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Chen HJ, Gardner EE, Shah Y, Zhang K, Thakur A, Zhang C, Elemento O, Varmus H. FORMATION OF MALIGNANT, METASTATIC SMALL CELL LUNG CANCERS THROUGH OVERPRODUCTION OF cMYC PROTEIN IN TP53 AND RB1 DEPLETED PULMONARY NEUROENDOCRINE CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.10.06.561244. [PMID: 37873210 PMCID: PMC10592623 DOI: 10.1101/2023.10.06.561244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm.
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Affiliation(s)
- Huanhuan Joyce Chen
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY
- The Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL
- The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL
| | | | - Yajas Shah
- Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY
| | - Kui Zhang
- The Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL
- The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL
| | - Abhimanyu Thakur
- The Pritzker School of Molecular Engineering, The University of Chicago, Chicago, IL
- The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL
| | - Chen Zhang
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY
| | - Olivier Elemento
- Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY
| | - Harold Varmus
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY
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2
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Chang CH, Liu F, Militi S, Hester S, Nibhani R, Deng S, Dunford J, Rendek A, Soonawalla Z, Fischer R, Oppermann U, Pauklin S. The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms. Nat Commun 2024; 15:3580. [PMID: 38678032 PMCID: PMC11055877 DOI: 10.1038/s41467-024-47680-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2022] [Accepted: 04/09/2024] [Indexed: 04/29/2024] Open
Abstract
The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.
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Affiliation(s)
- Chao-Hui Chang
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Feng Liu
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Stefania Militi
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Svenja Hester
- Target Discovery Institute, Nuffield Department of Medicine, Old Road, University of Oxford, Oxford, OX3 7FZ, UK
| | - Reshma Nibhani
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Siwei Deng
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - James Dunford
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Aniko Rendek
- Department of Histopathology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Zahir Soonawalla
- Department of Hepatobiliary and Pancreatic Surgery, Oxford University Hospitals NHS, Oxford, UK
| | - Roman Fischer
- Target Discovery Institute, Nuffield Department of Medicine, Old Road, University of Oxford, Oxford, OX3 7FZ, UK
| | - Udo Oppermann
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Siim Pauklin
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK.
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3
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Chang Y, Hummel SN, Jung J, Jin G, Deng Q, Bao X. Engineered hematopoietic and immune cells derived from human pluripotent stem cells. Exp Hematol 2023; 127:14-27. [PMID: 37611730 PMCID: PMC10615717 DOI: 10.1016/j.exphem.2023.08.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 08/09/2023] [Accepted: 08/17/2023] [Indexed: 08/25/2023]
Abstract
For the past decade, significant advances have been achieved in human hematopoietic stem cell (HSC) transplantation for treating various blood diseases and cancers. However, challenges remain with the quality control, amount, and cost of HSCs and HSC-derived immune cells. The advent of human pluripotent stem cells (hPSCs) may transform HSC transplantation and cancer immunotherapy by providing a cost-effective and scalable cell source for fundamental studies and translational applications. In this review, we discuss the current developments in the field of stem cell engineering for hematopoietic stem and progenitor cell (HSPC) differentiation and further differentiation of HSPCs into functional immune cells. The key advances in stem cell engineering include the generation of HSPCs from hPSCs, genetic modification of hPSCs, and hPSC-derived HSPCs for improved function, further differentiation of HPSCs into functional immune cells, and applications of cell culture platforms for hematopoietic cell manufacturing. Current challenges impeding the translation of hPSC-HSPCs and immune cells as well as further directions to address these challenges are also discussed.
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Affiliation(s)
- Yun Chang
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana; Purdue University Institute for Cancer Research, West Lafayette, Indiana
| | - Sydney N Hummel
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana; Purdue University Institute for Cancer Research, West Lafayette, Indiana
| | - Juhyung Jung
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana; Purdue University Institute for Cancer Research, West Lafayette, Indiana
| | - Gyuhyung Jin
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana; Purdue University Institute for Cancer Research, West Lafayette, Indiana
| | - Qing Deng
- Purdue University Institute for Cancer Research, West Lafayette, Indiana; Department of Biological Sciences, Purdue University, West Lafayette, Indiana
| | - Xiaoping Bao
- Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana; Purdue University Institute for Cancer Research, West Lafayette, Indiana.
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4
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Militi S, Nibhani R, Jalali M, Pauklin S. RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling. Cell Rep 2023; 42:113146. [PMID: 37725511 DOI: 10.1016/j.celrep.2023.113146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Revised: 05/30/2023] [Accepted: 08/31/2023] [Indexed: 09/21/2023] Open
Abstract
The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/β-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.
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Affiliation(s)
- Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Morteza Jalali
- Anne McLaren Laboratory for Regenerative Medicine, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK.
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5
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Fedorova V, Amruz Cerna K, Oppelt J, Pospisilova V, Barta T, Mraz M, Bohaciakova D. MicroRNA Profiling of Self-Renewing Human Neural Stem Cells Reveals Novel Sets of Differentially Expressed microRNAs During Neural Differentiation In Vitro. Stem Cell Rev Rep 2023:10.1007/s12015-023-10524-2. [PMID: 36918496 PMCID: PMC10366325 DOI: 10.1007/s12015-023-10524-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/28/2023] [Indexed: 03/16/2023]
Abstract
The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.
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Affiliation(s)
- Veronika Fedorova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Katerina Amruz Cerna
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Jan Oppelt
- Department of Pathology and Laboratory Medicine, Division of Neuropathology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Veronika Pospisilova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Tomas Barta
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Marek Mraz
- Central European Institute of Technology, Masaryk University, Brno, Czech Republic.,Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Dasa Bohaciakova
- Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. .,International Clinical Research Center (ICRC), St. Anne's University Hospital, Brno, Czech Republic.
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6
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MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors’ Efficacy in Neuroblastoma Cells. Int J Mol Sci 2023; 24:ijms24065408. [PMID: 36982482 PMCID: PMC10049239 DOI: 10.3390/ijms24065408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 03/07/2023] [Accepted: 03/08/2023] [Indexed: 03/14/2023] Open
Abstract
Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB’s mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB’s cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.
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7
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Field MG, Kuznetsoff JN, Zhang MG, Dollar JJ, Durante MA, Sayegh Y, Decatur CL, Kurtenbach S, Pelaez D, Harbour JW. RB1 loss triggers dependence on ESRRG in retinoblastoma. SCIENCE ADVANCES 2022; 8:eabm8466. [PMID: 35984874 PMCID: PMC9390996 DOI: 10.1126/sciadv.abm8466] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Accepted: 07/08/2022] [Indexed: 05/10/2023]
Abstract
Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 20 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify previously unknown Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death, which is exacerbated in hypoxia. These findings reveal a previously unidentified dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.
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Affiliation(s)
- Matthew G. Field
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA 52242, USA
| | - Jeffim N. Kuznetsoff
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Michelle G. Zhang
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - James J. Dollar
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Michael A. Durante
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Yoseph Sayegh
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Christina L. Decatur
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Stefan Kurtenbach
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Daniel Pelaez
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - J. William Harbour
- Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136, USA
- Department of Ophthalmology and Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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8
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Lossaint G, Horvat A, Gire V, Bacevic K, Mrouj K, Charrier-Savournin F, Georget V, Fisher D, Dulic V. Reciprocal regulation of p21 and Chk1 controls the Cyclin D1-RB pathway to mediate senescence onset after G2 arrest. J Cell Sci 2022; 135:274865. [PMID: 35343565 DOI: 10.1242/jcs.259114] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Accepted: 03/18/2022] [Indexed: 11/20/2022] Open
Abstract
Senescence is an irreversible proliferation withdrawal that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 and RB family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the CDK inhibitor p21 and Chk1 controls D-type cyclin-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting Cyclin D1-CDK2/CDK4. The resulting G2 exit, which precedes appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and DNA damage foci reduction. In p53/RB-proficient cancer cells, compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely Cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to Cyclin D1 and Cyclin E1-CDK complexes and down-regulating CDK6, whereas Chk2 knockdown enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting DNA damage-induced senescence onset in G2.
