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UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis

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UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis Published OnlineFirst February 6, 2020; DOI: 10.1158/0008-5472.CAN-19-1647 CANCER RESEARCH | MOLECULAR CELL BIOLOGY UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis Xi Qiao1,2,3, Ying Liu4,5, Maria Llamazares Prada6, Aravind K. Mohan7, Abhishekh Gupta8, Alok Jaiswal8, Mukund Sharma1,2,3, Joni Merisaari1,2,3, Heidi M. Haikala9, Kati Talvinen2, Laxman Yetukuri1,8, Joanna W. Pylvan€ ainen€ 10, Juha Klefstrom€ 9, Pauliina Kronqvist2, Annika Meinander7, Tero Aittokallio8,11, Ville Hietakangas4,5, Martin Eilers6, and Jukka Westermarck1,2 ABSTRACT ◥ For maximal oncogenic activity, cellular MYC protein levels need Graphical Abstract: http://cancerres.aacrjournals.org/content/ to be tightly controlled so that they do not induce apoptosis. Here, canres/80/7/1414/F1.large.jpg. we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC transcription MYC protein expression and regulated MYC target genes. Consis- Apoptosis tent with the tumor suppressor function of UBR5 (HYD) in Priming to drug-induced Drosophila, HYD suppressed dMYC-dependent overgrowth of apoptosis wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed Cancer cell UBR5 low MYC-dependent priming to therapy-induced apoptosis. Of direct proliferation fi Normal cell cancer relevance, MYC and UBR5 genes were coampli ed in MYC- UBR5 high proliferation driven human cancers. Functionally, UBR5 suppressed MYC- MYC protein levels Quiescence mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunoflu- UBR5 orescence analysis demonstrated reciprocal expression of UBR5 and 8q22.33 UBR5 MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC Breast cancer fi chemotherapy MYC activity. In MYC-ampli ed, and p53-mutant breast cancer 8q24.21 insensitivity cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. MYC Chr. 8 mRNA Protein Significance: These findings identify UBR5 as a novel MYC Coamplification of UBR5 with MYC is a cancer cell intrinsic mechanism regulator, the inactivation of which could be very important for to dampen MYC-mediated apoptosis in response to chemotherapy. understanding of MYC dysregulation on cancer cells. Introduction MYC regulates numerous physiologic and pathologic processes. Crit- ically, distinct protein expression levels dictate MYC's biological Emerging notion of overall poor concordance between gene ampli- output in vitro and in vivo (2–5). MYC protein levels are tightly fications and corresponding protein expression levels in cancer (1) regulated posttranslationally by phosphorylation, and by ubiquitin- highlights the need for better understanding of how protein expression mediated degradation (6–10). Especially in cancers, MYC is a classic levels are regulated at posttranslational levels. The transcription factor example of an oncoprotein, whose overexpression at protein level do 1Turku Bioscience Centre, University of Turku and Åbo Akademi University, Note: Supplementary data for this article are available at Cancer Research Turku, Finland. 2Institute of Biomedicine, University of Turku, Turku, Fin- Online (http://cancerres.aacrjournals.org/). land. 3TuDMM Doctoral Programme, University of Turku, Turku, Finland. Y. Liu and M.L. Prada contributed equally to this article. 4Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland. 5Institute of Biotechnology, University of Helsinki, Hel- Current address for M.L. Prada: BioMed X Innovation Center, Heidelberg, sinki, Finland. 6Theodor Boveri Institute and Comprehensive Cancer Center, Germany. Mainfranken, Biocenter, University of Wurzburg,€ Wurzburg,€ Germany. Corresponding Author: Jukka Westermarck, Turku Bioscience Centre, 7Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Tykistokatu€ 6A, Turku 20520, Finland. Phone: 358-29-450 2880; E-mail: Turku, Finland. 8Institute for Molecular Medicine Finland (FIMM), University jukka.westermarck@bioscience.fi of Helsinki, Helsinki, Finland. 9Research Programs Unit/Translational Can- cer Medicine & HiLIFE, University of Helsinki, Helsinki, Finland. 