this subpopulation, which could explain the lower thermogenic activity of human compared to mouse adipose tissue and suggests that targeting this pathway could be used to restore thermogenic activity.
  25. Cell. 2020 Oct 29. pii: S0092-8674(20)31300-3. [Epub ahead of print]183(3): 786-801.e19
      Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.
    Keywords:  cancer; checkpoint inhibitors; immunotherapy; innate immunity; melanoma; myeloid cells; nanobiologics; nanomedicine; nanotechnology; trained immunity
  26. Cells. 2020 Oct 25. pii: E2353. [Epub ahead of print]9(11):
      With a five-year survival rate under 9%, pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest tumors. Although the treatment options are slightly improving, PDAC is the second leading cause of cancer related death in 2020 in the US. In addition to a pronounced desmoplastic stroma reaction, pancreatic cancer is characterized by one of the lowest levels of oxygen availability within the tumor mass and these hypoxic conditions are known to contribute to tumor development and progression. In this context, the major hypoxia associated transcription factor family, HIF, regulates hundreds of genes involved in angiogenesis, metabolism, migration, invasion, immune escape and therapy resistance. Current research implications show, that hypoxia also modulates diverse areas of epigenetic mechanisms like non-coding RNAs, histone modifications or DNA methylation, which cooperate with the hypoxia-induced transcription factors as well as directly regulate the hypoxic response pathways. In this review, we will focus on hypoxia-mediated epigenetic alterations and their impact on pancreatic cancer.
    Keywords:  DNA methylation; HIF; epigenetics; histone modifications; hypoxia; non-coding RNA; pancreatic cancer
  27. Science. 2020 Oct 30. pii: eaaz0868. [Epub ahead of print]370(6516):
      The role of the physical microenvironment in tumor development, progression, metastasis, and treatment is gaining appreciation. The emerging multidisciplinary field of the physical sciences of cancer is now embraced by engineers, physicists, cell biologists, developmental biologists, tumor biologists, and oncologists attempting to understand how physical parameters and processes affect cancer progression and treatment. Discoveries in this field are starting to be translated into new therapeutic strategies for cancer. In this Review, we propose four physical traits of tumors that contribute to tumor progression and treatment resistance: (i) elevated solid stresses (compression and tension), (ii) elevated interstitial fluid pressure, (iii) altered material properties (for example, increased tissue stiffness, which historically has been used to detect cancer by palpation), and (iv) altered physical microarchitecture. After defining these physical traits, we discuss their causes, consequences, and how they complement the biological hallmarks of cancer.
  28. Proc Natl Acad Sci U S A. 2020 Oct 29. pii: 202020162. [Epub ahead of print]
  29. Nat Metab. 2020 Oct 26.
      Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.
  30. Adv Exp Med Biol. 2021 ;1270 57-71
      Pioneering experiments performed by Harold Varmus and Mike Bishop in 1976 led to one of the most influential discoveries in cancer research and identified the first cancer-causing oncogene called Src. Later experimental and clinical evidence suggested that Src kinase plays a significant role in promoting tumor growth and progression and its activity is associated with poor patient survival. Thus, several Src inhibitors were developed and approved by FDA for treatment of cancer patients. Tumor microenvironment (TME) is a highly complex and dynamic milieu where significant cross-talk occurs between cancer cells and TME components, which consist of tumor-associated macrophages, fibroblasts, and other immune and vascular cells. Growth factors and chemokines activate multiple signaling cascades in TME and induce multiple kinases and pathways, including Src, leading to tumor growth, invasion/metastasis, angiogenesis, drug resistance, and progression. Here, we will systemically evaluate recent findings regarding regulation of Src and significance of targeting Src in cancer therapy.