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Affiliation(s)
| | | | | | | | - Karim Mrouj
- IGMM, Univ. Montpellier, CNRS, Montpellier, France
| | | | - Virginie Georget
- CRBM, Univ. Montpellier, CNRS, Montpellier, France.,Montpellier Ressources Imagerie, BioCampus, University of Montpellier, CNRS, INSERM, Montpellier, France
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9
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Flores M, Goodrich DW. Retinoblastoma Protein Paralogs and Tumor Suppression. Front Genet 2022; 13:818719. [PMID: 35368709 PMCID: PMC8971665 DOI: 10.3389/fgene.2022.818719] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Accepted: 02/25/2022] [Indexed: 01/01/2023] Open
Abstract
The retinoblastoma susceptibility gene (RB1) is the first tumor suppressor gene discovered and a prototype for understanding regulatory networks that function in opposition to oncogenic stimuli. More than 3 decades of research has firmly established a widespread and prominent role for RB1 in human cancer. Yet, this gene encodes but one of three structurally and functionally related proteins that comprise the pocket protein family. A central question in the field is whether the additional genes in this family, RBL1 and RBL2, are important tumor suppressor genes. If so, how does their tumor suppressor activity overlap or differ from RB1. Here we revisit these questions by reviewing relevant data from human cancer genome sequencing studies that have been rapidly accumulating in recent years as well as pertinent functional studies in genetically engineered mice. We conclude that RBL1 and RBL2 do have important tumor suppressor activity in some contexts, but RB1 remains the dominant tumor suppressor in the family. Given their similarities, we speculate on why RB1 tumor suppressor activity is unique.
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Affiliation(s)
| | - David W. Goodrich
- Roswell Park Comprehensive Cancer Center, Department of Pharmacology and Therapeutics, Buffalo, NY, United States
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10
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Zhu Y, Cheng C, Chen L, Zhang L, Pan H, Hou L, Sun Z, Zhang L, Fu X, Chan KY, Zhang J. Cell cycle heterogeneity directs spontaneous 2C state entry and exit in mouse embryonic stem cells. Stem Cell Reports 2021; 16:2659-2673. [PMID: 34624246 PMCID: PMC8580870 DOI: 10.1016/j.stemcr.2021.09.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 09/07/2021] [Accepted: 09/08/2021] [Indexed: 12/01/2022] Open
Abstract
Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small number of two-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are unstable and they are prone to transiting back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA sequencing on isolated 2CLCs that underwent 2C-like state exit and re-entry, and revealed a step-by-step transitional process between 2C-like and pluripotent states. Mechanistically, we found that cell cycle played an important role in mediating these transitions by regulating assembly of the nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions.
The entry to and exit from the 2C-like state showed a step-by-step roadmap Cell cycle participates in mediating dynamic transitions between ESCs and 2CLCs G1/S phase arrest facilitates the Dux locus escape from heterochromatin Nucleolus-heterochromatin remodeling is involved in 2C activation
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Affiliation(s)
- Yuqing Zhu
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Haining, Zhejiang, China
| | - Chen Cheng
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Lang Chen
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Li Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Hongru Pan
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Linxiao Hou
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Zhen Sun
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China
| | - Ling Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China
| | - Xudong Fu
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
| | - Kuan Yoow Chan
- Zhejiang University-University of Edinburgh Institute, Zhejiang University School of Medicine, Haining, Zhejiang, China
| | - Jin Zhang
- Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Hangzhou, Zhejiang, China; Zhejiang Laboratory for Systems and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China; Institute of Hematology, Zhejiang University, Hangzhou, Zhejiang, China; Center of Gene/Cell Engineering and Genome Medicine, Hangzhou, Zhejiang, China.
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11
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Verdugo-Sivianes EM, Carnero A. Role of the Holoenzyme PP1-SPN in the Dephosphorylation of the RB Family of Tumor Suppressors During Cell Cycle. Cancers (Basel) 2021; 13:cancers13092226. [PMID: 34066428 PMCID: PMC8124259 DOI: 10.3390/cancers13092226] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 04/29/2021] [Accepted: 05/03/2021] [Indexed: 11/16/2022] Open
Abstract
Simple Summary Cell cycle progression is highly regulated by modulating the phosphorylation status of retinoblastoma (RB) family proteins. This process is controlled by a balance in the action of kinases, such as the complexes formed by cyclin-dependent kinases (CDKs) and cyclins, and phosphatases, mainly the protein phosphatase 1 (PP1). However, while the phosphorylation of the RB family has been largely studied, its dephosphorylation is less known. Recently, the PP1-Spinophilin (SPN) holoenzyme has been described as the main phosphatase responsible for the dephosphorylation of RB proteins during the G0/G1 transition and at the end of G1. Here, we describe the regulation of the phosphorylation status of RB family proteins, giving importance not only to their inactivation by phosphorylation but also to their dephosphorylation to restore the cell cycle. Abstract Cell cycle progression is highly regulated by modulating the phosphorylation status of the retinoblastoma protein (pRB) and the other two members of the RB family, p107 and p130. This process is controlled by a balance in the action of kinases, such as the complexes formed by cyclin-dependent kinases (CDKs) and cyclins, and phosphatases, mainly the protein phosphatase 1 (PP1). However, while the phosphorylation of the RB family has been largely studied, its dephosphorylation is less known. Phosphatases are holoenzymes formed by a catalytic subunit and a regulatory protein with substrate specificity. Recently, the PP1-Spinophilin (SPN) holoenzyme has been described as the main phosphatase responsible for the dephosphorylation of RB proteins during the G0/G1 transition and at the end of G1. Moreover, SPN has been described as a tumor suppressor dependent on PP1 in lung and breast tumors, where it promotes tumorigenesis by increasing the cancer stem cell pool. Therefore, a connection between the cell cycle and stem cell biology has also been proposed via SPN/PP1/RB proteins.
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Affiliation(s)
- Eva M. Verdugo-Sivianes
- Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocio, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Avda. Manuel Siurot s/n, 41013 Seville, Spain;
- CIBERONC, Instituto de Salud Carlos III, 28029 Madrid, Spain
| | - Amancio Carnero
- Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocio, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Avda. Manuel Siurot s/n, 41013 Seville, Spain;
- CIBERONC, Instituto de Salud Carlos III, 28029 Madrid, Spain
- Correspondence: ; Tel.: +34-955-92-31-11
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12
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Ter Huurne M, Stunnenberg HG. G1-phase progression in pluripotent stem cells. Cell Mol Life Sci 2021; 78:4507-4519. [PMID: 33884444 PMCID: PMC8195903 DOI: 10.1007/s00018-021-03797-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 01/19/2021] [Accepted: 02/19/2021] [Indexed: 11/10/2022]
Abstract
During early embryonic development both the rapid increase in cell number and the expression of genes that control developmental decisions are tightly regulated. Accumulating evidence has indicated that these two seemingly independent processes are mechanistically intertwined. The picture that emerges from studies on the cell cycle of embryonic stem cells is one in which proteins that promote cell cycle progression prevent differentiation and vice versa. Here, we review which transcription factors and signalling pathways play a role in both maintenance of pluripotency as well as cell cycle progression. We will not only describe the mechanism behind their function but also discuss the role of these regulators in different states of mouse pluripotency. Finally, we elaborate on how canonical cell cycle regulators impact on the molecular networks that control the maintenance of pluripotency and lineage specification.
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Affiliation(s)
- Menno Ter Huurne
- Department of Molecular Biology, Faculty of Science, Radboud University, 6525GA, Nijmegen, The Netherlands
- Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Rd, Parkville, Melbourne, VIC, 3052, Australia
| | - Hendrik G Stunnenberg
- Department of Molecular Biology, Faculty of Science, Radboud University, 6525GA, Nijmegen, The Netherlands.
- Princess Maxima Centre for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.
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13
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Huang Y, Su T, Wang C, Dong L, Liu S, Zhu Y, Hao K, Xia Y, Jiang Q, Qin J. Rbbp4 Suppresses Premature Differentiation of Embryonic Stem Cells. Stem Cell Reports 2021; 16:566-581. [PMID: 33606987 PMCID: PMC7940252 DOI: 10.1016/j.stemcr.2021.01.009] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 01/16/2021] [Accepted: 01/19/2021] [Indexed: 12/30/2022] Open
Abstract
Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages.
RBBP4 deficiency in ESCs leads to spontaneous differentiation into mesendodermal lineages Rbbp4 binding sites in ESCs substantially overlap with PRC2 binding Rbbp4 is absolutely essential for PRC2 chromatin occupancy Rbbp4 is necessary for sustaining the expression levels of Oct4 and Sox2
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Affiliation(s)
- Yikai Huang
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Ting Su
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Congcong Wang
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Lixia Dong
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Shuang Liu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Yaru Zhu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Kunying Hao
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China
| | - Yin Xia
- School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
| | - Qing Jiang
- Department of Sports Medicine and Adult Reconstructive Surgery, Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University, Nanjing, China.
| | - Jinzhong Qin
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, School of Medicine, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China.
