DOCSLIB.ORG

LATS1 Tumor Suppressor Regulates G2/M Transition and Apoptosis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
LATS1 Tumor Suppressor Regulates G2/M Transition and Apoptosis Oncogene (2002) 21, 1233 ± 1241 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc LATS1 tumor suppressor regulates G2/M transition and apoptosis Hong Xia1, Huilin Qi1, Yunfang Li1, Jing Pei1, James Barton1, Mark Blackstad1, Tian Xu2 and Wufan Tao*,1 1Stem Cell Institute, Division of Hematology, Oncology and Transplantation, Cancer Center, University of Minnesota, Minneapolis, Minnesota, MN 55455, USA; 2Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, CT 06536, USA The LATS1 gene is a mammalian member of the novel proliferation and form large tumors in many tissues in lats tumor suppressor family. Both lats mosaic ¯ies and mosaic ¯ies. Homozygous mutants display a wide LATS1 de®cient mice spontaneously develop tumors. range of developmental defects including embryonic Our previous studies have shown that inactivation of lethality (Xu et al., 1995). Drosophila lats leads to up-regulation of cyclin A in the LATS1 (Tao et al., 1999) or Warts1 (Nishiyama et al., ¯y, and the human LATS1 protein associates with 1999) is a mammalian homolog of Drosophila lats. CDC2 in early mitosis in HeLa cells, suggesting that the Expression of the human LATS1 gene in Drosophila lats gene family may negatively regulate cell prolifera- rescues all developmental defects in homozygous Lats tion by modulating CDC2/Cyclin A activity. We mutants and suppresses tumor formation in lats mosaic demonstrate here that transduction of the human breast ¯ies (Tao et al., 1999). Lats1-de®cient female mice cancer cell MCF-7 with recombinant LATS1 adenovirus spontaneously develop large soft tissue sarcomas and (Ad-LATS1), but not with EGFP adenovirus (Ad- ovarian stromal cell tumors, demonstrating that Lats1 EGFP), inhibits in vitro cell proliferation. Ectopic is, at least in mice, a tumor suppressor (St. John et al., expression of LATS1 in MCF-7 cells speci®cally down- 1999). The human LATS1 gene has been mapped to regulates Cyclin A and Cyclin B protein levels and chromosome 6q24 ± 25 (Nishiyama et al., 1999) where dramatically reduces CDC2 kinase activity, leading to a loss of heterozygosity has been observed in ovarian G2/M blockade. Furthermore, Ad-LATS1 suppresses (Cooke et al., 1996; Lee et al., 1990), cervical anchorage-independent growth of MCF-7 cells in soft (Mazurenko et al., 1999), and breast cancers (Fujii et agar and tumor formation in athymic nude mice. We al., 1996; Theile et al., 1996). Mutation analyses on 25 also demonstrate that ectopic expression of LATS1 in breast cancer samples found no mutation in the LATS1 MCF-7 cells and human lung cancer cell H460 up- coding region, but it is still possible that LATS1 may be regulates the level of BAX proteins and induces inactivated in human breast tumors by other epigenetic apoptosis. Finally, we show that LATS1 kinase activity mechanisms such as methylation (Morinaga et al., is required for its ability to inhibit cell growth and induce 2000). During mitosis LATS1 associates with zyxin, a apoptosis. The results indicate that the LATS1 tumor component of focal adhesion, in the mitotic apparatus. suppressor may play an important role in the control of Microinjection of a truncated human LATS1 protein human tumor development and that LATS1 suppresses (a.a 136 ± 700) interfered with the localization of zyxin tumorigenesis by negatively regulating cell proliferation to the mitotic apparatus and elongated mitosis (Hirota and modulating cell survival. et al., 2000). LATS2 (Yabuta et al., 2000), also known Oncogene (2002) 21, 1233 ± 1241. DOI: 10.1038/sj/ as KPM (Hori et al., 2000), is the second mammalian onc/1205174 member of the lats tumor suppressor gene family but the physiological function of LATS2 is unknown. Keywords: LATS1; tumor suppressor; G2/M transi- Although both Drosophila lats and mouse Lats1 tion; apoptosis suppress tumor formation in fruit ¯ies and mice, respectively, the role of LATS1 in human tumorigen- esis is not yet clear, nor are the