    Keywords:  Apoptosis; Cancer; Fibroblasts; Invasion; Kinase; Macrophages; Metastasis; Microenvironment; Migration; Motility; Oncogene; Pericytes; Proliferation; Src; Targeted therapy
  31. Nat Commun. 2020 10 28. 11(1): 5439
      Oncogenic KRAS mutations are the most frequent mutations in human cancer, but most difficult to target. While sustained proliferation caused by oncogenic KRAS-downstream signalling is a main driver of carcinogenesis, there is increasing evidence that it also mediates autocrine effects and crosstalk with the tumour microenvironment (TME). Here, we discuss recent reports connecting KRAS mutations with tumour-promoting inflammation and immune modulation caused by KRAS that leads to immune escape in the TME. We discuss the preclinical work on KRAS-induced inflammation and immune modulation in the context of currently ongoing clinical trials targeting cancer entities that carry KRAS mutations and strategies to overcome the oncogene-induced effects on the immune system.
  32. Turk J Biol. 2020 ;44(5): 230-237
      Pancreatic ductal adenocarcinoma (PDAC) pathology is known for its uncontrollable progress due to highly invasive characteristics and refractory behavior against existing chemotherapies. The aberrant expression of CDH1 (expresses the protein E-cadherin) is associated with increased overall survival in various cancers, however, E-cadherin expression in PDAC progression has remained elusive. We investigated the impact of exogenously elevated E-cadherin levels on the tumorigenicity of transduced low grade and metastatic PDAC cell lines, Panc-1 and AsPC-1, respectively. Constitutive expression of E-cadherin promoted a more hybrid E/M state in AsPC-1 cells, while it was associated with the acquisition of a more epithelial-like state in Panc1 cells. Our study suggests that E-cadherin may play differential roles in determining the metastatic characteristics of primary and metastatic pancreatic cancer cells.
    Keywords:  AsPC1; CRISPR/dCas9 activation; E-cadherin; Panc-1; pancreas cancer
  33. Nat Metab. 2020 Oct 26.
      The myocardium is metabolically flexible; however, impaired flexibility is associated with cardiac dysfunction in conditions including diabetes and heart failure. The mitochondrial pyruvate carrier (MPC) complex, composed of MPC1 and MPC2, is required for pyruvate import into the mitochondria. Here we show that MPC1 and MPC2 expression is downregulated in failing human and mouse hearts. Mice with cardiac-specific deletion of Mpc2 (CS-MPC2-/-) exhibited normal cardiac size and function at 6 weeks old, but progressively developed cardiac dilation and contractile dysfunction, which was completely reversed by a high-fat, low-carbohydrate ketogenic diet. Diets with higher fat content, but enough carbohydrate to limit ketosis, also improved heart failure, while direct ketone body provisioning provided only minor improvements in cardiac remodelling in CS-MPC2-/- mice. An acute fast also improved cardiac remodelling. Together, our results reveal a critical role for mitochondrial pyruvate use in cardiac function, and highlight the potential of dietary interventions to enhance cardiac fat metabolism to prevent or reverse cardiac dysfunction and remodelling in the setting of MPC deficiency.
  34. STAR Protoc. 2020 Sep 18. 1(2): 100079
      Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent's Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping. For complete details on the use and execution of this protocol, please refer to Brunton et al. (2020).
  35. Nat Rev Cancer. 2020 Oct 30.
      Platinum (Pt) compounds entered the clinic as anticancer agents when cisplatin was approved in 1978. More than 40 years later, even in the era of precision medicine and immunotherapy, Pt drugs remain among the most widely used anticancer drugs. As Pt drugs mainly target DNA, it is not surprising that recent insights into alterations of DNA repair mechanisms provide a useful explanation for their success. Many cancers have defective DNA repair, a feature that also sheds new light on the mechanisms of secondary drug resistance, such as the restoration of DNA repair pathways. In addition, genome-wide functional screening approaches have revealed interesting insights into Pt drug uptake. About half of cisplatin and carboplatin but not oxaliplatin may enter cells through the widely expressed volume-regulated anion channel (VRAC). The analysis of this heteromeric channel in tumour biopsies may therefore be a useful biomarker to stratify patients for initial Pt treatments. Moreover, Pt-based approaches may be improved in the future by the optimization of combinations with immunotherapy, management of side effects and use of nanodelivery devices. Hence, Pt drugs may still be part of the standard of care for several cancers in the coming years.