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14
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Verdugo-Sivianes EM, Rojas AM, Muñoz-Galván S, Otero-Albiol D, Carnero A. Mutation of SPINOPHILIN (PPP1R9B) found in human tumors promotes the tumorigenic and stemness properties of cells. Am J Cancer Res 2021; 11:3452-3471. [PMID: 33537097 PMCID: PMC7847670 DOI: 10.7150/thno.53572] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 12/20/2020] [Indexed: 12/17/2022] Open
Abstract
Rationale: SPINOPHILIN (SPN, PPP1R9B) is an important tumor suppressor involved in the progression and malignancy of different tumors depending on its association with protein phosphatase 1 (PP1) and the ability of the PP1-SPN holoenzyme to dephosphorylate retinoblastoma (pRB). Methods: We performed a mutational analysis of SPN in human tumors, focusing on the region of interaction with PP1 and pRB. We explored the effect of the SPN-A566V mutation in an immortalized non-tumorigenic cell line of epithelial breast tissue, MCF10A, and in two different p53-mutated breast cancer cells lines, T47D and MDA-MB-468. Results: We characterized an oncogenic mutation of SPN found in human tumor samples, SPN-A566V, that affects both the SPN-PP1 interaction and its phosphatase activity. The SPN-A566V mutation does not affect the interaction of the PP1-SPN holoenzyme with pocket proteins pRB, p107 and p130, but it affects its ability to dephosphorylate them during G0/G1 and G1, indicating that the PP1-SPN holoenzyme regulates cell cycle progression. SPN-A566V also promoted stemness, establishing a connection between the cell cycle and stem cell biology via pocket proteins and PP1-SPN regulation. However, only cells with both SPN-A566V and mutant p53 have increased tumorigenic and stemness properties. Conclusions: SPN-A566V, or other equivalent mutations, could be late events that promote tumor progression by increasing the CSC pool and, eventually, the malignant behavior of the tumor.
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15
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Rehman SK, Haynes J, Collignon E, Brown KR, Wang Y, Nixon AML, Bruce JP, Wintersinger JA, Singh Mer A, Lo EBL, Leung C, Lima-Fernandes E, Pedley NM, Soares F, McGibbon S, He HH, Pollet A, Pugh TJ, Haibe-Kains B, Morris Q, Ramalho-Santos M, Goyal S, Moffat J, O'Brien CA. Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy. Cell 2021; 184:226-242.e21. [PMID: 33417860 PMCID: PMC8437243 DOI: 10.1016/j.cell.2020.11.018] [Citation(s) in RCA: 289] [Impact Index Per Article: 72.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Revised: 08/25/2020] [Accepted: 11/10/2020] [Indexed: 12/22/2022]
Abstract
Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.
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Affiliation(s)
- Sumaiyah K Rehman
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Jennifer Haynes
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Evelyne Collignon
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada
| | - Kevin R Brown
- Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada
| | - Yadong Wang
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Allison M L Nixon
- Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Jeffrey P Bruce
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Jeffrey A Wintersinger
- Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Computer Science, University of Toronto, Toronto, ON M5T 3A1, Canada; Vector Institute, Toronto, ON M5G 1M1, Canada
| | - Arvind Singh Mer
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Edwyn B L Lo
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Cherry Leung
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | | | - Nicholas M Pedley
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Fraser Soares
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada
| | - Sophie McGibbon
- Department of Physics, University of Toronto, Toronto, ON M5S 1A7, Canada
| | - Housheng Hansen He
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Aaron Pollet
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada
| | - Trevor J Pugh
- Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; Clinical Genomics Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2C1, Canada
| | - Benjamin Haibe-Kains
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Computer Science, University of Toronto, Toronto, ON M5T 3A1, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Quaid Morris
- Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Computer Science, University of Toronto, Toronto, ON M5T 3A1, Canada; Vector Institute, Toronto, ON M5G 1M1, Canada; Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Miguel Ramalho-Santos
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
| | - Sidhartha Goyal
- Department of Physics, University of Toronto, Toronto, ON M5S 1A7, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1, Canada.
| | - Jason Moffat
- Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3E1, Canada.
| | - Catherine A O'Brien
- Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Surgery, University Health Network, Toronto, ON M5G 1L7, Canada.
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Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin. Proc Natl Acad Sci U S A 2020; 117:33628-33638. [PMID: 33318192 PMCID: PMC7776986 DOI: 10.1073/pnas.2011780117] [Citation(s) in RCA: 88] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
As a genetic malignancy, retinoblastoma (Rb) is caused by RB1 mutations; however, its developmental origin and drug agents for human Rb remain largely unexplored. Here we describe an innovative Rb organoid model derived from human embryonic stem cells with a biallelic mutagenesis of the RB1 gene. We identify tumorigenic growth in the Rb organoids, as well as properties consistent with human primary Rb. We confirm that the Rb cell of origin stemmed from ARR3+ maturing cone precursor cells and SYK inhibitors displaying a significant therapeutic response. Our elegant in-dish Rb organoid model can be used to efficiently and effectively dissect the origin of Rb and mechanisms of Rb tumorigenesis, as well as screen novel therapies. Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate <30%. We have developed a cancerous model of Rb in retinal organoids derived from genetically engineered human embryonic stem cells (hESCs) with a biallelic mutagenesis of the RB1 gene. These organoid Rbs exhibit properties highly consistent with Rb tumorigenesis, transcriptome, and genome-wide methylation. Single-cell sequencing analysis suggests that Rb originated from ARR3-positive maturing cone precursors during development, which was further validated by immunostaining. Notably, we found that the PI3K-Akt pathway was aberrantly deregulated and its activator spleen tyrosine kinase (SYK) was significantly up-regulated. In addition, SYK inhibitors led to remarkable cell apoptosis in cancerous organoids. In conclusion, we have established an organoid Rb model derived from genetically engineered hESCs in a dish that has enabled us to trace the cell of origin and to test novel candidate therapeutic agents for human Rb, shedding light on the development and therapeutics of other malignancies.
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Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2. Proc Natl Acad Sci U S A 2020; 117:15673-15683. [PMID: 32571933 PMCID: PMC7355020 DOI: 10.1073/pnas.1922535117] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization of Rb1 -/- primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at the S18-2 promoter region and that the S18-2 expression is positively correlated with KLF4 levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.
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18
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Padgett J, Santos SDM. From clocks to dominoes: lessons on cell cycle remodelling from embryonic stem cells. FEBS Lett 2020; 594:2031-2045. [PMID: 32535913 DOI: 10.1002/1873-3468.13862] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 05/01/2020] [Accepted: 05/27/2020] [Indexed: 12/21/2022]
Abstract
Cell division is a fundamental cellular process and the evolutionarily conserved networks that control cell division cycles adapt during development, tissue regeneration, cell de-differentiation and reprogramming, and a variety of pathological conditions. Embryonic development is a prime example of such versatility: fast, clock-like divisions hallmarking embryonic cells at early developmental stages become slower and controlled during cellular differentiation and lineage specification. In this review, we compare and contrast the unique cell cycle of mouse and human embryonic stem cells with that of early embryonic cells and of differentiated cells. We propose that embryonic stem cells provide an extraordinarily useful model system to understand cell cycle remodelling during embryonic-to-somatic transitions. We discuss how cell cycle networks help sustain embryonic stem cell pluripotency and self-renewal and how they safeguard cell identity and proper cell number in differentiated cells. Finally, we highlight the incredible diversity in cell cycle regulation within mammals and discuss the implications of studying cell cycle remodelling for understanding healthy and disease states.
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Affiliation(s)
- Joe Padgett
- Quantitative Cell Biology Lab, The Francis Crick Institute, London, UK
| | - Silvia D M Santos
- Quantitative Cell Biology Lab, The Francis Crick Institute, London, UK
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19
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Kafer GR, Cesare AJ. A Survey of Essential Genome Stability Genes Reveals That Replication Stress Mitigation Is Critical for Peri-Implantation Embryogenesis. Front Cell Dev Biol 2020; 8:416. [PMID: 32548123 PMCID: PMC7274024 DOI: 10.3389/fcell.2020.00416] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Accepted: 05/05/2020] [Indexed: 12/16/2022] Open
Abstract
Murine development demands that pluripotent epiblast stem cells in the peri-implantation embryo increase from approximately 120 to 14,000 cells between embryonic days (E) 4.5 and E7.5. This is possible because epiblast stem cells can complete cell cycles in under 3 h in vivo. To ensure conceptus fitness, epiblast cells must undertake this proliferative feat while maintaining genome integrity. How epiblast cells maintain genome health under such an immense proliferation demand remains unclear. To illuminate the contribution of genome stability pathways to early mammalian development we systematically reviewed knockout mouse data from 347 DDR and repair associated genes. Cumulatively, the data indicate that while many DNA repair functions are dispensable in embryogenesis, genes encoding replication stress response and homology directed repair factors are essential specifically during the peri-implantation stage of early development. We discuss the significance of these findings in the context of the unique proliferative demands placed on pluripotent epiblast stem cells.