mechanism(s) by which Introduction LATS1 controls tumor development. Our previous studies showed that loss of function of lats in lats (also known as warts), a novel tumor suppressor Drosophila results in up-regulation of Cyclin A and family, encodes a class of putative Ser/Thr protein that LATS1-associated CDC2 lacks kinase activity in kinases. The lats gene was ®rst identi®ed as a tumor HeLa cells (Tao et al., 1999; Turenchalk et al., 1999), suppressor in Drosophila (Justice et al., 1995; Xu et al., suggesting that the lats gene family may control tumor 1995). Somatic cell mutants for lats undergo extensive development by negatively regulating cell proliferation. Here, we further demonstrate that LATS1 inhibits cell proliferation by blocking the G2/M transition. Our studies reveal that the ectopic expression of LATS1 in *Correspondence: W Tao; E-mail: [email protected] Received 31 July 2001; revised 2 November 2001; accepted 7 the human breast cancer cell line, MCF-7, speci®cally November 2001 down-regulates mitotic cyclins and CDC2/Cyclin LATS1, tumor suppressor, G2/M arrest, apoptosis H Xia et al 1234 kinase activity. Our results also show that the Ad- cancer cell line, MCF-7, was infected with either Ad- LATS1-transduced MCF-7 cell lose tumorigenicity. In LATS1 or control virus Ad-EGFP. Anchorage-inde- addition, we show that ectopic expression of LATS1 in pendent growth of MCF-7 cells expressing exogenous MCF-7 cells and human lung cancer cell H460 up- LATS1 was measured in soft agar and tumor regulates the level of BAX proteins and induces development in nude mice was examined. As shown apoptosis. Finally, we demonstrate that LATS1 kinase in Figure 2a, Ad-LATS1 signi®cantly suppressed activity is required for its ability to inhibit cell growth colony growth of MCF-7 cells in soft agar as compared and induce apoptosis. to control virus Ad-EGFP. Six of six mice in which the Ad-EGFP-transduced cells were injected displayed tumor growth within 10 weeks. In contrast, six of six mice injected with Ad-LATS1 transduced cells were Results tumor free 10 weeks after inoculation. These studies demonstrate that ectopic expression of LATS1 sup- The expression of exogenous LATS1 gene in the human presses the anchorage-independent growth of MCF-7 breast cancer cell MCF-7 cells in soft agar and tumorigenicity of MCF-7 cells in To express the human LATS1 gene in MCF-7 cells and vivo, indicating the LATS1 may play a role in human easily monitor the cells expressing LATS1,we tumor development control. generated one control adenovirus, Ad-EGFP, and three recombinant adenoviruses which express LATS1 and Expression of LATS1 inhibits the growth of MCF7 cells its mutants by modifying a pre-existing adenovirus and blocks cells at G2/M expression system (Figure 1a, also see Materials and methods). The LATS1 and EGFP genes are expressed The eects of ectopic expression of LATS1 on cell as a bicistron, separated by IRES. The infection growth were examined to investigate the mechanism(s) eciency of the adenovirus can be easily monitored by which the expression of LATS1 suppresses tumor- by EGFP, and transduced cells can be enriched by igenicity of MCF-7 cells. The growth curve of Ad- FACS for analysis. Expression of the endogenous LATS1 protein in MCF-7 cells is very low and can only be detected by immunoprecipitation/Western blot with anti-LATS1 antibodies (lanes 1 and 2, Figure 1b). However, the exogenous human LATS1 protein is easily detected by direct Western blot in Ad-LATS1- transduced MCF-7 cells (lane 4, Figure 1b). LATS1 suppresses tumorigenicity of MCF-7 cells To test whether expression of LATS1 suppresses tumorigenicity of human tumor cells, the human breast Figure 2 LATS1 suppresses the tumorigenicity of MCF-7 cells and inhibits cell proliferation. (a) Lats1 suppresses anchorage- independent growth of MCF-7 cells in soft agar. Colonies derived from cells infected by Ad-EGFP (left) or Ad-LATS1 (right) were grown for 5 weeks in the soft agar. (b) Growth inhibition assay. Figure 1 Expression of the LATS1 protein in MCF-7 cells. (a) 104 of Ad-LATS1-infected MCF-7 cells or control cells were The schematic drawing represents a newly modi®ed replication- plated in a 3.5 cm dish. Total cell numbers were counted daily for defective adenovirus (Ad-EGFP) and the recombinant adeno- 10 days. Experiments were repeated three times. (c) Cell cycle viruses that express human LATS1 and mutant LATS1.