bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒12‒11
twenty-one papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute
  1. The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC.
  2. PIK3CA gain-of-function mutation in adipose tissue induces metabolic reprogramming with Warburg-like effect and severe endocrine disruption.
  3. AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.
  4. Unveiling molecular insights into the mechanism of activation of oncogenic phosphoinositide 3-kinase mutants.
  5. Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis.
  6. Impact of the Human Cell Atlas on medicine.
  7. Cross-talk between TSC2 and the extracellular matrix controls pulmonary vascular proliferation and pulmonary hypertension.
  8. The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance.
  9. Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.
  10. Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells.
  11. Nodal signaling establishes a competency window for stochastic cell fate switching.
  12. Glycolytic flux-signaling controls mouse embryo mesoderm development.
  13. Breast cancer prevention by short-term inhibition of TGFβ signaling.
  14. Tracing the origin of everything.
  15. Phosphatidylinositol (3,5)-bisphosphate machinery regulates neurite thickness through neuron-specific endosomal protein NSG1/NEEP21.
  16. Intracellular galectin-3 is a lipopolysaccharide sensor that promotes glycolysis through mTORC1 activation.
  17. Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress.
  18. A multi-omics longitudinal aging dataset in primary human fibroblasts with mitochondrial perturbations.
  19. Hallmarks of cancer: The insulin-like growth factors perspective.
  20. Full-Length Single-Cell RNA-Sequencing with FLASH-seq.
  21. Embryonic origins of adult pluripotent stem cells.
  1. Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01642-4. [Epub ahead of print]41(10): 111759
    The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC.
    Ji-Sun Lee, Michael W Lero, Jose Mercado-Matos, Sha Zhu, Minjeong Jo, Claire E Tocheny, Jennifer S Morgan, Leslie M Shaw.
      Despite the strong association of the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway with tumor initiation, recurrence, and metastasis, the mechanism by which this pathway regulates cancer progression is not well understood. Here, we report that IIS supports breast cancer stem cell (CSC) self-renewal in an IRS2-phosphatidylinositol 3-kinase (PI3K)-dependent manner that involves the activation and stabilization of MYC. IRS2-PI3K signaling enhances MYC expression through the inhibition of GSK3β activity and suppression of MYC phosphorylation on threonine 58, thus reducing proteasome-mediated degradation of MYC and sustaining active pS62-MYC function. A stable T58A-Myc mutant rescues CSC function in Irs2-/- cells, supporting the role of this MYC stabilization in IRS2-dependent CSC regulation. These findings establish a mechanistic connection between the IIS pathway and MYC and highlight a role for IRS2-dependent signaling in breast cancer progression.
    Keywords:  CP: Cancer; CSC; IGF; IRS2; MYC; PI3K; breast cancer; cancer stem cell; insulin; insulin receptor substrate; insulin-like growth factor
    DOI:  https://doi.org/10.1016/j.celrep.2022.111759
  2. Sci Adv. 2022 Dec 09. 8(49): eade7823
    PIK3CA gain-of-function mutation in adipose tissue induces metabolic reprogramming with Warburg-like effect and severe endocrine disruption.
    Sophia Ladraa, Lola Zerbib, Charles Bayard, Antoine Fraissenon, Quitterie Venot, Gabriel Morin, Alexandre P Garneau, Pierre Isnard, Célia Chapelle, Clément Hoguin, Sylvie Fraitag, Jean-Paul Duong, Laurent Guibaud, Alix Besançon, Sophie Kaltenbach, Patrick Villarese, Vahid Asnafi, Christine Broissand, Nicolas Goudin, Michael Dussiot, Ivan Nemazanyy, Thomas Viel, Gwennhael Autret, Céline Cruciani-Guglielmacci, Jessica Denom, Julie Bruneau, Bertrand Tavitian, Christophe Legendre, Julien Dairou, Jean-Marc Lacorte, Pacifique Levy, Mario Pende, Michel Polak, Guillaume Canaud.