  36. Br J Cancer. 2020 Oct 26.
      BACKGROUND: The Phase 2 SCALOP trial compared gemcitabine with capecitabine-based consolidation chemoradiotherapy (CRT) in locally advanced pancreatic cancer (LAPC).METHODS: Thirty-five systematically identified circulating biomarkers were analysed in plasma samples from 60 patients enroled in SCALOP. Each was measured in triplicate at baseline (prior to three cycles of gemcitabine-capecitabine induction chemotherapy) and, for a subset, prior to CRT. Association with overall survival (OS) was determined using univariable Cox regression and optimal thresholds delineating low to high values identified using time-dependent ROC curves. Independence from known prognostic factors was assessed using Spearman correlation and the Wilcoxon rank sum test prior to multivariable Cox regression modelling including independent biomarkers and known prognostic factors.
    RESULTS: Baseline circulating levels of C-C motif chemokine ligand 5 (CCL5) were significantly associated with OS, independent of other clinicopathological characteristics. Patients with low circulating CCL5 (CCL5low) had a median OS of 18.5 (95% CI 11.76-21.32) months compared to 11.3 (95% CI 9.86-15.51) months in CCL5high; hazard ratio 1.95 (95% CI 1.04-8.65; p = 0.037).
    CONCLUSIONS: CCL5 is an independent prognostic biomarker in LAPC. Given the known role of CCL5 in tumour invasion, metastasis and the induction of an immunosuppressive micro-environment, targeting of CCL5-mediated pathways may offer therapeutic potential in pancreatic cancer.
    CLINICAL TRIAL REGISTRATION: The SCALOP trial was registered with ISRCTN, number 96169987 (registered 29 May 2008).
  37. Nat Struct Mol Biol. 2020 Oct 26.
      The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
  38. Nat Metab. 2020 Oct 26.
      Hypothalamic agouti-related peptide (AgRP) and neuropeptide Y-expressing neurons have a critical role in driving food intake, but also in modulating complex, non-feeding behaviours1. We interrogated whether AgRP neurons are relevant to the emergence of anorexia nervosa symptomatology in a mouse model. Here we show, using in vivo fibre photometry, a rapid inhibition of AgRP neuronal activity following voluntary cessation of running. All AgRP neuron-ablated, food-restricted mice die within 72 h of compulsive running, while daily activation of AgRP neurons using a chemogenetic tool increases voluntary running with no lethality of food-restricted animals. Animals with impaired AgRP neuronal circuits are unable to properly mobilize fuels during food-restriction-associated exercise; however, when provided with elevated fat content through diet, their death is completely prevented. Elevated fat content in the diet also prevents the long-term behavioural impact of food-restricted fit mice with elevated exercise volume. These observations elucidate a previously unsuspected organizational role of AgRP neurons, via the mediation of the periphery, in the regulation of compulsive exercise and its related lethality with possible implications for psychiatric conditions, such as anorexia nervosa.
  39. Sci Immunol. 2020 Oct 30. pii: eaba5962. [Epub ahead of print]5(52):
      The presence of polyfunctional CD4+ T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4+ T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4+ T cells to undergo robust expansion, infiltrate tumors vigorously, and elicit antitumor CD8+ T cell responses in a CD4+ T cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4+ T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4+ T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to coexpress CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells in a mouse B cell lymphoma model. The optimal therapeutic outcome was obtained when both CD4+ and CD8+ CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8+ T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4+ T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4+ T cells to potentiate adoptive T cell therapy for cancer.