(b) pro®le analysis. Ad-LATS1-transduced (right) and control (left) Expression of LATS1 in MCF-7 cells. Endogenous LATS1 cells were harvested at days 3 post infection for DNA content proteins detected by Western blot (1) and IP-Western blot (2) analysis by FACS. The histogram presented is one of three and exogenous LATS1 proteins in MCF-7 cells infected by Ad- experimental results and the cell populations (percentages) in each LATS1 (4) and Ad-EGFP (3) detected by Western blot. IRES: cell cycle stage are the average of three experiments. PI: internal ribosome entry site; IP: immunoprecipitation propidium iodide Oncogene LATS1, tumor suppressor, G2/M arrest, apoptosis H Xia et al 1235 LATS1-transduced MCF-7 cells was compared with that of Ad-EGFP-transduced cells. We found that the number of Ad-EGFP-transduced MCF-7 cells in- creased almost threefold by day 10. In contrast, the total number of Ad-LATS1-transduced cells remained unchanged by day 10 (Figure 2b). This result suggests that the growth of Ad-LATS1-transdcued MCF-7 cells was strongly inhibited by the transiently expressed LATS1 protein. The eect of ectopic expression of LATS1 on the cell cycle progression of MCF-7 cells was then examined by FACS. As shown in Figure 2c, expression of LATS1 resulted in a dramatic decrease (from 59%+6 to 31%+3) in the G1 cell population and a signi®cant increase (from 27%+4 to 36%+4) in the G2/M cell population at day 3 post-virus infection.
Load more

Recommended publications
  • Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling
    G C A T T A C G G C A T genes Review Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling Aoife A. Nolan 1, Nourhan K. Aboud 1, Walter Kolch 1,2,* and David Matallanas 1,* 1 Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland; [email protected] (A.A.N.); [email protected] (N.K.A.) 2 Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland * Correspondence: [email protected] (W.K.); [email protected] (D.M.) Abstract: Oncogenic RAS (Rat sarcoma) mutations drive more than half of human cancers, and RAS inhibition is the holy grail of oncology. Thirty years of relentless efforts and harsh disappointments have taught us about the intricacies of oncogenic RAS signalling that allow us to now get a pharma- cological grip on this elusive protein. The inhibition of effector pathways, such as the RAF-MEK-ERK pathway, has largely proven disappointing. Thus far, most of these efforts were aimed at blocking the activation of ERK. Here, we discuss RAF-dependent pathways that are regulated through RAF functions independent of catalytic activity and their potential role as targets to block oncogenic RAS signalling. We focus on the now well documented roles of RAF kinase-independent functions in apoptosis, cell cycle progression and cell migration. Keywords: RAF kinase-independent; RAS; MST2; ASK; PLK; RHO-α; apoptosis; cell cycle; cancer therapy Citation: Nolan, A.A.; Aboud, N.K.; Kolch, W.; Matallanas, D. Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling.
    [Show full text]
  • Establishment and Genomic Characterization of a Sporadic Malignant Peripheral Nerve Sheath Tumor Cell Line Jody Fromm Longo1, Stephanie N
    www.nature.com/scientificreports OPEN Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line Jody Fromm Longo1, Stephanie N. Brosius3,5,7, Iya Znoyko1, Victoria A. Alers1, Dorea P. Jenkins1, Robert C. Wilson1,2, Andrew J. Carroll4, Daynna J. Wolf1, Kevin A. Roth6 & Steven L. Carroll1,2,3* Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofbromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodefcient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identifed in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identifed mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive neoplasms derived from the Schwann cell lineage1,2.
