923 SUMOylation of HMGA2: selective destabilization of promyelocytic leukemia protein via proteasome Xuefei Cao,1 Carlos Clavijo,1 Xu Li,1,2 H. Helen Lin,2 Collectively, our results unveil a previously unrecognized Yuan Chen,3 Hsiu-Ming Shih,4 and David K. Ann1,2 effect by HMGA2 on the modulation of PML protein level, providing a novel mechanism underlying HMGA2 function 1Department of Pharmacology and Pharmaceutical Sciences, and underscoring the molecular basis for oncogenic pro- University of Southern California, Los Angeles, California; gression by HMGA2. [Mol Cancer Ther 2008;7(4):923–34] Departments of 2Molecular and Clinical Pharmacology, and 3Immunology, City of Hope, Duarte National Medical Center, Duarte, California; and 4Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China Introduction Members of the high mobility group A (HMGA1a, Abstract HMGA1b, and HMGA2) family of architectural transcrip- tion factors are distinguished by three reiterated ‘‘AT The HMGA2 architectural protein functions in a variety hooks’’ of 10 to 12 residues in each DNA binding motif, of cellular processes, such as cell growth, transcription and they are critical for a variety of cellular processes, regulation, neoplastic transformation, and progression. including gene transcription, induction of neoplastic Up-regulation of HMGA2 protein is observed in many transformation, and promotion of metastatic progression tumors and is associated with advanced cancers with poor (1, 2). The expression level of HMGA2 is maximal during prognoses. Although the expression and biochemical early stages of fetal development but is low in terminally properties of HMGA2 protein are regulated by microRNA differentiated cells (1). A strong correlation has been and phosphorylation, it is unknown whether HMGA2 established between HMGA2 protein expression and the activity can also be regulated by SUMOylation, and that relevant oncogenic phenotypes. First, HMGA2 expression is what is investigated in this report. We identified HMGA2 is implicated in malignant phenotypes of mesenchymal as as a SUMOylation target and showed that the expression well as epithelial origin (3–5). Second, the evidence for its of wild-type HMGA2, but not SUMOylation-defective direct role in tumorigenesis came from transfection of an HMGA2(2K/R), selectively lowered the steady-state level antisense DNA against HMGA2 in the normal thyroid cells of PML protein. Consequently, the HMGA2-elicited PML that prevented the neoplastic transformation induced by down-regulation rendered a reduction in the average myeloproliferative sarcoma virus and Kristen murine number of PML nuclear bodies per cell and the volume sarcoma virus (6). In addition, the transgenic mice over- of PML assembled per PML nuclear body. Using small expressing HMGA2 developed pituitary adenomas (7). interfering RNA to suppress endogenous ubiquitin expres- Third, the expression of HMGA2 in oral squamous cell sion and proteasome inhibitor to repress ubiquitin-mediated carcinoma is associated with increased disease recurrence protein degradation, we showed that HMGA2 confers and metastasis, along with a reduced survival rate PML down-regulation through ubiquitin-proteasome– manifested by a facilitated epithelial-mesenchymal transi- dependent protein degradation. Importantly, arsenic triox- tion (8). Moreover, HMGA2 expression is associated with ide treatment stimulated HMGA2 SUMOylation, leading the poor prognosis and metastasis in patients with breast to the formation of HMGA2 nuclear foci surrounding PML cancer (9). nuclear bodies and the stimulation of PML degradation. Covalent attachment of ubiquitin and small ubiquitin- like modifier (SUMO) to proteins plays a major role in regulating cellular functions (10–12). Both modifications depend on similar, but distinct, enzyme cascades. Briefly, Received 8/7/07; revised 11/28/07; accepted 2/15/08. ubiquitin and SUMO are activated in an ATP-dependent Grant support: NIH research grants R01 DE 10742 and DE 14183 (D.K. manner by individual E1-activating enzymes, transferred to Ann), and CA 94595 (Y. Chen); and Taiwan National Health Research Institutes grant MG-092-PP-03 (H.M. Shih). The imaging studies in this the respective E2-conjugating enzymes and subsequently work were supported in part by NIH P30 DK48522 (Confocal Microscopy attached to the Lys acceptor sites of target proteins by Subcore, University of Southern California Center for Liver Diseases). distinct E3 ligases. Whereas ubiquitination generally The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked promotes protein degradation, SUMOylation largely serves advertisement in accordance with 18 U.S.C. Section 1734 solely to to regulate diverse cellular