  40. Front Oncol. 2020 ;10 520330
      Glucose metabolism and systemic inflammation have been associated with cancer aggressiveness and patient prognosis in various malignancies. This study aimed to evaluate the prognostic significance of pretreatment GLR(glucose to lymphocyte ratio) and systemic immune inflammation in patients with pancreatic cancer. We studied 360 patients with pathologically diagnosed pancreatic adenocarcinoma that was clinically unresectable. Baseline clinicopathological characteristics and laboratory investigations including fasting blood glucose, platelet count, lymphocyte count, neutrophil count, carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA199), and follow-up data were collected for further analysis. The patients were randomly divided into a training cohort (n = 238) and a validation cohort (n = 122). Univariate and multivariate Cox proportional hazard regression analyses were performed to identify the prognostic value of GLR, systemic immune-inflammation markers, and tumor biomarkers. A nomogram model was developed based on the identified prognostic factors, and we used the C-index to evaluate the accuracy of the Cox regression model prediction. Multivariate analysis revealed that GLR [hazard ratio (HR): 2.597; 95% confidence interval (CI): 1.728-3.904)] and CA199 (HR: 2.484; 95% CI: 1.295-4.765) are independent predictors of poor overall survival in the training cohort and were incorporated into the nomogram for OS as independent factors. Moreover, the C-index analyses demonstrated that the C-indexes in the training cohort and the validation cohort were 0.674 and 0.671, respectively. The nomogram model predicts overall survival relatively accurately. We found that the baseline GLR is an independent prognostic factor for patients with pancreatic cancer, and the proposed nomogram can be used as an effective tool for predicting the outcomes of prognosis of patients with pancreatic cancer.
    Keywords:  glucose to lymphocyte ratio; nomogram; pancreatic cancer; prognosis; tumor biomarker
  41. Sci Transl Med. 2020 Oct 28. pii: eabb8969. [Epub ahead of print]12(567):
      Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post-allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.
  42. Sci Signal. 2020 Oct 27. pii: eaaz3975. [Epub ahead of print]13(655):
      In its natural habitat in the forest soil, the cellular slime mold Dictyostelium discoideum is exposed to obstacles. Starving Dictyostelium cells secrete cAMP, which is the key extracellular signaling molecule that promotes the aggregation process required for their long-term survival. Here, we investigated the influence of environmental inhomogeneities on the signaling and pattern formation of Dictyostelium cells. We present experimental data and numerical simulations on the pattern formation of signaling Dictyostelium cells in the presence of periodic arrays of millimeter-sized pillars. We observed concentric cAMP waves that initiated almost synchronously at the pillars and propagated outward. In response to these circular waves, the Dictyostelium cells streamed toward the pillars, forming aggregates arranged in patterns that reflected the periodicity of the lattice of pillars. Our results suggest that, in nature, the excitability threshold and synchronization level of the cells are two key parameters that control the nature of the interaction between cells and spatial heterogeneities in their environment.
  43. Sci Adv. 2020 Oct;pii: eabb9200. [Epub ahead of print]6(44):
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked fibrosis and low immunogenicity, features that are linked to treatment resistance and poor clinical outcomes. Therefore, understanding how PDAC regulates the desmoplastic and immune stromal components is of great clinical importance. We found that acyl-CoA synthetase long-chain 3 (ACSL3) is up-regulated in PDAC and correlates with increased fibrosis. Our in vivo results show that Acsl3 knockout hinders PDAC progression, markedly reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells, and increases cytotoxic T cell infiltration. This effect is, at least in part, due to decreased plasminogen activator inhibitor-1 (PAI-1) secretion from tumor cells. Accordingly, PAI-1 expression in PDAC positively correlates with markers of fibrosis and immunosuppression and predicts poor patient survival. We found that PAI-1 pharmacological inhibition strongly enhances chemo- and immunotherapeutic response against PDAC, increasing survival of mice. Thus, our results unveil ACSL3-PAI-1 signaling as a requirement for PDAC progression with druggable attributes.