    [Show full text]
  • Inhibition of Polo-Like Kinase 1 During the DNA Damage Response Is Mediated Through Loss of Aurora a Recruitment by Bora
    OPEN Oncogene (2017) 36, 1840–1848 www.nature.com/onc ORIGINAL ARTICLE Inhibition of Polo-like kinase 1 during the DNA damage response is mediated through loss of Aurora A recruitment by Bora W Bruinsma1,2,4,5, M Aprelia1,2,5, I García-Santisteban1,3, J Kool2,YJXu2 and RH Medema1,2 When cells in G2 phase are challenged with DNA damage, several key mitotic regulators such as Cdk1/Cyclin B, Aurora A and Plk1 are inhibited to prevent entry into mitosis. Here we have studied how inhibition of Plk1 is established after DNA damage. Using a Förster resonance energy transfer (FRET)-based biosensor for Plk1 activity, we show that inhibition of Plk1 after DNA damage occurs with relatively slow kinetics and is entirely dependent on loss of Plk1-T210 phosphorylation. As T210 is phosphorylated by the kinase Aurora A in conjunction with its co-factor Bora, we investigated how they are affected by DNA damage. Interestingly, we find that the interaction between Bora and Plk1 remains intact during the early phases of the DNA damage response (DDR), whereas Plk1 activity is already inhibited at this stage. Expression of an Aurora A mutant that is refractory to inhibition by the DDR failed to prevent inhibition of Plk1 and loss of T210 phosphorylation, suggesting that inhibition of Plk1 may be established by perturbing recruitment of Aurora A by Bora. Indeed, expression of a fusion in which Aurora A was directly coupled to Bora prevented DNA damage-induced inhibition of Plk1 activity, as well as inhibition of T210 phosphorylation. Taken together, these data demonstrate that DNA damage affects the function of Aurora A at multiple levels: both by direct inhibition of Aurora A activity, as well as by perturbing the interaction with its co-activator Bora.
    [Show full text]
  • Photoreceptor Proliferation and Dysregulation of Cell Cycle Genes in Early Onset Inherited Retinal Degenerations
    University of Pennsylvania ScholarlyCommons Departmental Papers (Vet) School of Veterinary Medicine 3-11-2016 Photoreceptor Proliferation and Dysregulation of Cell Cycle Genes in Early Onset Inherited Retinal Degenerations Kristin L. Gardiner University of Pennsylvania Louise Downs University of Pennsylvania Agnes I. Berta-Antalics University of Pennsylvania Evelyn Santana University of Pennsylvania Gustavo D. Aguirre University of Pennsylvania, [email protected] See next page for additional authors Follow this and additional works at: https://repository.upenn.edu/vet_papers Part of the Veterinary Medicine Commons Recommended Citation Gardiner, K. L., Downs, L., Berta-Antalics, A. I., Santana, E., Aguirre, G. D., & Genini, S. (2016). Photoreceptor Proliferation and Dysregulation of Cell Cycle Genes in Early Onset Inherited Retinal Degenerations. BMC Genomics, 17 (221), http://dx.doi.org/10.1186/s12864-016-2477-9 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/vet_papers/152 For more information, please contact [email protected]. Photoreceptor Proliferation and Dysregulation of Cell Cycle Genes in Early Onset Inherited Retinal Degenerations Abstract Background Mitotic terminally differentiated photoreceptors (PRs) are observed in early retinal degeneration (erd), an inherited canine retinal disease driven by mutations in the NDR kinase STK38L (NDR2). Results We demonstrate that a similar proliferative response, but of lower magnitude, occurs in two other early onset disease models, X-linked progressive retinal atrophy 2 (xlpra2) and rod cone dysplasia 1 (rcd1). Proliferating cells are rod PRs, and not microglia or Müller cells. Expression of the cell cycle related genes RB1 and E2F1 as well as CDK2,4,6 was up-regulated, but changes were mutation-specific.