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Affiliation(s)
| | - Anthony J. Cesare
- Genome Integrity Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
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20
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Abstract
Thymus regenerative therapy implementation is severely obstructed by the limited number and expansion capacity in vitro of tissue-specific thymic epithelial stem cells (TESC). Current solutions are mostly based on growth factors that can drive differentiation of pluripotent stem cells toward tissue-specific TESC. Target-specific small chemical compounds represent an alternative solution that could induce and support the clonal expansion of TESC and reversibly block their differentiation into mature cells. These compounds could be used both in the composition of culture media designed for TESC expansion in vitro, and in drugs development for thymic regeneration in vivo. It should allow reaching the ultimate objective - autologous thymic tissue regeneration in paediatric patients who had their thymus removed in the course of cardiac surgery.
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21
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The cell cycle in stem cell proliferation, pluripotency and differentiation. Nat Cell Biol 2019; 21:1060-1067. [PMID: 31481793 DOI: 10.1038/s41556-019-0384-4] [Citation(s) in RCA: 255] [Impact Index Per Article: 42.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Accepted: 07/24/2019] [Indexed: 12/30/2022]
Abstract
Cyclins, cyclin-dependent kinases and other components of the core cell cycle machinery drive cell division. Growing evidence indicates that this machinery operates in a distinct fashion in some mammalian stem cell types, such as pluripotent embryonic stem cells. In this Review, we discuss our current knowledge of how cell cycle proteins mechanistically link cell proliferation, pluripotency and cell fate specification. We focus on embryonic stem cells, induced pluripotent stem cells and embryonic neural stem/progenitor cells.
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22
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Shcherbina A, Li J, Narayanan C, Greenleaf W, Kundaje A, Chetty S. Brief Report: Cell Cycle Dynamics of Human Pluripotent Stem Cells Primed for Differentiation. Stem Cells 2019; 37:1151-1157. [PMID: 31135093 PMCID: PMC6711778 DOI: 10.1002/stem.3041] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Revised: 04/26/2019] [Accepted: 05/14/2019] [Indexed: 01/09/2023]
Abstract
Understanding the molecular properties of the cell cycle of human pluripotent stem cells (hPSCs) is critical for effectively promoting differentiation. Here, we use the Fluorescence Ubiquitin Cell Cycle Indicator system adapted into hPSCs and perform RNA sequencing on cell cycle sorted hPSCs primed and unprimed for differentiation. Gene expression patterns of signaling factors and developmental regulators change in a cell cycle‐specific manner in cells primed for differentiation without altering genes associated with pluripotency. Furthermore, we identify an important role for PI3K signaling in regulating the early transitory states of hPSCs toward differentiation. stem cells2019;37:1151–1157
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Affiliation(s)
- Anna Shcherbina
- Department of Biomedical Informatics, Stanford University, Stanford, California, USA
| | - Jingling Li
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, USA
| | - Cyndhavi Narayanan
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, USA
| | - William Greenleaf
- Department of Genetics, Stanford University, Stanford, California, USA
| | - Anshul Kundaje
- Department of Genetics, Stanford University, Stanford, California, USA.,Department of Computer Science, Stanford University, Stanford, California, USA
| | - Sundari Chetty
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, USA.,Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA
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23
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Hatzistergos KE, Williams AR, Dykxhoorn D, Bellio MA, Yu W, Hare JM. Tumor Suppressors RB1 and CDKN2a Cooperatively Regulate Cell-Cycle Progression and Differentiation During Cardiomyocyte Development and Repair. Circ Res 2019; 124:1184-1197. [PMID: 30744497 DOI: 10.1161/circresaha.118.314063] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
RATIONALE Although rare cardiomyogenesis is reported in the adult mammalian heart, whether this results from differentiation or proliferation of cardiomyogenic cells remains controversial. The tumor suppressor genes RB1 (retinoblastoma) and CDKN2a (cyclin-dependent kinase inhibitor 2a) are critical cell-cycle regulators, but their roles in human cardiomyogenesis remains unclear. OBJECTIVE We hypothesized that developmental activation of RB1 and CDKN2a cooperatively cause permanent cell-cycle withdrawal of human cardiac precursors (CPCs) driving terminal differentiation into mature cardiomyocytes, and that dual inactivation of these tumor suppressor genes promotes myocyte cell-cycle reentry. METHODS AND RESULTS Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes revealed that RB1 and CDKN2a are upregulated at the onset of cardiac precursor specification, simultaneously with GATA4 (GATA-binding protein 4) homeobox genes PBX1 (pre-B-cell leukemia transcription factor 1) and MEIS1 (myeloid ecotropic viral integration site 1 homolog), and remain so until terminal cardiomyocyte differentiation. In both GATA4+ hPSC cardiac precursors and postmitotic hPSC-cardiomyocytes, RB1 is hyperphosphorylated and inactivated. Transient, stage-specific, depletion of RB1 during hPSC differentiation enhances cardiomyogenesis at the cardiac precursors stage, but not in terminally differentiated hPSC-cardiomyocytes, by transiently upregulating GATA4 expression through a cell-cycle regulatory pathway involving CDKN2a. Importantly, cytokinesis in postmitotic hPSC-cardiomyocytes can be induced with transient, dual RB1, and CDKN2a silencing. The relevance of this pathway in vivo was suggested by findings in a porcine model of cardiac cell therapy post-MI, whereby dual RB1 and CDKN2a inactivation in adult GATA4+ cells correlates with the degree of scar size reduction and endogenous cardiomyocyte mitosis, particularly in response to combined transendocardial injection of adult human hMSCs (bone marrow-derived mesenchymal stromal cells) and cKit+ cardiac cells. CONCLUSIONS Together these findings reveal an important and coordinated role for RB1 and CDKN2a in regulating cell-cycle progression and differentiation during human cardiomyogenesis. Moreover, transient, dual inactivation of RB1 and CDKN2a in endogenous adult GATA4+ cells and cardiomyocytes mediates, at least in part, the beneficial effects of cell-based therapy in a post-MI large mammalian model, a finding with potential clinical implications.
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Affiliation(s)
- Konstantinos E Hatzistergos
- From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL
- Department of Cell Biology (K.E.H.), University of Miami, Miller School of Medicine, FL
| | - Adam R Williams
- From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL
- Department of Surgery (A.R.W.), University of Miami, Miller School of Medicine, FL
- Department of Surgery, Duke University School of Medicine, Durham, NC (A.R.W.)
| | - Derek Dykxhoorn
- Department of Human Genetics (D.D.), University of Miami, Miller School of Medicine, FL
- John P. Hussman Institute for Human Genomics (D.D.), University of Miami, Miller School of Medicine, FL
| | - Michael A Bellio
- From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL
| | - Wendou Yu
- From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL
- Department of Pediatrics (W.Y.), University of Miami, Miller School of Medicine, FL
| | - Joshua M Hare
- From the Interdisciplinary Stem Cell Institute (K.E.H., A.R.W., M.A.B., W.Y., J.M.H.), University of Miami, Miller School of Medicine, FL
- Department of Molecular and Cellular Pharmacology (J.M.H.), University of Miami, Miller School of Medicine, FL
- Cardiology Division, Department of Medicine (J.M.H.), University of Miami, Miller School of Medicine, FL
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24
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Chen HJ, Poran A, Unni AM, Huang SX, Elemento O, Snoeck HW, Varmus H. Generation of pulmonary neuroendocrine cells and SCLC-like tumors from human embryonic stem cells. J Exp Med 2019; 216:674-687. [PMID: 30737256 PMCID: PMC6400536 DOI: 10.1084/jem.20181155] [Citation(s) in RCA: 70] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2018] [Revised: 10/04/2018] [Accepted: 12/17/2018] [Indexed: 12/23/2022] Open
Abstract
By blocking an important signaling pathway (called NOTCH) and interfering with expression of two tumor suppressor genes in cells derived from human embryonic stem cells, Chen et al. have developed a model for studying small cell lung cancers. Cancer models based on cells derived from human embryonic stem cells (hESCs) may reveal why certain constellations of genetic changes drive carcinogenesis in specialized lineages. Here we demonstrate that inhibition of NOTCH signaling induces up to 10% of lung progenitor cells to form pulmonary neuroendocrine cells (PNECs), putative precursors to small cell lung cancers (SCLCs), and we can increase PNECs by reducing levels of retinoblastoma (RB) proteins with inhibitory RNA. Reducing levels of TP53 protein or expressing mutant KRAS or EGFR genes did not induce or expand PNECs, but tumors resembling early-stage SCLC grew in immunodeficient mice after subcutaneous injection of PNEC-containing cultures in which expression of both RB and TP53 was blocked. Single-cell RNA profiles of PNECs are heterogeneous; when RB levels are reduced, the profiles resemble those from early-stage SCLC; and when both RB and TP53 levels are reduced, the transcriptome is enriched with cell cycle–specific RNAs. Our findings suggest that genetic manipulation of hESC-derived pulmonary cells will enable studies of this recalcitrant cancer.