processes, including nuclear indicate this fact. transport, genome integrity, signal transduction, and trans- Note: X. Li and C. Clavijo contributed equally to this work. criptional regulation (10–12). The list of known SUMO Requests for reprints: David K. Ann, Department of Clinical and Molecular targets has grown substantially in recent years, and over Pharmacology, City of Hope, 1500 East Duarte Road, Duarte, CA 91010. Phone: 626-359-8111, ext. 64967; Fax: 626-471-7204. half of the presently identified SUMO targets are transcrip- E-mail: [email protected] tion factors or coregulators, attenuating transcriptional Copyright C 2008 American Association for Cancer Research. activation (10–12). Additionally, SUMOylation has been doi:10.1158/1535-7163.MCT-07-0540 shown to affect the dynamic interaction between transcription Mol Cancer Ther 2008;7(4). April 2008 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2008 American Association for Cancer Research. 924 HMGA2-Facilitated PML Degradation regulator(s) and promyelocytic leukemia (PML) protein resulting pCMV-Tag2A-SUMO-1 between HindIII and (13, 14). PML is essential for the formation of a nuclear XhoI. The sequences of derived constructs were verified matrix–associated multiprotein complex, namely the PML by DNA sequencing. The ubiquitin siRNA (h) was nuclear body (15). PML and PML nuclear bodies have purchased from Santa Cruz Biotechnology, Inc. The sense been implicated in governing a variety of cellular processes, and antisense oligonucleotides were individually dissolved including transcriptional regulation, tumor suppression, in RNase-free water at 100 Amol/L and subsequently cellular senescence, apoptosis, and immune responses annealed by heating at 90jC for 1 min, and incubating at (16–18). 37jC for 1 h at a concentration of 20 Amol/L. Transfection In searching for HMGA2-interacting proteins and possi- of siRNA was done with Lipofectamine 2000 (Invitrogen) ble molecular mechanisms by which HMGA2 functions according to the manufacturer’s instruction. as an oncogene, we identified SUMO E2 conjugase Ubc9 Rat deSUMOylase DAxam was cloned by reverse and SUMO-1 as such partners, suggesting HMGA2 as a transcription-PCR using total RNA extracted from rat potential target for SUMO conjugation. In this study, we parotid Pa-4 cells. Oligo-dT primer was used in the reverse show that the expression of HMGA2 resulted in the transcription reaction, whereas the deSUMOylase-specific SUMOylation-dependent down-regulation of PML, but primer pairs used in the PCR reactions were designed not RanGAP-1 and GR, protein level and a concordant according to rat DAxam sequence in Genbank(21). The reduction in the average size and number of PML nuclear resulting PCR products were first cloned into TA-cloning bodies. Moreover, the observed HMGA2-dependent PML vector pCR2.1-TOPO (Invitrogen) and subsequently into down-regulation was partially reversed by small interfering the FLAG-tagged expression vector pCMV-Tag3B (Strata- RNA (siRNA) against ubiquitin or proteasome inhibitor, gene). Expression of the resulting constructs was confirmed suggesting the involvement of ubiquitin-proteasome– by transient transfection into Pa-4 cells followed by dependent protein degradation. Together, these observa- Western analysis using anti-FLAG antibody. As compared tions led us to propose a novel function for HMGA2, with the mock-transfected control, a unique and intense which uses a SUMO-dependent mechanism(s) to selec- protein band at 60 kDa, corresponding to DAxam, was tively promote the degradation of PML protein, perturbing observed in total cell lysates prepared from transfected cell the dynamic networkand multiple functions associated lysates (data not shown). with PML. Cell Lysis and Western Analyses Cells were washed twice in ice-cold 1Â PBS and lysed in SDS sample loading buffer. Equal amounts of proteins from Materials and Methods total cell extracts were fractionated by electrophoresis on Cell Culture SDS-PAGE and were electroblotted onto an Immobilon-P The rat parotid epithelial cell line Pa-4, also known as membrane (Millipore). The membrane was blocked with parotid C5 cells, was plated on Primaria culture dishes 5% nonfat milkand 0.1% Tween 20 in TBS for 1 h at (Falcon) in DMEM/Ham’s F-12 (1:1) medium supple- room temperature and incubated with the respective mented with 2.5% FCS, insulin (5 Ag/mL), transferrin primary antibodies, including anti-GR (Santa Cruz Bio- (5 Ag/mL), epidermal growth factor (25 ng/mL), hydro- technology), anti-HA (Covance), anti-PML (Santa Cruz cortisone (1.1 Amol/L), glutamine (5 mmol/L), and Biotechnology), anti-GMP (Zymed Laboratories, Inc.), anti- kanamycin monosulfate (60 Ag/mL) and maintained in