      PIK3CA-related overgrowth syndrome (PROS) is a genetic disorder caused by somatic mosaic gain-of-function mutations of PIK3CA. Clinical presentation of patients is diverse and associated with endocrine disruption. Adipose tissue is frequently involved, but its role in disease development and progression has not been elucidated. Here, we created a mouse model of PIK3CA-related adipose tissue overgrowth that recapitulates patient phenotype. We demonstrate that PIK3CA mutation leads to GLUT4 membrane accumulation with a negative feedback loop on insulin secretion, a burst of liver IGFBP1 synthesis with IGF-1 sequestration, and low circulating levels. Mouse phenotype was mainly driven through AKT2. We also observed that PIK3CA mutation induces metabolic reprogramming with Warburg-like effect and protein and lipid synthesis, hallmarks of cancer cells, in vitro, in vivo, and in patients. We lastly show that alpelisib is efficient at preventing and improving PIK3CA-adipose tissue overgrowth and reversing metabolomic anomalies in both animal models and patients.
    DOI:  https://doi.org/10.1126/sciadv.ade7823
  3. Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01611-4. [Epub ahead of print]41(10): 111733
    AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.
    Ehud Wainstein, Galia Maik-Rachline, John Blenis, Rony Seger.
      AKT is a central signaling protein kinase that plays a role in the regulation of cellular survival metabolism and cell growth, as well as in pathologies such as diabetes and cancer. Human AKT consists of three isoforms (AKT1-3) that may fulfill different functions. Here, we report that distinct subcellular localization of the isoforms directly influences their activity and function. AKT1 is localized primarily in the cytoplasm, AKT2 in the nucleus, and AKT3 in the nucleus or nuclear envelope. None of the isoforms actively translocates into the nucleus upon stimulation. Interestingly, AKT3 at the nuclear envelope is constitutively phosphorylated, enabling a constant phosphorylation of TSC2 at this location. Knockdown of AKT3 induces moderate attenuation of cell proliferation of breast cancer cells. We suggest that in addition to the stimulation-induced activation of the lysosomal/cytoplasmic AKT1-TSC2 pathway, a subpopulation of TSC2 is constitutively inactivated by AKT3 at the nuclear envelope of transformed cells.
    Keywords:  AKT; CP: Cell biology; Tsc2; nuclear envelope; phosphorylation; subcellular localization
    DOI:  https://doi.org/10.1016/j.celrep.2022.111733
  4. Proc Natl Acad Sci U S A. 2022 Dec 13. 119(50): e2218237119
    Unveiling molecular insights into the mechanism of activation of oncogenic phosphoinositide 3-kinase mutants.
    Stefan G Krimmer, Joseph Schlessinger.
      
    DOI:  https://doi.org/10.1073/pnas.2218237119
  5. Development. 2022 Dec 01. pii: dev200754. [Epub ahead of print]149(23):
    Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis.
    Jie Liu, Mingming Zhang, Haojian Dong, Jingwen Liu, Aihua Mao, Guozhu Ning, Yu Cao, Yiyue Zhang, Qiang Wang.
      Developmentally, the great vessels of the heart originate from the pharyngeal arch arteries (PAAs). During PAA vasculogenesis, PAA precursors undergo sequential cell fate decisions that are accompanied by proliferative expansion. However, how these two processes are synchronized remains poorly understood. Here, we find that the zebrafish chemokine receptor Cxcr4a is expressed in PAA precursors, and genetic ablation of either cxcr4a or the ligand gene cxcl12b causes PAA stenosis. Cxcr4a is required for the activation of the downstream PI3K/AKT cascade, which promotes not only PAA angioblast proliferation, but also differentiation. AKT has a well-known role in accelerating cell-cycle progression through the activation of cyclin-dependent kinases. Despite this, we demonstrate that AKT phosphorylates Etv2 and Scl, the key regulators of angioblast commitment, on conserved serine residues, thereby protecting them from ubiquitin-mediated proteasomal degradation. Altogether, our study reveals a central role for chemokine signaling in PAA vasculogenesis through orchestrating angioblast proliferation and differentiation.