    [Show full text]
  • Comprehensive Identification of Proteins in Hodgkin Lymphoma
    Laboratory Investigation (2007) 87, 1113–1124 & 2007 USCAP, Inc All rights reserved 0023-6837/07 $30.00 Comprehensive identification of proteins in Hodgkin lymphoma-derived Reed–Sternberg cells by LC-MS/MS Jeremy C Wallentine1, Ki Kwon Kim1, Charles E Seiler III1, Cecily P Vaughn2, David K Crockett2, Sheryl R Tripp2, Kojo SJ Elenitoba-Johnson1,2 and Megan S Lim1,2 Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed–Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed–Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse- phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUESTs against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and im- munohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identi- fications per protein and an error rate of o5.0%.
    [Show full text]
  • PDF Hosted at the Radboud Repository of the Radboud University Nijmegen
    PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/85871 Please be advised that this information was generated on 2021-09-29 and may be subject to change. ISOFORMS IN MUSCLE AND BRAIN CELLS localization and function • ralph j.a. oude ophuis • 2011 9 789088 912344 > ISBN 978-90-8891234,-4 DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION Voor het bijwonen van de openbare verdediging van het proefschrift van RALPH J.A. OUDE OPHUIS DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION op vrijdag 1 april 2011 om 13:00u precies in de Aula van de Radboud Universiteit Nijmegen aan de Comeniuslaan 2 te Nijmegen Na afloop van de verdediging is er een receptie ter plaatse PARANIMFEN Susan Mulders [email protected] Rinske van de Vorstenbosch r.vandevorstenbosch(§) ncmls.ru.nl DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION ISBN-13 978-90-8891234-4 ISBN-10 90-8891-234-3 Printed by Proefsohriftmaken.nl || Printyourthesis.com Published by Uitgeverij BOXPress, Oisterwijk DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op vrijdag 1 april 2011 om 13:00 uur precies door Raphaël Johannes Antonius Oude Ophuis geboren op 24 oktober 1978 te Sint-Oedenrode Promotor Prof.
    [Show full text]
  • Androgen Receptor
    RALTITREXED Dihydrofolate reductase BORTEZOMIB IsocitrateCannabinoid dehydrogenase CB1EPIRUBICIN receptor HYDROCHLORIDE [NADP] cytoplasmic VINCRISTINE SULFATE Hypoxia-inducible factor 1 alpha DOXORUBICINAtaxin-2 HYDROCHLORIDENIFENAZONEFOLIC ACID PYRIMETHAMINECellular tumor antigen p53 Muscleblind-likeThyroidVINBURNINEVINBLASTINETRIFLURIDINE protein stimulating 1 DEQUALINIUM SULFATEhormone receptor CHLORIDE Menin/Histone-lysine N-methyltransferasePHENELZINE MLLLANATOSIDE SULFATE C MELATONINDAUNORUBICINBETAMETHASONEGlucagon-like HYDROCHLORIDEEndonuclease peptide 4 1 receptor NICLOSAMIDEDIGITOXINIRINOTECAN HYDROCHLORIDE HYDRATE BISACODYL METHOTREXATEPaired boxAZITHROMYCIN protein Pax-8 ATPase family AAA domain-containing proteinLIPOIC 5 ACID, ALPHA Nuclear receptorCLADRIBINEDIGOXIN ROR-gammaTRIAMTERENE CARMUSTINEEndoplasmic reticulum-associatedFLUOROURACIL amyloid beta-peptide-binding protein OXYPHENBUTAZONEORLISTAT IDARUBICIN HYDROCHLORIDE 6-phospho-1-fructokinaseHeat shockSIMVASTATIN protein beta-1 TOPOTECAN HYDROCHLORIDE AZACITIDINEBloom syndromeNITAZOXANIDE protein Huntingtin Human immunodeficiency virus typeTIPRANAVIR 1 protease VitaminCOLCHICINE D receptorVITAMIN E FLOXURIDINE TAR DNA-binding protein 43 BROMOCRIPTINE MESYLATEPACLITAXEL CARFILZOMIBAnthrax lethalFlap factorendonucleasePrelamin-A/C 1 CYTARABINE Vasopressin V2 receptor AMITRIPTYLINEMicrotubule-associated HYDROCHLORIDERetinoidTRIMETHOPRIM proteinMothers X receptor tau against alpha decapentaplegic homolog 3 Histone-lysine N-methyltransferase-PODOFILOX H3 lysine-9OXYQUINOLINE
    [Show full text]
  • Activation of Diverse Signalling Pathways by Oncogenic PIK3CA Mutations