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Affiliation(s)
| | - Asaf Poran
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY.,Caryl and Israel Englander Institute for Precision Medicine and Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY
| | - Arun M Unni
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY
| | - Sarah Xuelian Huang
- Columbia Center for Human Development, Department of Medicine, Columbia University Irving Medical Center, New York, NY.,Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX
| | - Olivier Elemento
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY.,Caryl and Israel Englander Institute for Precision Medicine and Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY
| | - Hans-Willem Snoeck
- Columbia Center for Human Development, Department of Medicine, Columbia University Irving Medical Center, New York, NY
| | - Harold Varmus
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY
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25
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Fabbrizi MR, Warshowsky KE, Zobel CL, Hallahan DE, Sharma GG. Molecular and epigenetic regulatory mechanisms of normal stem cell radiosensitivity. Cell Death Discov 2018; 4:117. [PMID: 30588339 PMCID: PMC6299079 DOI: 10.1038/s41420-018-0132-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 11/01/2018] [Accepted: 11/20/2018] [Indexed: 12/14/2022] Open
Abstract
Ionizing radiation (IR) therapy is a major cancer treatment modality and an indispensable auxiliary treatment for primary and metastatic cancers, but invariably results in debilitating organ dysfunctions. IR-induced depletion of neural stem/progenitor cells in the subgranular zone of the dentate gyrus in the hippocampus where neurogenesis occurs is considered largely responsible for deficiencies such as learning, memory, and spatial information processing in patients subjected to cranial irradiation. Similarly, IR therapy-induced intestinal injuries such as diarrhea and malabsorption are common side effects in patients with gastrointestinal tumors and are believed to be caused by intestinal stem cell drop out. Hematopoietic stem cell transplantation is currently used to reinstate blood production in leukemia patients and pre-clinical treatments show promising results in other organs such as the skin and kidney, but ethical issues and logistic problems make this route difficult to follow. An alternative way to restore the injured tissue is to preserve the stem cell pool located in that specific tissue/organ niche, but stem cell response to ionizing radiation is inadequately understood at the molecular mechanistic level. Although embryonic and fetal hypersensity to IR has been very well known for many decades, research on embryonic stem cell models in culture concerning molecular mechanisms have been largely inconclusive and often in contradiction of the in vivo observations. This review will summarize the latest discoveries on stem cell radiosensitivity, highlighting the possible molecular and epigenetic mechanism(s) involved in DNA damage response and programmed cell death after ionizing radiation therapy specific to normal stem cells. Finally, we will analyze the possible contribution of stem cell-specific chromatin's epigenetic constitution in promoting normal stem cell radiosensitivity.
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Affiliation(s)
- Maria Rita Fabbrizi
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108 USA
| | - Kacie E. Warshowsky
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108 USA
| | - Cheri L. Zobel
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108 USA
| | - Dennis E. Hallahan
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108 USA
- Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO 63108 USA
| | - Girdhar G. Sharma
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108 USA
- Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO 63108 USA
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26
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Li J, Narayanan C, Bian J, Sambo D, Brickler T, Zhang W, Chetty S. A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family. PLoS One 2018; 13:e0208110. [PMID: 30540809 PMCID: PMC6291069 DOI: 10.1371/journal.pone.0208110] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Accepted: 11/12/2018] [Indexed: 01/01/2023] Open
Abstract
The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO’s capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.
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Affiliation(s)
- Jingling Li
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Cyndhavi Narayanan
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Jing Bian
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Danielle Sambo
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Thomas Brickler
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Wancong Zhang
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
| | - Sundari Chetty
- Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, United States of America
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, United States of America
- * E-mail:
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27
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Abstract
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.
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Affiliation(s)
- Michelle M J Mens
- Department of Epidemiology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands
| | - Mohsen Ghanbari
- Department of Epidemiology, Erasmus University Medical Center, P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands. .,Department of Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
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28
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Edwards NA, Watson AJ, Betts DH. Knockdown of p66Shc Alters Lineage-Associated Transcription Factor Expression in Mouse Blastocysts. Stem Cells Dev 2018; 27:1479-1493. [PMID: 30091687 PMCID: PMC6209429 DOI: 10.1089/scd.2018.0131] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2018] [Accepted: 08/08/2018] [Indexed: 11/12/2022] Open
Abstract
The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM.
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Affiliation(s)
- Nicole A. Edwards
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada
| | - Andrew J. Watson
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada
- Department of Obstetrics and Gynecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada
- The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, Canada
| | - Dean H. Betts
- Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada
- Department of Obstetrics and Gynecology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada
- The Children's Health Research Institute (CHRI), Lawson Health Research Institute, London, Canada
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29
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Abstract
The canonical model of RB-mediated tumour suppression developed over the past 30 years is based on the regulation of E2F transcription factors to restrict cell cycle progression. Several additional functions have been proposed for RB, on the basis of which a non-canonical RB pathway can be described. Mechanistically, the non-canonical RB pathway promotes histone modification and regulates chromosome structure in a manner distinct from cell cycle regulation. These functions have implications for chemotherapy response and resistance to targeted anticancer agents. This Opinion offers a framework to guide future studies of RB in basic and clinical research.
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Affiliation(s)
- Frederick A Dick
- London Regional Cancer Program, Children's Health Research Institute, Western University, London, Ontario, Canada.
- London Regional Cancer Program, Department of Biochemistry, Western University, London, Ontario, Canada.
| | - David W Goodrich
- Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA
| | - Julien Sage
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Nicholas J Dyson
- Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Harvard Medical School, Charlestown, MA, USA
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30
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Zaveri L, Dhawan J. Cycling to Meet Fate: Connecting Pluripotency to the Cell Cycle. Front Cell Dev Biol 2018; 6:57. [PMID: 29974052 PMCID: PMC6020794 DOI: 10.3389/fcell.2018.00057] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Accepted: 05/14/2018] [Indexed: 01/26/2023] Open
Abstract
Pluripotent stem cells are characterized by their high proliferative rates, their ability to self-renew and their potential to differentiate to all the three germ layers. This rapid proliferation is brought about by a highly modified cell cycle that allows the cells to quickly shuttle from DNA synthesis to cell division, by reducing the time spent in the intervening gap phases. Many key regulators that define the somatic cell cycle are either absent or exhibit altered behavior, allowing the pluripotent cell to bypass cell cycle checkpoints typical of somatic cells. Experimental analysis of this modified stem cell cycle has been challenging due to the strong link between rapid proliferation and pluripotency, since perturbations to the cell cycle or pluripotency factors result in differentiation. Despite these hurdles, our understanding of this unique cell cycle has greatly improved over the past decade, in part because of the availability of new technologies that permit the analysis of single cells in heterogeneous populations. This review aims to highlight some of the recent discoveries in this area with a special emphasis on different states of pluripotency. We also discuss the highly interlinked network that connects pluripotency factors and key cell cycle genes and review evidence for how this interdependency may promote the rapid cell cycle. This issue gains translational importance since disruptions in stem cell proliferation and differentiation can impact disorders at opposite ends of a spectrum, from cancer to degenerative disease.