    Keywords:   cxcl12b ; cxcr4a ; Angioblast differentiation; Cell proliferation; Pharyngeal arch artery; Zebrafish
    DOI:  https://doi.org/10.1242/dev.200754
  6. Nat Med. 2022 Dec 08.
    Impact of the Human Cell Atlas on medicine.
    Jennifer E Rood, Aidan Maartens, Anna Hupalowska, Sarah A Teichmann, Aviv Regev.
      Single-cell atlases promise to provide a 'missing link' between genes, diseases and therapies. By identifying the specific cell types, states, programs and contexts where disease-implicated genes act, we will understand the mechanisms of disease at the cellular and tissue levels and can use this understanding to develop powerful disease diagnostics; identify promising new drug targets; predict their efficacy, toxicity and resistance mechanisms; and empower new kinds of therapies, from cancer therapies to regenerative medicine. Here, we lay out a vision for the potential of cell atlases to impact the future of medicine, and describe how advances over the past decade have begun to realize this potential in common complex diseases, infectious diseases (including COVID-19), rare diseases and cancer.
    DOI:  https://doi.org/10.1038/s41591-022-02104-7
  7. Sci Signal. 2022 Dec 06. 15(763): eabn2743
    Cross-talk between TSC2 and the extracellular matrix controls pulmonary vascular proliferation and pulmonary hypertension.
    Yuanjun Shen, Dmitry A Goncharov, Andressa Pena, Jeffrey Baust, Andres Chavez Barragan, Arnab Ray, Analise Rode, Timothy N Bachman, Baojun Chang, Lifeng Jiang, Paul Dieffenbach, Laura E Fredenburgh, Mauricio Rojas, Horace DeLisser, Ana L Mora, Tatiana V Kudryashova, Elena A Goncharova.
      Increased proliferation and survival of cells in small pulmonary arteries (PAs) drive pulmonary arterial hypertension (PAH). Because cell growth mediated by the mTOR-containing mTORC1 complex is inhibited by tuberous sclerosis complex 2 (TSC2), we investigated the role of this GTPase-activating protein in PAH pathology. TSC2 abundance was decreased in remodeled small PAs and PA vascular smooth muscle cells (PAVSMCs) from patients with PAH or from rodent pulmonary hypertension (PH) models, as well as PAVSMCs maintained on substrates that reproduced pathology-induced stiffness. Accordingly, mice with smooth muscle-specific reduction in TSC2 developed PH. At the molecular level, decreased TSC2 abundance led to stiffness-induced PAVSMC proliferation, increased abundance of the mechanosensitive transcriptional coactivators YAP/TAZ, and enhanced mTOR kinase activity. Moreover, extracellular matrix (ECM) produced by TSC2-deficient PAVSMCs stimulated the proliferation of nondiseased PA adventitial fibroblasts and PAVSMCs through fibronectin and its receptor, the α5β1 integrin. Reconstituting TSC2 in PAVSMCs from patients with PAH through overexpression or treatment with the SIRT1 activator SRT2104 decreased YAP/TAZ abundance, mTOR activity, and ECM production, as well as inhibited proliferation and induced apoptosis. In two rodent models of PH, SRT2104 treatment restored TSC2 abundance, attenuated pulmonary vascular remodeling, and ameliorated PH. Thus, TSC2 in PAVSMCs integrates ECM composition and stiffness with pro-proliferative and survival signaling, and restoring TSC2 abundance could be an attractive therapeutic option to treat PH.
    DOI:  https://doi.org/10.1126/scisignal.abn2743
  8. Nat Commun. 2022 Dec 06. 13(1): 7522
    The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance.
    Alessandra Dall'Agnese, Jesse M Platt, Ming M Zheng, Max Friesen, Giuseppe Dall'Agnese, Alyssa M Blaise, Jessica B Spinelli, Jonathan E Henninger, Erin N Tevonian, Nancy M Hannett, Charalampos Lazaris, Hannah K Drescher, Lea M Bartsch, Henry R Kilgore, Rudolf Jaenisch, Linda G Griffith, Ibrahim I Cisse, Jacob F Jeppesen, Tong I Lee, Richard A Young.
      Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41467-022-35176-7
  9. J Biol Chem. 2022 Dec 02. pii: S0021-9258(22)01209-1. [Epub ahead of print] 102766
    Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.
    David J Wisniewski, Mariya S Liyasova, Soumya Korrapati, Xu Zhang, Shashikala Ratnayake, Qingrong Chen, Samuel F Gilbert, Alexis Catalano, Donna Voeller, Daoud Meerzaman, Udayan Guha, Natalie Porat-Shliom, Christina M Annunziata, Stanley Lipkowitz.
      Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Additionally, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.
    Keywords:  Cbl; EGFR; FLOT2; MAPK; cancer biology; cell proliferation; mass spectrometry; phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2022.102766
  10. Nat Commun. 2022 Dec 03. 13(1): 7470
    Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells.
    Patricia Gerdes, Sue Mei Lim, Adam D Ewing, Michael R Larcombe, Dorothy Chan, Francisco J Sanchez-Luque, Lucinda Walker, Alexander L Carleton, Cini James, Anja S Knaupp, Patricia E Carreira, Christian M Nefzger, Ryan Lister, Sandra R Richardson, Jose M Polo, Geoffrey J Faulkner.
      Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
    DOI:  https://doi.org/10.1038/s41467-022-35180-x
  11. Dev Cell. 2022 Dec 05. pii: S1534-5807(22)00808-5. [Epub ahead of print]57(23): 2604-2622.e5
    Nodal signaling establishes a competency window for stochastic cell fate switching.
    Andrew D Economou, Luca Guglielmi, Philip East, Caroline S Hill.
      Specification of the germ layers by Nodal signaling has long been regarded as an archetype of how graded morphogens induce different cell fates. However, this deterministic model cannot explain why only a subset of cells at the early zebrafish embryo margin adopt the endodermal fate, whereas their immediate neighbours, experiencing a similar signaling environment, become mesoderm. Combining pharmacology, quantitative imaging and single cell transcriptomics, we demonstrate that sustained Nodal signaling establishes a bipotential progenitor state from which cells can switch to an endodermal fate or differentiate into mesoderm. Switching is a random event, the likelihood of which is modulated by Fgf signaling. This inherently imprecise mechanism nevertheless leads to robust endoderm formation because of buffering at later stages. Thus, in contrast to previous deterministic models of morphogen action, Nodal signaling establishes a temporal window when cells are competent to undergo a stochastic cell fate switch, rather than determining fate itself.
    Keywords:  FGF; Nodal; endoderm specification; mesoderm specification; morphogen gradient; stochastic cell switching
    DOI:  https://doi.org/10.1016/j.devcel.2022.11.008
  12. Elife. 2022 Dec 05. pii: e83299. [Epub ahead of print]11
    Glycolytic flux-signaling controls mouse embryo mesoderm development.
    Hidenobu Miyazawa, Marteinn T Snaebjornsson, Nicole Prior, Eleni Kafkia, Henrik M Hammarén, Nobuko Tsuchida-Straeten, Kiran R Patil, Martin Beck, Alexander Aulehla.
      How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.
    Keywords:  developmental biology; mouse
    DOI:  https://doi.org/10.7554/eLife.83299
  13. Nat Commun. 2022 Dec 07. 13(1): 7558
    Breast cancer prevention by short-term inhibition of TGFβ signaling.
    Maša Alečković, Simona Cristea, Carlos R Gil Del Alcazar, Pengze Yan, Lina Ding, Ethan D Krop, Nicholas W Harper, Ernesto Rojas Jimenez, Donghao Lu, Anushree C Gulvady, Pierre Foidart, Marco Seehawer, Benedetto Diciaccio, Katherine C Murphy, Jason Pyrdol, Jayati Anand, Kodie Garza, Kai W Wucherpfennig, Rulla M Tamimi, Franziska Michor, Kornelia Polyak.
      Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFβ signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFβ in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy.