    ARTICLE Received 14 Feb 2014 | Accepted 12 Aug 2014 | Published 23 Sep 2014 DOI: 10.1038/ncomms5961 Activation of diverse signalling pathways by oncogenic PIK3CA mutations Xinyan Wu1, Santosh Renuse2,3, Nandini A. Sahasrabuddhe2,4, Muhammad Saddiq Zahari1, Raghothama Chaerkady1, Min-Sik Kim1, Raja S. Nirujogi2, Morassa Mohseni1, Praveen Kumar2,4, Rajesh Raju2, Jun Zhong1, Jian Yang5, Johnathan Neiswinger6, Jun-Seop Jeong6, Robert Newman6, Maureen A. Powers7, Babu Lal Somani2, Edward Gabrielson8, Saraswati Sukumar9, Vered Stearns9, Jiang Qian10, Heng Zhu6, Bert Vogelstein5, Ben Ho Park9 & Akhilesh Pandey1,8,9 The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets. 1 McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 527, Baltimore, Maryland 21205, USA.
    [Show full text]
  • The Role of Zyxin and LIMD1 in Mitosis and Cancer
    University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-4-2019 The Role of Zyxin and LIMD1 in Mitosis and Cancer Jiuli Zhou University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Cancer Biology Commons, and the Cell Biology Commons Recommended Citation Zhou, Jiuli, "The Role of Zyxin and LIMD1 in Mitosis and Cancer" (2019). Theses & Dissertations. 353. https://digitalcommons.unmc.edu/etd/353 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. THE ROLE OF ZYXIN AND LIMD1 IN MITOSIS AND CANCER by Jiuli Zhou A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Pathology & Microbiology Graduate Program Under the Supervision of Professor Jixin Dong University of Nebraska Medical Center Omaha, Nebraska May, 2019 Supervisory Committee: Jennifer D. Black, Ph.D. Robert E. Lewis, Ph.D. Kaihong Su, Ph.D. i ACKNOWLEDGEMENTS Funding support: The China Scholarship Council; University of Nebraska Medical Center (UNMC) Graduate Studies Office Fellowship; National Cancer Institute/National Institutes of Health (NCI/NIH); Department of Defense; the COBRE Grant from the Nebraska Center for Cell Signaling/National Institute of General Medical Sciences (NIGMS/NIH). More important than the funding support, I would like to acknowledge the support and encouragement I received during my doctoral research.
    [Show full text]
  • SS18-SSX–Dependent YAP/TAZ Signaling in Synovial Sarcoma
    Published OnlineFirst February 27, 2019; DOI: 10.1158/1078-0432.CCR-17-3553 Translational Cancer Mechanisms and Therapy Clinical Cancer Research SS18-SSX–Dependent YAP/TAZ Signaling in Synovial Sarcoma Ilka Isfort1,2, Magdalene Cyra1,2, Sandra Elges2, Sareetha Kailayangiri3, Bianca Altvater3, Claudia Rossig3,4, Konrad Steinestel2,5, Inga Grunewald€ 1,2, Sebastian Huss2, Eva Eßeling6, Jan-Henrik Mikesch6, Susanne Hafner7, Thomas Simmet7, Agnieszka Wozniak8,9, Patrick Schoffski€ 8,9, Olle Larsson10, Eva Wardelmann2, Marcel Trautmann1,2, and Wolfgang Hartmann1,2 Abstract Purpose: Synovial sarcoma is a soft tissue malignancy Results: Asignificant subset of synovial sarcoma characterized by a reciprocal t(X;18) translocation. The chi- showed nuclear positivity for YAP/TAZ and their tran- meric SS18-SSX fusion protein acts as a transcriptional dysre- scriptional targets FOXM1 and PLK1. In synovial sarco- gulator representing the major driver of the disease; however, ma cells, RNAi-mediated knockdown of SS18-SSX led to the signaling pathways activated by SS18-SSX remain to be significant reduction of YAP/TAZ-TEAD transcriptional elucidated to define innovative therapeutic strategies. activity. Conversely, SS18-SSX overexpression in SCP-1 Experimental Design: Immunohistochemical evaluation cells induced aberrant YAP/TAZ-dependent signals, mech- of the Hippo signaling pathway effectors YAP/TAZ was per- anistically mediated by an IGF-II/IGF-IR signaling loop formed in a large cohort of synovial sarcoma tissue specimens. leading to dysregulation of the Hippo effectors LATS1 SS18-SSX dependency and biological function of the YAP/TAZ and MOB1. Modulation of YAP/TAZ-TEAD–mediated Hippo signaling cascade were analyzed in five synovial sarco- transcriptional activity by RNAi or verteporfintreatment ma cell lines and a mesenchymal stem cell model in vitro.