  44. Mol Metab. 2020 Oct 23. pii: S2212-8778(20)30180-0. [Epub ahead of print] 101106
      OBJECTIVE: Skeletal muscle regeneration relies on muscle-specific, adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic disease and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally, non-essential amino acids, is altered in humans with these pathologies and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine on MuSC/MPC function.METHODS: For in vitro experiments, primary, human MPCs (hMPCs) were used and for in vivo experiments young (4-6 mo) and old (>20 mo) mice were used. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses.
    RESULTS: We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine, in a mouse model of skeletal muscle regeneration, lowered the abundance of MuSCs 3 days post injury. Stable-isotope tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine were necessary to support glutathione and global protein synthesis in hMPCs. Finally, using an aged mouse model we find that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post injury.
    CONCLUSIONS: These studies demonstrate that despite an absolute requirement of serine/glycine for MuSC/MPC proliferation, de novo synthesis is inadequate to support these demands making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration.
    Keywords:  Muscle; glycine metabolism; muscle metabolism; muscle progenitor cell; muscle regeneration; muscle stem cell; proliferation; protein synthesis; serine metabolism
  45. Adv Exp Med Biol. 2020 ;1277 63-74
      Caveolin-1 (Cav-1), a major structural component of cell membrane caveolae, is involved in a variety of intracellular signaling pathways as well as transmembrane transport. Cav-1, as a scaffolding protein, modulates signal transduction associated with cell cycle progression, cellular senescence, cell proliferation and death, lipid homeostasis, etc. Cav-1 is also thought to regulate the expression or activity of oncoproteins, such as Src family kinases, H-Ras, protein kinase C, epidermal growth factor, extracellular signal-regulated kinase, and endothelial nitric oxide synthase. Because of its frequent overexpression or mutation in various tumor tissues and cancer cell lines, Cav-1 has been speculated to play a role as an oncoprotein in cancer development and progression. In contrast, Cav-1 may also function as a tumor suppressor, depending on the type of cancer cells and/or surrounding stromal cells in the tumor microenvironment as well as the stage of tumors.
    Keywords:  Cancer progression; Cancer stem-like cells; Cancer-associated fibroblasts; Caveolae; Caveolin-1; Epithelial-mesenchymal transition; Metastasis; Stem cells; Stromal cells; Tumor microenvironment
  46. Cancer Lett. 2020 Oct 27. pii: S0304-3835(20)30573-5. [Epub ahead of print]
      Recent research on genomic profiling of pancreatic ductal adenocarcinoma (PDAC) has identified many potentially actionable alterations. However, the feasibility of using genomic profiling to guide routine clinical decision making for PDAC patients remains unclear. We retrospectively reviewed PDAC patients between October 2013 and December 2017, who underwent treatment at the Johns Hopkins Hospital and had clinical tumor next-generation sequencing (NGS) through commercial resources. Ninety-two patients with 93 tumors tested were included. Forty-eight (52%) patients had potentially curative surgeries. The median time from the tissue available to the NGS testing ordered was 229 days (interquartile range 62-415). A total of three (3%) patients had matched targeted therapies based on genomic profiling results. Genomic profiling guided personalized treatment for PDAC patients is feasible, but the percentage of patients who receive targeted therapy is low. The main challenges are ordering NGS testing early in the clinical course of the disease and the limited evidence of using a targeted approach in these patients. A real-time department level genomic testing ordering system in combination with an evidence-based flagging system for potentially actionable alterations could help address these shortcomings.
    Keywords:  Actionable alteration; Clinical genomic testing; Matched therapy
  47. STAR Protoc. 2020 Sep 18. 1(2): 100104
      Fluorescent in situ hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).