    DOI:  https://doi.org/10.1038/s41467-022-35043-5
  14. Cell. 2022 Dec 08. pii: S0092-8674(22)01458-1. [Epub ahead of print]185(25): 4677-4679
    Tracing the origin of everything.
    Erin L Davies.
      Highly potent adult stem cells fuel lifelong tissue homeostasis and regeneration in many aquatic invertebrates, yet their developmental backstories remain obscure. In this issue of Cell, Kimura and colleagues reveal the cellular origin of adult pluripotent stem cells and propose a molecular trajectory for their specification during acoel embryogenesis.
    DOI:  https://doi.org/10.1016/j.cell.2022.11.015
  15. J Biol Chem. 2022 Dec 06. pii: S0021-9258(22)01218-2. [Epub ahead of print] 102775
    Phosphatidylinositol (3,5)-bisphosphate machinery regulates neurite thickness through neuron-specific endosomal protein NSG1/NEEP21.
    Lijuan Qi, Chen Sun, Shenqing Sun, Aiqing Li, Qiuming Hu, Yaobo Liu, Yanling Zhang.
      Phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P2] is a critical signaling phospholipid involved in endo-lysosome homeostasis. It is synthesized by a protein complex composed of PIKfyve, Vac14, and Fig4. Defects in PtdIns(3,5)P2 synthesis underlie a number of human neurological disorders, including Charcot-Marie-Tooth disease, child onset progressive dystonia, and others. However, neuron-specific functions of PtdIns(3,5)P2 remain less understood. Here we show that PtdIns(3,5)P2 pathway is required to maintain neurite thickness. Suppression of PIKfyve activities using either pharmacological inhibitors or RNA silencing resulted in decreased neurite thickness. We further find that the regulation of neurite thickness by PtdIns(3,5)P2 is mediated by NSG1/NEEP21, a neuron-specific endosomal protein. Knockdown of NSG1 expression also led to thinner neurites. mCherry tagged NSG1 co-localized and interacted with proteins in the PtdIns(3,5)P2 machinery. Perturbation of PtdIns(3,5)P2 dynamics by overexpressing Fig4 or a PtdIns(3,5)P2 binding domain resulted in mis-localization of NSG1 to non-endosomal locations, and suppressing PtdIns(3,5)P2 synthesis resulted in an accumulation of NSG1 in EEA1-positive early endosomes. Importantly, overexpression of NSG1 rescued neurite thinning in PtdIns(3,5)P2 deficient CAD neurons and primary cortical neurons. Our study uncovered the role of PtdIns(3,5)P2 in the morphogenesis of neurons, which revealed a novel aspect of the pathogenesis of PtdIns(3,5)P2 related neuropathies. We also identified NSG1 as an important downstream protein of PtdIns(3,5)P2, which may provide a novel therapeutic target in neurological diseases.
    Keywords:  NSG1; PIKfyve; PtdIns(3,5)P(2); neurite outgrowth; neurite thickness
    DOI:  https://doi.org/10.1016/j.jbc.2022.102775
  16. Nat Commun. 2022 Dec 08. 13(1): 7578
    Intracellular galectin-3 is a lipopolysaccharide sensor that promotes glycolysis through mTORC1 activation.
    Xing Chen, Chunyu Yu, Xinhua Liu, Beibei Liu, Xiaodi Wu, Jiajing Wu, Dong Yan, Lulu Han, Zifan Tang, Xinyi Yuan, Jianqiu Wang, Yue Wang, Shumeng Liu, Lin Shan, Yongfeng Shang.