    [Show full text]
  • Human LATS1 Is a Mitotic Exit Network Kinase
    Research Article Human LATS1 Is a Mitotic Exit Network Kinase John Bothos,1,2 Robyn L. Tuttle,1 Michelle Ottey,3 Francis C. Luca,3 and Thanos D. Halazonetis1,4 1The Wistar Institute; 2Program in Cell and Molecular Biology, Biomedical Graduate Studies; 3Department of Animal Biology, School of Veterinary Medicine; and 4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Abstract cerevisiae, there are three members in the NDR family. Dbf2 and The kinase LATS/WARTS is a tumor suppressor protein its nearly identical and redundant Dbf20 kinase are key components of the mitotic exit network (MEN), a signaling pathway that conserved in evolution, but its function at the molecular level is not well understood. We report here that human LATS1 coordinates cyclin-dependent kinase (CDK) inactivation, sister interacts with MOB1A, a protein whose homologue in budding chromatid decondensation, and cytokinesis at the end of mitosis yeast associates with kinases involved in mitotic exit. This (13–15); on the other hand, Cbk1, the third member, promotes suggested that LATS1 may be a component of the previously polarized growth and activates daughter-specific gene transcription uncharacterized mitotic exit network in higher eukaryotes. after cytokinesis (16–19). In Schizosaccharomyces pombe, there Indeed, moderate overexpression of human LATS1 in cells are two members in the NDR family. Sid2, the orthologue of exposed to microtubule poisons facilitated mitotic exit, and Dbf2/Dbf20, functions in the septation initiation network (SIN), this activity required MOB1A. Reciprocally, small interfering which is analogous to the S. cerevisiae MEN (13–15); on the other RNA–mediated suppression of LATS1 or MOB1A prolonged hand, Orb6, like its orthologue Cbk1, is required for maintenance of cell polarity after completion of mitosis (20).
    [Show full text]
  • Inhibition of ERK 1/2 Kinases Prevents Tendon Matrix Breakdown Ulrich Blache1,2,3, Stefania L
    www.nature.com/scientificreports OPEN Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown Ulrich Blache1,2,3, Stefania L. Wunderli1,2,3, Amro A. Hussien1,2, Tino Stauber1,2, Gabriel Flückiger1,2, Maja Bollhalder1,2, Barbara Niederöst1,2, Sandro F. Fucentese1 & Jess G. Snedeker1,2* Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fbrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants. Tendon is a musculoskeletal tissue that transmits muscle force to bone. To accomplish its biomechanical function, tendon tissues adopt a specialized extracellular matrix (ECM) structure1. Te load-bearing tendon compart- ment consists of highly aligned collagen-rich fascicles that are interspersed with tendon stromal cells. Tendon is a mechanosensitive tissue whereby physiological mechanical loading is vital for maintaining tendon archi- tecture and homeostasis2. Mechanical unloading of the tissue, for instance following tendon rupture or more localized micro trauma, leads to proteolytic breakdown of the tissue with severe deterioration of both structural and mechanical properties3–5.
    [Show full text]