  48. Cell. 2020 Oct 29. pii: S0092-8674(20)31389-1. [Epub ahead of print]
      Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
    Keywords:  Cas9; chromosome loss; double strand break repair; embryonic stem cells; germ-line gene editing; homologous recombination; human embryo; interhomolog recombination; microhomology mediated end joining; mitosis
  49. Oncoimmunology. 2020 Oct 19. 9(1): 1830513
      Lymph-node metastasis is a prognosis factor for poor clinical outcome of breast cancer patients. Currently, how breast cancer cells establish pre-metastatic niche in the tumor-draining lymph nodes (TDLNs) is still unclear. To address this question, we isolated heterogeneous cells including immune and stromal cells from naive lymph nodes (LNs) of the FVB/NJ mice and TDLNs of the MMTV-PyMT mice. Single-cell RNA sequencing was performed to investigate the transcriptome of the cells and various bioinformatics analyses were used to identify the altered pathways. Our results revealed several significant changes between naïve LNs and TDLNs. First, according to immunologic signature and pathway analysis, CD4+ and CD8 + T cells showed upregulated angiogenesis pathway genes and higher regulatory T (Treg)-associated genes while they demonstrated downregulation of interferon response and inflammatory response gene signatures, concurrently suggesting an immunosuppressive microenvironment in the TDLNs. Second, profiling of B cells showed down-regulation of marginal zone B lymphocytes in the TDLNs, which was validated by flow cytometric analysis. Third, we found the enhancement of oxidative phosphorylation pathway in the fibroblastic reticular cells (FRCs) of the MMTV-PyMT mice and the elevation of related genes including Prdx3, Ndufa4 and Uqcrb, suggesting massive ATP consumption and TCA cycle metabolism in the FRCs. Collectively, our results reveal the reprogramming of TDLNs during breast cancer progression at single-cell level in a spontaneous breast cancer model and suggest the changes in immune modulation and metabolic switch are key alterations in the preparation of pre-metastatic niche by breast cancer cells.
    Keywords:  Single-cell RNA sequencing; fibroblastic reticular cells; mmtv-PyMT mice; oxidative phosphorylation; tumor-draining lymph node
  50. Cancer Res. 2020 Oct 26. pii: canres.2410.2020. [Epub ahead of print]
      Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolved protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZPs) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, pre-malignant Hi-Myc prostatic glands. Systemic administration of an in vivo imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types.
  51. Mol Cell. 2020 Oct 15. pii: S1097-2765(20)30686-9. [Epub ahead of print]
      The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.
    Keywords:  lipid kinase; lipid phosphatase; phosphoinositide homeostasis
  52. FASEB J. 2020 Oct 26.
      All-trans-retinoic acid (atRA), the active metabolite of vitamin A, is an essential signaling molecule in all chordates. Global knockouts of the atRA clearing enzymes Cyp26a1 or Cyp26b1 are embryonic lethal. In adult rodents, inhibition of Cyp26a1 and Cyp26b1 increases atRA concentrations and signaling. However, postnatal knockout of Cyp26a1 does not cause a severe phenotype. We hypothesized that Cyp26b1 is the main atRA clearing Cyp in postnatal mammals. This hypothesis was tested by generating tamoxifen-inducible knockout mouse models of Cyp26b1 alone or with Cyp26a1. Both mouse models showed dermatitis, blepharitis, and splenomegaly. Histology showed infiltration of inflammatory cells including neutrophils and T lymphocytes into the skin and hyperkeratosis/hyperplasia of the nonglandular stomach. The mice lacking both Cyp26a1 and Cyp26b1 also had a reduced lifespan, failed to gain weight, and showed fat atrophy. There were significant changes in vitamin A homeostasis. Postnatal knockout of Cyp26b1 resulted in increased atRA concentrations in the skin while the postnatal knockout of both Cyp26a1 and Cyp26b1 resulted in increased atRA concentrations in the liver, serum, skin, spleen, and intestines. This study demonstrates the paramount role of Cyp26b1 in regulating retinoid homeostasis in postnatal life.