      How the carbohydrate binding protein galectin-3 might act as a diabetogenic and tumorogenic factor remains to be investigated. Here we report that intracellular galectin-3 interacts with Rag GTPases and Ragulator on lysosomes. We show that galectin-3 senses lipopolysaccharide (LPS) to facilitate the interaction of Rag GTPases and Ragulator, leading to the activation of mTORC1. We find that the lipopolysaccharide/galectin-3-Rag GTPases/Ragulator-mTORC1 axis regulates a cohort of genes including GLUT1, and HK2, and PKM2 that are critically involved in glucose uptake and glycolysis. Indeed, galectin-3 deficiency severely compromises LPS-promoted glycolysis. Importantly, the expression of HK2 is significantly reduced in diabetes patients. In multiple types of cancer including hepatocellular carcinoma (HCC), galectin-3 is highly expressed, and its level of expression is positively correlated with that of HK2 and PKM2 and negatively correlated with the prognosis of HCC patients. Our study unravels that galectin-3 is a sensor of LPS, an important modulator of the mTORC1 signaling, and a critical regulator of glucose metabolism.
    DOI:  https://doi.org/10.1038/s41467-022-35334-x
  17. Mol Syst Biol. 2022 Dec;18(12): e11401
    Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress.
    Ryan L Hanson, Eric Batchelor.
      In response to different cellular stresses, the transcription factor p53 undergoes different dynamics. p53 dynamics, in turn, control cell fate. However, distinct stresses can generate the same p53 dynamics but different cell fate outcomes, suggesting integration of dynamic information from other pathways is important for cell fate regulation. To determine how MAPK activities affect p53-mediated responses to DNA breaks and oxidative stress, we simultaneously tracked p53 and either ERK, JNK, or p38 activities in single cells. While p53 dynamics were comparable between the stresses, cell fate outcomes were distinct. Combining MAPK dynamics with p53 dynamics was important for distinguishing between the stresses and for generating temporal ordering of cell fate pathways. Furthermore, cross-talk between MAPKs and p53 controlled the balance between proliferation and cell death. These findings provide insight into how cells integrate signaling pathways with distinct temporal patterns of activity to encode stress specificity and drive different cell fate decisions.
    Keywords:  MAPKs; cell stress responses; dynamics; p53; single cells
    DOI:  https://doi.org/10.15252/msb.202211401
  18. Sci Data. 2022 Dec 03. 9(1): 751
    A multi-omics longitudinal aging dataset in primary human fibroblasts with mitochondrial perturbations.
    Gabriel Sturm, Anna S Monzel, Kalpita R Karan, Jeremy Michelson, Sarah A Ware, Andres Cardenas, Jue Lin, Céline Bris, Balaji Santhanam, Michael P Murphy, Morgan E Levine, Steve Horvath, Daniel W Belsky, Shuang Wang, Vincent Procaccio, Brett A Kaufman, Michio Hirano, Martin Picard.
      Aging is a process of progressive change. To develop biological models of aging, longitudinal datasets with high temporal resolution are needed. Here we report a multi-omics longitudinal dataset for cultured primary human fibroblasts measured across their replicative lifespans. Fibroblasts were sourced from both healthy donors (n = 6) and individuals with lifespan-shortening mitochondrial disease (n = 3). The dataset includes cytological, bioenergetic, DNA methylation, gene expression, secreted proteins, mitochondrial DNA copy number and mutations, cell-free DNA, telomere length, and whole-genome sequencing data. This dataset enables the bridging of mechanistic processes of aging as outlined by the "hallmarks of aging", with the descriptive characterization of aging such as epigenetic age clocks. Here we focus on bridging the gap for the hallmark mitochondrial metabolism. Our dataset includes measurement of healthy cells, and cells subjected to over a dozen experimental manipulations targeting oxidative phosphorylation (OxPhos), glycolysis, and glucocorticoid signaling, among others. These experiments provide opportunities to test how cellular energetics affect the biology of cellular aging. All data are publicly available at our webtool: https://columbia-picard.shinyapps.io/shinyapp-Lifespan_Study/.
    DOI:  https://doi.org/10.1038/s41597-022-01852-y
  19. Front Oncol. 2022 ;12 1055589
    Hallmarks of cancer: The insulin-like growth factors perspective.
    Haim Werner, Derek LeRoith.