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Affiliation(s)
- Lamuk Zaveri
- Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India.,CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India.,Manipal Academy of Higher Education, Manipal, India
| | - Jyotsna Dhawan
- Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India.,CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India
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31
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Yang S, Zhu Z, Zhang X, Zhang N, Yao Z. Idelalisib induces PUMA-dependent apoptosis in colon cancer cells. Oncotarget 2018; 8:6102-6113. [PMID: 28008149 PMCID: PMC5351616 DOI: 10.18632/oncotarget.14043] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 12/12/2016] [Indexed: 01/22/2023] Open
Abstract
Idelalisib, a PI3K inhibitor, specifically targeting p110δ, has been approved for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma and follicular lymphoma. However, the mechanisms of action of idelalisib in colon cancer cells are not well understood. We investigated how idelalisib suppresses colon cancer cells growth and potentiates effects of other chemotherapeutic drugs. In this study, we found that idelalisib treatment induces PUMA in colon cancer cells irrespective of p53 status through the p65 pathway following AKT inhibition and glycogen synthase kinase 3β (GSK3β) activation. PUMA is necessary for idelalisib-induced apoptosis in colon cancer cells. Idelalisib also synergized with 5-FU or regorafenib to induce marked apoptosis via PUMA in colon cancer cells. Furthermore, PUMA deficiency suppressed apoptosis and antitumor effect of idelalisib in xenograft model. These results demonstrate a critical role of PUMA in mediating the anticancer effects of idelalisib in colon cancer cells and suggest that PUMA induction can be used as an indicator of idelalisib sensitivity, and also have important implications for it clinical applications.
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Affiliation(s)
- Shida Yang
- Department of Laboratory Medicine, The People's Hospital of Liaoning Province, Shenyang, China
| | - Zhiyong Zhu
- Department of Orthopedics, The People's Hospital of Liaoning Province, Shenyang, China
| | - Xiaobing Zhang
- Department of Laboratory Medicine, The People's Hospital of Liaoning Province, Shenyang, China
| | - Ning Zhang
- Department of Laboratory Medicine, The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, China
| | - Zhicheng Yao
- Department of Neurology, The People's Hospital of Liaoning Province, Shenyang, China
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32
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She S, Wei Q, Kang B, Wang YJ. Cell cycle and pluripotency: Convergence on octamer‑binding transcription factor 4 (Review). Mol Med Rep 2017; 16:6459-6466. [PMID: 28901500 PMCID: PMC5865814 DOI: 10.3892/mmr.2017.7489] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2016] [Accepted: 07/14/2017] [Indexed: 12/14/2022] Open
Abstract
Embryonic stem cells (ESCs) have unlimited expansion potential and the ability to differentiate into all somatic cell types for regenerative medicine and disease model studies. Octamer-binding transcription factor 4 (OCT4), encoded by the POU domain, class 5, transcription factor 1 gene, is a transcription factor vital for maintaining ESC pluripotency and somatic reprogramming. Many studies have established that the cell cycle of ESCs is featured with an abbreviated G1 phase and a prolonged S phase. Changes in cell cycle dynamics are intimately associated with the state of ESC pluripotency, and manipulating cell-cycle regulators could enable a controlled differentiation of ESCs. The present review focused primarily on the emerging roles of OCT4 in coordinating the cell cycle progression, the maintenance of pluripotency and the glycolytic metabolism in ESCs.
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Affiliation(s)
- Shiqi She
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Qucheng Wei
- Cardiovascular Key Lab of Zhejiang, Department of Cardiology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China
| | - Bo Kang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
| | - Ying-Jie Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China
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33
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Kitajima S, Takahashi C. Intersection of retinoblastoma tumor suppressor function, stem cells, metabolism, and inflammation. Cancer Sci 2017; 108:1726-1731. [PMID: 28865172 PMCID: PMC5581511 DOI: 10.1111/cas.13312] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Revised: 06/28/2017] [Accepted: 06/30/2017] [Indexed: 12/27/2022] Open
Abstract
The Retinoblastoma (RB) tumor suppressor regulates G1/S transition during cell cycle progression by modulating the activity of E2F transcription factors. The RB pathway plays a central role in the suppression of most cancers, and RB mutation was initially discovered by virtue of its role in tumor initiation. However, as cancer genome sequencing has evolved to profile more advanced and treatment‐resistant cancers, it has become increasingly clear that, in the majority of cancers, somatic RB inactivation occurs during tumor progression. Furthermore, despite the presence of deregulation of cell cycle control due to an INK4A deletion, additional CCND amplification and/or other mutations in the RB pathway, mutation or deletion of the RB gene is often observed during cancer progression. Of note, RB inactivation during cancer progression not only facilitates G1/S transition but also enhances some characteristics of malignancy, including altered drug sensitivity and a return to the undifferentiated state. Recently, we reported that RB inactivation enhances pro‐inflammatory signaling through stimulation of the interleukin‐6/STAT3 pathway, which directly promotes various malignant features of cancer cells. In this review, we highlight the consequences of RB inactivation during cancer progression, and discuss the biological and pathological significance of the interaction between RB and pro‐inflammatory signaling.
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Affiliation(s)
- Shunsuke Kitajima
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
| | - Chiaki Takahashi
- Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, Japan
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34
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Mello SS, Sinow C, Raj N, Mazur PK, Bieging-Rolett K, Broz DK, Imam JFC, Vogel H, Wood LD, Sage J, Hirose T, Nakagawa S, Rinn J, Attardi LD. Neat1 is a p53-inducible lincRNA essential for transformation suppression. Genes Dev 2017; 31:1095-1108. [PMID: 28698299 PMCID: PMC5538433 DOI: 10.1101/gad.284661.116] [Citation(s) in RCA: 171] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2016] [Accepted: 05/26/2017] [Indexed: 12/12/2022]
Abstract
Mello et al. identify Neat1, a ncRNA constituent of paraspeckles, as a p53 target gene that plays a crucial role in suppressing transformation in response to oncogenic signals. The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1−/− mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in KrasG12D-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.
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Affiliation(s)
- Stephano S Mello
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Carolyn Sinow
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Nitin Raj
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Pawel K Mazur
- Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Kathryn Bieging-Rolett
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Daniela Kenzelmann Broz
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Jamie F Conklin Imam
- Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA.,Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Hannes Vogel
- Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Laura D Wood
- Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA
| | - Julien Sage
- Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA.,Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
| | - Tetsuro Hirose
- Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
| | - Shinichi Nakagawa
- RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
| | - John Rinn
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA
| | - Laura D Attardi
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305, USA.,Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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35
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Avior Y, Lezmi E, Yanuka D, Benvenisty N. Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells. Stem Cell Reports 2017; 8:1354-1365. [PMID: 28392220 PMCID: PMC5425613 DOI: 10.1016/j.stemcr.2017.03.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2016] [Revised: 03/06/2017] [Accepted: 03/07/2017] [Indexed: 12/18/2022] Open
Abstract
Human embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1−/− hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1−/− cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1–/–-specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1.
RB1-null hESCs were generated using CRISPR/Cas9 RB1−/− hESCs generate large, neural-enriched teratomas, possibly by ZEB1 activation RB1 inactivation triggers aberrant mitochondrial abundance and function Unbiased drug screening found that carboplatin specifically targets RB1-null cells
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Affiliation(s)
- Yishai Avior
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Dorit Yanuka
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel.
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36
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Pluripotency of embryo-derived stem cells from rodents, lagomorphs, and primates: Slippery slope, terrace and cliff. Stem Cell Res 2017; 19:104-112. [DOI: 10.1016/j.scr.2017.01.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Revised: 01/01/2017] [Accepted: 01/13/2017] [Indexed: 12/14/2022] Open
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Matsui T, Nieto-Estévez V, Kyrychenko S, Schneider JW, Hsieh J. Retinoblastoma protein controls growth, survival and neuronal migration in human cerebral organoids. Development 2017; 144:1025-1034. [PMID: 28087635 DOI: 10.1242/dev.143636] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2016] [Accepted: 12/16/2016] [Indexed: 01/22/2023]
Abstract
The tumor suppressor retinoblastoma protein (RB) regulates S-phase cell cycle entry via E2F transcription factors. Knockout (KO) mice have shown that RB plays roles in cell migration, differentiation and apoptosis, in developing and adult brain. In addition, the RB family is required for self-renewal and survival of human embryonic stem cells (hESCs). Since little is known about the role of RB in human brain development, we investigated its function in cerebral organoids differentiated from gene-edited hESCs lacking RB. We show that RB is abundantly expressed in neural stem and progenitor cells in organoids at 15 and 28 days of culture. RB loss promoted S-phase entry in DCX+ cells and increased apoptosis in Sox2+ neural stem and progenitor cells, and in DCX+ and Tuj1+ neurons. Associated with these cell cycle and pro-apoptotic effects, we observed increased CCNA2 and BAX gene expression, respectively. Moreover, we observed aberrant Tuj1+ neuronal migration in RB-KO organoids and upregulation of the gene encoding VLDLR, a receptor important in reelin signaling. Corroborating the results in RB-KO organoids in vitro, we observed ectopically localized Tuj1+ cells in RB-KO teratomas grown in vivo Taken together, these results identify crucial functions for RB in the cerebral organoid model of human brain development.