    Keywords:  cytochrome P450; dermatitis; inflammation; retinoic acid; vitamin A
  53. Proc Natl Acad Sci U S A. 2020 Oct 26. pii: 202016388. [Epub ahead of print]
      Magnetically actuated miniature soft robots are capable of programmable deformations for multimodal locomotion and manipulation functions, potentially enabling direct access to currently unreachable or difficult-to-access regions inside the human body for minimally invasive medical operations. However, magnetic miniature soft robots are so far mostly based on elastomers, where their limited deformability prevents them from navigating inside clustered and very constrained environments, such as squeezing through narrow crevices much smaller than the robot size. Moreover, their functionalities are currently restricted by their predesigned shapes, which is challenging to be reconfigured in situ in enclosed spaces. Here, we report a method to actuate and control ferrofluid droplets as shape-programmable magnetic miniature soft robots, which can navigate in two dimensions through narrow channels much smaller than their sizes thanks to their liquid properties. By controlling the external magnetic fields spatiotemporally, these droplet robots can also be reconfigured to exhibit multiple functionalities, including on-demand splitting and merging for delivering liquid cargos and morphing into different shapes for efficient and versatile manipulation of delicate objects. In addition, a single-droplet robot can be controlled to split into multiple subdroplets and complete cooperative tasks, such as working as a programmable fluidic-mixing device for addressable and sequential mixing of different liquids. Due to their extreme deformability, in situ reconfigurability and cooperative behavior, the proposed ferrofluid droplet robots could open up a wide range of unprecedented functionalities for lab/organ-on-a-chip, fluidics, bioengineering, and medical device applications.
    Keywords:  cargo delivery; ferrofluid droplet; multifunctional; shape-programmable; soft robot
  54. Support Care Cancer. 2020 Oct 26.
      PURPOSE: The combination of gemcitabine (GEM) and nanoparticle albumin-bound paclitaxel (nab-PTX) is an effective chemotherapeutic regimen for locally advanced and metastatic pancreatic cancer. The dose-limiting toxicities (DLTs) of this treatment are sepsis and neutropenia, while the relative dose intensity (RDI) of GEM is approximately 75% and of nab-PTX is 70-80%. In this study, we evaluated the risk factor(s) regarding treatment suspension, which leads to reduction in the RDI of these agents, enabling appropriate schedule management.METHODS: Two hundred patients with pancreatic cancer who received GEM + nab-PTX were retrospectively investigated. Frequency and risk factor(s) of suspension of the treatment and grade 3/4 neutropenia in the first course were evaluated.
    RESULTS: The frequency of treatment suspension in the first course was 61%. The frequency of grade 3/4 neutropenia was 51%, while that of thrombocytopenia was 7.5%. The RDI was 78.0% for GEM and 77.7% for nab-PTX. Univariate and multivariate analyses to identify risk or preventive factors related to treatment suspension suggested that low platelet count at baseline was a risk factor, whereas dose reduction from the treatment initiation was a preventive factor. The most common cause of abeyance was grade 3/4 neutropenia (83.6%), the risk factors of which were low platelet count and age ≥ 65 years at baseline, while dose reduction was a preventive factor.
    CONCLUSION: We found that a low platelet level at baseline was a risk factor, whereas dose reduction from initiation was a preventive factor in regard to treatment suspension and severe neutropenia occurrence in GEM + nab-PTX treatment.
    Keywords:  Gemcitabine; Neutropenia; Relative dose intensity; Risk factor; Treatment suspension; nab-paclitaxel
  55. Cell Cycle. 2020 Oct 28. 1-3
      Ferroptosis is a newly identified form of cell death that is regulated by many metabolic pathways, including iron, lipid and amino acids. Recent two studies reveal that the mitochondria and energy stress could also mediate ferroptosis. Gao et al. report that mitochondria play an essential role in ferroptosis induced by cysteine deprivation. In addition, Lee et al. show that energy stress depressed ferroptosis partly through activation of AMP-activated protein kinase. These findings provide potential therapeutic strategies for treating ferroptosis-related diseases, such as cancer, tissue injury and neurodegenerative diseases.