      The identification of a series of attributes or hallmarks that are shared by virtually all cancer cells constitutes a true milestone in cancer research. The conceptualization of a catalogue of common genetic, molecular, biochemical and cellular events under a unifying Hallmarks of Cancer idea had a major impact in oncology. Furthermore, the fact that different types of cancer, ranging from pediatric tumors and leukemias to adult epithelial cancers, share a large number of fundamental traits reflects the universal nature of the biological events involved in oncogenesis. The dissection of a complex disease like cancer into a finite directory of hallmarks is of major basic and translational relevance. The role of insulin-like growth factor-1 (IGF1) as a progression/survival factor required for normal cell cycle transition has been firmly established. Similarly well characterized are the biochemical and cellular activities of IGF1 and IGF2 in the chain of events leading from a phenotypically normal cell to a diseased one harboring neoplastic traits, including growth factor independence, loss of cell-cell contact inhibition, chromosomal abnormalities, accumulation of mutations, activation of oncogenes, etc. The purpose of the present review is to provide an in-depth evaluation of the biology of IGF1 at the light of paradigms that emerge from analysis of cancer hallmarks. Given the fact that the IGF1 axis emerged in recent years as a promising therapeutic target, we believe that a careful exploration of this signaling system might be of critical importance on our ability to design and optimize cancer therapies.
    Keywords:  IGF1 receptor (IGF1R); apoptosis; cancer hallmarks; cell cycle; insulin-like growth factor-1 (IGF1); p53; tumor suppressors
    DOI:  https://doi.org/10.3389/fonc.2022.1055589
  20. Methods Mol Biol. 2023 ;2584 123-164
    Full-Length Single-Cell RNA-Sequencing with FLASH-seq.
    Vincent Hahaut, Simone Picelli.
      The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009 (Tang et al. Nat Methods 6:377-382, 2009). While the first methods analyzed just a handful of cells, the throughput and performance rapidly increased over a very short time span. However, it was not until the introduction of emulsion droplets methods, such as the well-known kits commercialized by 10x Genomics, that the robust and reproducible analysis of thousands of cells became feasible (Zheng et al Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049, 2017). Despite generating data at a speed and a cost per cell that remains unmatched for full-length protocols like Smart-seq (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708-714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096-1098, 2013), scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the entire transcriptome.Building upon the existing Smart-seq2/3 workflows (Hagemann-Jensen et al Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 38:708-714, 2020; Picelli et al Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10:1096-1098, 2013), we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than previous versions, requiring limited hands-on time and with a great potential for customization (Hahaut et al. Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq. http://biorxiv.org/lookup/doi/10.1101/2021.07.14.452217. https://doi.org/10.1101/2021.07.14.452217, 2021). Here, we present three variants of the FS protocol.Standard FLASH-seq (FS), which builds upon Smart-seq2 developed in the past, is non-stranded and does not use unique molecular identifiers (UMIs) but still remains the easiest method to measure gene expression in a cell population.FLASH-seq low-amplification (FS-LA) represents the fastest method, which generates sequencing-ready libraries in 4.5 h, without sacrificing performance.FLASH-seq with UMIs (FS-UMI) builds upon the same principle as Smart-seq3 and introduces UMIs for molecule counting and isoform reconstruction. The newly designed template-switching oligonucleotide (TSO) contains a 5-bp spacer, which allows the generation of high-quality data while minimizing the amount of strand-invasion artifacts.
    Keywords:  FLASH-seq; FLASH-seq low-amplification; FLASH-seq with UMI; RNA-seq; automation; full-length; high-throughput; single cell
    DOI:  https://doi.org/10.1007/978-1-0716-2756-3_5
  21. Cell. 2022 Dec 08. pii: S0092-8674(22)01420-9. [Epub ahead of print]185(25): 4756-4769.e13
    Embryonic origins of adult pluripotent stem cells.
    Julian O Kimura, D Marcela Bolaños, Lorenzo Ricci, Mansi Srivastava.
      Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.
    Keywords:  Hofstenia; acoels; embryonic development; fate map; lineage tracing; pluripotency; regeneration; single-cell transcriptomics; stem cells
    DOI:  https://doi.org/10.1016/j.cell.2022.11.008
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