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Affiliation(s)
- Takeshi Matsui
- Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Vanesa Nieto-Estévez
- Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Sergii Kyrychenko
- Department of Internal Medicine and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jay W Schneider
- Department of Internal Medicine and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jenny Hsieh
- Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA
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38
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He H, Wang C, Dai Q, Li F, Bergholz J, Li Z, Li Q, Xiao ZX. p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation. Stem Cell Reports 2016; 7:1087-1098. [PMID: 27866875 PMCID: PMC5161534 DOI: 10.1016/j.stemcr.2016.10.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2016] [Revised: 10/21/2016] [Accepted: 10/24/2016] [Indexed: 01/23/2023] Open
Abstract
Embryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.
p53/p73 are key for DNA damage-induced apoptosis but not G2/M arrest in mESCs Both p53 and p73 are required for differentiation-induced apoptosis in mESCs Doxorubicin induces RB via p53-mediated suppression of miR-17-92 and miR-106a-363 p73 expression is induced upon differentiation in mESCs
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Affiliation(s)
- Hanbing He
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Cheng Wang
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qian Dai
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China
| | - Fengtian Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Johann Bergholz
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Zhonghan Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qintong Li
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China.
| | - Zhi-Xiong Xiao
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China.
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Abstract
The E2F family of transcription factors is a key determinant of cell proliferation in response to extra- and intra-cellular signals. Within this family, E2F4 is a transcriptional repressor whose activity is critical to engage and maintain cell cycle arrest in G0/G1 in conjunction with members of the retinoblastoma (RB) family. However, recent observations challenge this paradigm and indicate that E2F4 has a multitude of functions in cells besides this cell cycle regulatory role, including in embryonic and adult stem cells, during regenerative processes, and in cancer. Some of these new functions are independent of the RB family and involve direct activation of target genes. Here we review the canonical functions of E2F4 and discuss recent evidence expanding the role of this transcription factor, with a focus on cell fate decisions in tissue homeostasis and regeneration.
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Affiliation(s)
- Jenny Hsu
- a Departments of Pediatrics and Genetics , Stanford University , Stanford , CA , USA
| | - Julien Sage
- a Departments of Pediatrics and Genetics , Stanford University , Stanford , CA , USA
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40
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Wang XQ, Lo CM, Chen L, Ngan ESW, Xu A, Poon RY. CDK1-PDK1-PI3K/Akt signaling pathway regulates embryonic and induced pluripotency. Cell Death Differ 2016; 24:38-48. [PMID: 27636107 PMCID: PMC5260505 DOI: 10.1038/cdd.2016.84] [Citation(s) in RCA: 81] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2016] [Revised: 07/18/2016] [Accepted: 07/19/2016] [Indexed: 02/08/2023] Open
Abstract
The mechanisms of how signaling pathways are coordinated and integrated for the
maintenance of the self-renewal of human embryonic stem cells (hESCs) and the
acquisition of pluripotency in reprogramming are still only partly understood.
CDK1 is a key regulator of mitosis. Recently, CDK1 has been shown to be involved
in regulating self-renewal of stem cells, even though the mechanistic role of
how CDK1 regulates pluripotency is unknown. In this report, we aim to understand
how CDK1 can control pluripotency by reducing CDK1 activity to a level that has
no effect on cell cycle progression. We demonstrated that high levels of CDK1 is
associated with the pluripotency stage of hESCs; and decreased CDK1 activity to
a level without perturbing the cell cycle is sufficient to induce
differentiation. CDK1 specifically targets the phosphorylation of PDK1 and
consequently the activity of PI3K/Akt and its effectors ERK and
GSK3β. Evidence of the reversion of inactive CDK1-mediated
differentiation by the inhibition of Akt signaling effectors suggests that the
CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the
pluripotency of hESCs. Moreover, cyclin B1-CDK1 complexes promote somatic
reprogramming efficiency, probably by regulating the maturation of induced
pluripotent stem cells (iPSCs), as cyclin B1 stimulates a higher cellular level
of LIN28A, suggesting that monitoring iPSC factors could be a new path for the
enhancement of reprogramming efficiency. Together, we demonstrate an essential
role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation
of self-renewal, differentiation, and somatic reprogramming, which provides a
novel kinase cascade mechanism for pluripotency control and acquisition.
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Affiliation(s)
- Xiao Qi Wang
- Department of Surgery, The University of Hong Kong, Hong Kong, China.,State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
| | - Chung Mau Lo
- Department of Surgery, The University of Hong Kong, Hong Kong, China
| | - Lin Chen
- Department of Surgery, The University of Hong Kong, Hong Kong, China
| | - Elly S-W Ngan
- Department of Surgery, The University of Hong Kong, Hong Kong, China
| | - Aimin Xu
- Department of Medicine, The University of Hong Kong, Hong Kong, China
| | - Randy Yc Poon
- Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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41
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An integrative approach predicted co-expression sub-networks regulating properties of stem cells and differentiation. Comput Biol Chem 2016; 64:250-262. [PMID: 27475236 DOI: 10.1016/j.compbiolchem.2016.07.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2016] [Revised: 06/22/2016] [Accepted: 07/15/2016] [Indexed: 12/18/2022]
Abstract
The differentiation of human Embryonic Stem Cells (hESCs) is accompanied by the formation of different intermediary cells, gradually losing its stemness and acquiring differentiation. The precise mechanisms underlying hESCs integrity and its differentiation into fibroblast (Fib) are still elusive. Here, we aimed to assess important genes and co-expression sub-networks responsible for stemness, early differentiation of hESCs into embryoid bodies (EBs) and its lineage specification into Fibs. To achieve this, we compared transcriptional profiles of hESCs-EBs and EBs-Fibs and obtained differentially expressed genes (DEGs) exclusive to hESCs-EBs (early differentiation), EBs-Fibs (late differentiation) and common DEGs in hESCs-EBs and EBs-Fibs. Then, we performed gene set enrichment analysis (GSEA) followed by overrepresentation study and identified key genes for each gene category. The regulations of these genes were studied by integrating ChIP-Seq data of core transcription factors (TFs) and histone methylation marks in hESCs. Finally, we identified co-expression sub-networks from key genes of each gene category using k-clique sub-network extraction method. Our study predicted seven genes edicting core stemness properties forming a co-expression network. From the pathway analysis of sub-networks of hESCs-EBs, we hypothesize that FGF2 is contributing to pluripotent transcription network of hESCs in association with DNMT3B and JARID2 thereby facilitating cell proliferation. On the contrary, FGF2 is found to promote cell migration in Fibs along with DDR2, CAV1, DAB2, and PARVA. Moreover, our study identified three k-clique sub-networks regulating TGF-β signaling pathway thereby promoting EBs to Fibs differentiation by: (i) modulating extracellular matrix involving ITGB1, TGFB1I1 and GBP1, (ii) regulating cell cycle remodeling involving CDKN1A, JUNB and DUSP1 and (iii) helping in epithelial to mesenchymal transition (EMT) involving THBS1, INHBA and LOX. This study put forward the unexplored genes and co-expression sub-networks regulating stemness and different stages of differentiation of hESCs which will undoubtedly add to the comprehensive understanding of hESCs biology.
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42
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Kramer N, Rosner M, Kovacic B, Hengstschläger M. Full biological characterization of human pluripotent stem cells will open the door to translational research. Arch Toxicol 2016; 90:2173-2186. [PMID: 27325309 DOI: 10.1007/s00204-016-1763-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2016] [Accepted: 06/13/2016] [Indexed: 12/13/2022]
Abstract
Since the discovery of human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), great hopes were held for their therapeutic application including disease modeling, drug discovery screenings, toxicological screenings and regenerative therapy. hESC and hiPSC have the advantage of indefinite self-renewal, thereby generating an inexhaustible pool of cells with, e.g., specific genotype for developing putative treatments; they can differentiate into derivatives of all three germ layers enabling autologous transplantation, and via donor-selection they can express various genotypes of interest for better disease modeling. Furthermore, drug screenings and toxicological screenings in hESC and hiPSC are more pertinent to identify drugs or chemical compounds that are harmful for human, than a mouse model could predict. Despite continuing research in the wide field of therapeutic applications, further understanding of the underlying basic mechanisms of stem cell function is necessary. Here, we summarize current knowledge concerning pluripotency, self-renewal, apoptosis, motility, epithelial-to-mesenchymal transition and differentiation of pluripotent stem cells.