10Turku Cancer Res 2020;80:1414–27 BioImaging, University of Turku and Åbo Akademi University, Turku, Fin- doi: 10.1158/0008-5472.CAN-19-1647 land. 11Department of Mathematics and Statistics, University of Turku, Turku, Finland. Ó2020 American Association for Cancer Research. AACRJournals.org | 1414 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 6, 2020; DOI: 10.1158/0008-5472.CAN-19-1647 Ubiquitin Ligase UBR5 Controls MYC in Development and Cancer not match neither with the extent of mRNA overexpression nor with Institut fur€ Rechtsmedizin, Universit€at Wurzburg€ (Wurzburg,€ Ger- the frequency of genetic amplifications (7, 11). many). All the cell lines were tested negative for Mycoplasma during In addition to cell culture and mouse models (3, 4, 8, 12, 13), the role the period of this study. Drosophila S2 cells were cultured in Schneider of MYC has been widely studied in Drosophila, where the conserved Drosophila Medium (Thermo Fisher Scientific). All growth mediums homolog of MYC, dMYC regulates tissue growth and animal were supplemented with 10% heat-inactivated FBS (Gibco), 2 mmol/L size (14, 15). In particular, overexpression of dMYC induces growth L-glutamine, and penicillin (50 U/mL)/streptomycin (50 mg/mL). of cells of the imaginal discs, which are the organs giving rise to wings GFP-UBR5, GFP-UBR5-DHECT, Flag-UBR5, and Flag-UBR5- in adult fly (14). Importantly, dMYC can complement mammalian DHECT were kind gifts from Darren Saunders & Charles MYC in vivo (13) implying high degree of functional conservation. In Watts (28, 29). V5-MYC, V5-MYCT58A and have been described humans, MYC is a master regulator of malignant growth. However, previously (10). Flag-MYC plasmids and HA-MYC (1–262) were MYC protein levels exceeding the optimal proliferation promoting from Prof. Bruno Amati (European Institute of Oncology, Milan, levels primes both normal and cancer cells to apoptosis (2, 3, 5, 12). The Italy). HA-MYC-WT and HA-MYC mutants were kind gifts from principle of MYC-mediated apoptosis priming was originally revealed Prof. William P. Tansey (Vanderbilt University School of Medicine, by findings showing that highly overexpressed transgenic MYC initi- Nashville, TN). HA-ubiqiuitin was a kind gift from Prof. Lea Sistonen ates both proliferation and apoptosis programs in pancreatic islet cells. (Åbo Akademi University, Turku, Finland). Plasmids were transfected However, MYC overexpression was capable to drive tumorigenesis with Lipofectamine 2000 Transfection Reagent (Thermo Fisher Sci- only in the context of efficient apoptosis suppression (12). More entific) according to the manufacturer's instructions. After 48-hour recently, the in vivo importance of MYC expression levels in defining transfection, cell lysate was collected. Small interfering RNA (siRNA) the balance between proliferation and apoptosis was validated by allelic transfections were performed with Oligofectamine Transfection series of inducible MYC expression (3). In addition to spontaneous Reagent (Thermo Fisher Scientific) following to the manufacturer's apoptosis, high MYC levels prime tumor cells to apoptosis induction protocol. Three days after transfections, cells were harvested for by drugs that interfere with replication and cell division, such as analysis. siRNA target sequences are in supplementary table 1. For topoisomerase inhibitors or taxanes (2, 16, 17). Indeed, MYC is an double-stranded RNA (dsRNA)-mediated RNAi in Drosophila S2 important determinant of in vivo cell survival both during develop- cells, the DNA fragment of hyd was amplified with primers flanked ment and in response to cancer therapy (4). However, how MYC by T7 promoter, and dsRNA was produced using the TranscriptAid T7 protein levels are controlled endogenously to maintain an optimal High Yield Transcription Kit (Thermo Fisher Scientific). To knock MYC balance is incompletely understood. down Hyd, S2 cells were cultured with 5 mg/mL dsRNA for 120 hours. UBR5 (Ubiquitin protein ligase E3 component n-recognin 5) is an evolutionary conserved E3 ubiquitin ligase that destabilizes proteins Immunoblotting and immunoprecipitation with N-terminal recognition sequences exposed by proteolytic cleav- The following antibodies were used for Western blot analysis: UBR5 age (18, 19). Recently, UBR5 has been shown to target substrates also (sc-515494, Santa Cruz Biotechnology), MYC (ab32072, Abcam), through other recognition mechanisms than N-terminal recogni- cleaved PARP (ab32064, Abcam), BIM (2933, Cell Signaling Tech- tion (20). UBR5 is essential for mammalian development (21), and nology), vinculin (sc-25336, Santa Cruz Biotechnology), GAPDH has been linked to both protumorigenic and tumor-suppressing (5G4-6C5, HyTest Ltd), HA (3724, Cell Signaling Technology), Flag activities (19, 21–23). However, the mechanism by which UBR5 would (F3165, Sigma), GFP (sc-9996, Santa Cruz Biotechnology), GST promote tumor growth are unclear. It is also unclear how UBR5 may (sc-138, Santa Cruz Biotechnology),
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