    Keywords:  Ferroptosis; energy stress; mitochondria
  56. Proc Natl Acad Sci U S A. 2020 Oct 26. pii: 202003524. [Epub ahead of print]
      The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.
    Keywords:  PERIOD; bistable phospho-switch; circadian rhythm; cytoplasmic trafficking; negative feedback loop
  57. Phys Biol. 2020 Oct 28.
      The nucleus of eukaryotic cells harbors active and out of equilibrium environment which is conducive for diverse gene regulatory processes. On a molecular scale, gene regulatory processes take place within hierarchically compartmentalized sub-nuclear bodies. While the impact of nuclear structure on gene regulation is widely appreciated it has remained much less clear weather and how gene regulation is impacting nuclear order itself. Recently the liquid-liquid phase separation emerged as a fundemtnal mechanism driving the formation of biomolecular condensates including membraneless organelles, chromatin territories, and transcriptional domains. The transience and environmental sensitivity of biomolecular condensation is strongly suggestive of kinetic gene-regulatory control of phase separation. To this end we have constructed a minimalist model of reactive nucleoplasm based on Cahn-Hilliard formulation of ternary protein-RNA-nucleoplasm components coupled to non-equilibrium and spatially dependent gene expression. Through an extensive set of simulations, we find a broad range of kinetic regimes where the interplay of phase separation and reactive time-scales can generate heterogeneous multi-modal gene expression patterns. Furthermore, the significance of this finding is that heterogeneity of gene expression is linked directly with the heterogeneity of length-scales in phase-separated condensates.
    Keywords:  Eukaryotic nucleus; Gene regulation; LLPS; Nonequilibrium; Nucleoplasm; Phase separation
  58. Nat Rev Endocrinol. 2020 Oct 26.
      The metabolic syndrome is prevalent in developed nations and accounts for the largest burden of non-communicable diseases worldwide. The metabolic syndrome has direct effects on health and increases the risk of developing cancer. Lifestyle factors that are known to promote the metabolic syndrome generally cause pro-inflammatory alterations in microbiota communities in the intestine. Indeed, alterations to the structure and function of intestinal microbiota are sufficient to promote the metabolic syndrome, inflammation and cancer. Among the lifestyle factors that are associated with the metabolic syndrome, disruption of the circadian system, known as circadian dysrhythmia, is increasingly common. Disruption of the circadian system can alter microbiome communities and can perturb host metabolism, energy homeostasis and inflammatory pathways, which leads to the metabolic syndrome. This Perspective discusses the role of intestinal microbiota and microbial metabolites in mediating the effects of disruption of circadian rhythms on human health.
  59. Cancer Metastasis Rev. 2020 Oct 28.
      Traditional laboratory model organisms are indispensable for cancer research and have provided insight into numerous mechanisms that contribute to cancer development and progression in humans. However, these models do have some limitations, most notably related to successful drug translation, because traditional model organisms are often short-lived, small-bodied, genetically homogeneous, often immunocompromised, are not exposed to natural environments shared with humans, and usually do not develop cancer spontaneously. We propose that assimilating information from a variety of long-lived, large, genetically diverse, and immunocompetent species that live in natural environments and do develop cancer spontaneously (or do not develop cancer at all) will lead to a more comprehensive understanding of human cancers. These non-traditional model organisms can also serve as sentinels for environmental risk factors that contribute to human cancers. Ultimately, expanding the range of animal models that can be used to study cancer will lead to improved insights into cancer development, progression and metastasis, tumor microenvironment, as well as improved therapies and diagnostics, and will consequently reduce the negative impacts of the wide variety of cancers afflicting humans overall.
    Keywords:  Cancer; Cancer resistance; Metastasis; Non-traditional model organisms; Sentinel; Spontaneous tumors; Tumor microenvironment