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Affiliation(s)
- Nina Kramer
- Institute of Medical Genetics, Medical University of Vienna, Währingerstrasse 10, 1090, Vienna, Austria
| | - Margit Rosner
- Institute of Medical Genetics, Medical University of Vienna, Währingerstrasse 10, 1090, Vienna, Austria
| | - Boris Kovacic
- Institute of Medical Genetics, Medical University of Vienna, Währingerstrasse 10, 1090, Vienna, Austria
| | - Markus Hengstschläger
- Institute of Medical Genetics, Medical University of Vienna, Währingerstrasse 10, 1090, Vienna, Austria.
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Abstract
Human retinoblastoma gene RB1 is the first tumor suppressor gene (TSG) isolated by positional cloning in 1986. RB is extensively studied for its ability to regulate cell cycle by binding to E2F1 and inhibiting the transcriptional activity of the latter. In human embryonic stem cells (ESCs), only a minute trace of RB is found in complex with E2F1. Increased activity of RB triggers differentiation, cell cycle arrest, and cell death. On the other hand, inactivation of the entire RB family (RB1, RBL1, and RBL2) in human ESC induces G2/M arrest and cell death. These observations indicate that both loss and overactivity of RB could be lethal for the stemness of cells. A question arises why inactive RB is required for the survival and stemness of cells? To shed some light on this question, we analyzed the RB-binding proteins. In this review we have focused on 27 RB-binding partners that may have potential roles in different aspects of stem cell biology.
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Affiliation(s)
- M Mushtaq
- Karolinska Institutet, Stockholm, Sweden
| | | | - E V Kashuba
- Karolinska Institutet, Stockholm, Sweden; R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU, Kyiv, Ukraine.
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44
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Pauklin S, Madrigal P, Bertero A, Vallier L. Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D. Genes Dev 2016; 30:421-33. [PMID: 26883361 PMCID: PMC4762427 DOI: 10.1101/gad.271452.115] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1-3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions.
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Affiliation(s)
- Siim Pauklin
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Madingley, Cambridge CB2 0SZ, United Kingdom
| | - Pedro Madrigal
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Madingley, Cambridge CB2 0SZ, United Kingdom; Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom
| | - Alessandro Bertero
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Madingley, Cambridge CB2 0SZ, United Kingdom
| | - Ludovic Vallier
- Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Madingley, Cambridge CB2 0SZ, United Kingdom; Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom
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45
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Kohno S, Kitajima S, Sasaki N, Takahashi C. Retinoblastoma tumor suppressor functions shared by stem cell and cancer cell strategies. World J Stem Cells 2016; 8:170-84. [PMID: 27114748 PMCID: PMC4835675 DOI: 10.4252/wjsc.v8.i4.170] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Revised: 12/30/2015] [Accepted: 02/14/2016] [Indexed: 02/06/2023] Open
Abstract
Carcinogenic transformation of somatic cells resembles nuclear reprogramming toward the generation of pluripotent stem cells. These events share eternal escape from cellular senescence, continuous self-renewal in limited but certain population of cells, and refractoriness to terminal differentiation while maintaining the potential to differentiate into cells of one or multiple lineages. As represented by several oncogenes those appeared to be first keys to pluripotency, carcinogenesis and nuclear reprogramming seem to share a number of core mechanisms. The retinoblastoma tumor suppressor product retinoblastoma (RB) seems to be critically involved in both events in highly complicated manners. However, disentangling such complicated interactions has enabled us to better understand how stem cell strategies are shared by cancer cells. This review covers recent findings on RB functions related to stem cells and stem cell-like behaviors of cancer cells.
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Affiliation(s)
- Susumu Kohno
- Susumu Kohno, Chiaki Takahashi, Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
| | - Shunsuke Kitajima
- Susumu Kohno, Chiaki Takahashi, Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
| | - Nobunari Sasaki
- Susumu Kohno, Chiaki Takahashi, Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
| | - Chiaki Takahashi
- Susumu Kohno, Chiaki Takahashi, Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
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46
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Kitajima S, Kohno S, Kondoh A, Sasaki N, Nishimoto Y, Li F, Abdallah Mohammed MS, Muranaka H, Nagatani N, Suzuki M, Kido Y, Takahashi C. Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts. Stem Cells 2016; 33:1657-69. [PMID: 25694388 DOI: 10.1002/stem.1971] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2014] [Accepted: 01/14/2015] [Indexed: 01/08/2023]
Abstract
Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.
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Affiliation(s)
- Shunsuke Kitajima
- Division of Oncology and Molecular Biology, Stem Cell Research Program, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, Japan
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47
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Peiris TH, Ramirez D, Barghouth PG, Ofoha U, Davidian D, Weckerle F, Oviedo NJ. Regional signals in the planarian body guide stem cell fate in the presence of genomic instability. Development 2016; 143:1697-709. [PMID: 27013241 DOI: 10.1242/dev.131318] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2015] [Accepted: 03/10/2016] [Indexed: 12/28/2022]
Abstract
Cellular fate decisions are influenced by their topographical location in the adult body. For instance, tissue repair and neoplastic growth are greater in anterior than in posterior regions of adult animals. However, the molecular underpinnings of these regional differences are unknown. We identified a regional switch in the adult planarian body upon systemic disruption of homologous recombination with RNA-interference of Rad51 Rad51 knockdown increases DNA double-strand breaks (DSBs) throughout the body, but stem cells react differently depending on their location along the anteroposterior axis. In the presence of extensive DSBs, cells in the anterior part of the body resist death, whereas cells in the posterior region undergo apoptosis. Furthermore, we found that proliferation of cells with DNA damage is induced in the presence of brain tissue and that the retinoblastoma pathway enables overproliferation of cells with DSBs while attending to the demands of tissue growth and repair. Our results implicate both autonomous and non-autonomous mechanisms as key mediators of regional cell behavior and cellular transformation in the adult body.
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Affiliation(s)
- T Harshani Peiris
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA
| | - Daniel Ramirez
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA
| | - Paul G Barghouth
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA
| | - Udokanma Ofoha
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA
| | - Devon Davidian
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA
| | - Frank Weckerle
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA
| | - Néstor J Oviedo
- Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA 95343, USA Quantitative and Systems Biology Graduate Program, University of California, Merced, CA 95343, USA Health Sciences Research Institute, University of California, Merced, CA 95343, USA
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Crosstalk between stem cell and cell cycle machineries. Curr Opin Cell Biol 2015; 37:68-74. [PMID: 26520682 DOI: 10.1016/j.ceb.2015.10.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Revised: 09/03/2015] [Accepted: 10/06/2015] [Indexed: 12/22/2022]
Abstract
Pluripotent stem cells, defined by an unlimited self-renewal capacity and an undifferentiated state, are best typified by embryonic stem cells. These cells have a unique cell cycle compared to somatic cells as defined by a rapid progression through the cell cycle and a minimal time spent in G1. Recent reports indicate that pluripotency and cell cycle regulation are mechanistically linked. In this review, we discuss the reciprocal co-regulation of these processes, how this co-regulation may prevent differentiation, and how cellular reprogramming can re-establish the unique cell cycle regulation in induced pluripotent stem cells.
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Tanaka T, Kanatsu-Shinohara M, Shinohara T. The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage. J Reprod Dev 2015; 61:305-16. [PMID: 25959802 PMCID: PMC4547988 DOI: 10.1262/jrd.2015-027] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Spermatogonial stem cells (SSCs) undergo self-renewal divisions to provide the foundation for spermatogenesis. Although Rb1 deficiency is reportedly essential for SSC self-renewal, its mechanism has remained unknown. Here we report that Rb1 is critical for cell cycle progression and protection of SSCs from DNA double-strand breaks (DSBs). Cultured SSCs depleted of Cdkn1b proliferated poorly and showed diminished expression of CDK4 and RB1, thereby leading to hypophosphorylation of RB1. Rb1 deficiency induced cell cycle arrest and apoptosis in cultured SSCs, which expressed markers for DNA DSBs. This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency. Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell
cycle arrest and apoptosis. These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.
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Affiliation(s)
- Takashi Tanaka
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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Julian LM, Blais A. Transcriptional control of stem cell fate by E2Fs and pocket proteins. Front Genet 2015; 6:161. [PMID: 25972892 PMCID: PMC4412126 DOI: 10.3389/fgene.2015.00161] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2014] [Accepted: 04/08/2015] [Indexed: 01/04/2023] Open
Abstract
E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs.
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Affiliation(s)
- Lisa M Julian
- Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON Canada
| | - Alexandre Blais
- Ottawa Institute of Systems Biology, Ottawa, ON Canada ; Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON Canada
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