Published OnlineFirst February 9, 2010; DOI: 10.1158/0008-5472.CAN-09-2453 Tumor and Stem Cell Biology Cancer Research Tumor Suppressor HLJ1 Binds and Functionally Alters Nucleophosmin via Activating Enhancer Binding Protein 2α Complex Formation Tzu-Pei Chang1,3, Sung-Liang Yu3,4, Sheng-Yi Lin1, Yi-Jing Hsiao1, Gee-Chen Chang1,2, Pan-Chyr Yang3,5, and Jeremy J.W. Chen1,3 Abstract HLJ1, a member of the heat shock protein 40 chaperone family, is a newly identified tumor suppressor that has been implicated in tumorigenesis and metastasis in non–small cell lung cancer. However, the mechanism of HLJ1 action is presently obscure. In this study, we report that HLJ1 specifically interacts with the nuclear protein nucleophosmin (NPM1), forming a multiprotein complex that alters the nucleolar distribution and oligomerization state of NPM1. Enforced accumulation of NPM1 oligomers by overexpression in weakly inva- sive but high HLJ1-expressing cells induced the activity of signal transducer and activator of transcription 3 (STAT3) and increased cellular migration, invasiveness, and colony formation. Furthermore, silencing HLJ1 accelerated NPM1 oligomerization, inhibited the activity of transcription corepressor activating enhancer binding protein 2α (AP-2α), and increased the activities of matrix metalloproteinase-2 (MMP-2) and STAT3. Our findings suggest that HLJ1 switches the role of NPM1, which can act as tumor suppressor or oncogene, by modulating the oligomerization of NPM1 via HLJ1-NPM1 heterodimer formation and recruiting AP-2α to the MMP-2 promoter. Cancer Res; 70(4); 1656–67. ©2010 AACR. Introduction and identified a panel of metastasis-associated genes, includ- ing human liver DnaJ-like protein (HLJ1, also known as With its high relapse and low cure rates, lung cancer is the DNAJB4; ref. 4). most common cause of cancer deaths worldwide (1). Molec- We subsequently showed that HLJ1 is a newly identified tu- ular-targeted therapy (MTT), which aims at specific molecu- mor suppressor in non–small cell lung cancer (NSCLC) that lar derangements in cancer cells or their microenvironment, can inhibit tumorigenesis and metastasis of lung cancer cells has emerged recently as one of the most important modali- in vitro and in vivo and whose expression correlates with sur- ties of cancer treatment (2, 3). Characterization of the puta- vival of patients (5). We also discovered a new mechanism tive genes involved and understanding the molecular whereby HLJ1 is transcriptionally upregulated via enhancer pathogenesis of tumor development are needed urgently activator protein-1 binding to promoter Yin Yang-1 (YY1) for identifying new molecular-targeted genes in lung cancer. and the coactivator p300 (6, 7). However, the mechanisms un- In a previous study, we used microarrays to screen a series of derlying the role of HLJ1 in tumor progression are unknown. lung cancer model cell lines with varying invasive capabilities Nucleophosmin (NPM1) is a nucleolar phosphoprotein that localizes in granular regions of the nucleolus and is highly expressed in malignant and actively dividing cells. Authors' Affiliations:1Institutes of Biomedical Sciences and Molecular Biology, National Chung Hsing University; 2Division of Chest Medicine, NPM1 shuttles continuously between the nucleus and cyto- Department of Internal Medicine, Taichung Veterans General Hospital, plasm (8) and acts as a multifunctional protein that plays an Taichung, Taiwan and 3NTU Center for Genomic Medicine, 4Department of Clinical Laboratory Sciences and Medical Biotechnology, and important role in the increased nucleolar activity needed for 5Department of Internal Medicine, National Taiwan University College of cell proliferation (9). Previous reports have revealed that Medicine, Taipei, Taiwan NPM1 has DNA-binding activity (10), can bind nuclear and Note: Supplementary data for this article are available at Cancer nucleolar localization signals (11), regulates the stability Research Online (http://cancerres.aacrjournals.org/). and transcriptional activity of p53 (12), and interacts with T-P. Chang and S-L. Yu contributed equally to this work and should be several transcription factors including transcription factor considered joint first authors. activating enhancer binding protein 2α (AP-2α), IFN regula- P-C. Yang and J.J.W. Chen codirected the project and contributed tory factor-1, alternative reading frame, NF-κB, and YY1 equally. (13–16). The NPM1 protein has been proposed to have both Corresponding Author: Jeremy J.W. Chen, Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan 40227, oncogenic and tumor-suppressing activities depending on its ROC. Phone: 886-4-22840896 ext. 125; Fax: 886-4-22853469; E-mail: level of expression and cellular localization (17, 18). [email protected]. The objective of this study was to identify the molecular doi: 10.1158/0008-5472.CAN-09-2453 mechanism responsible for the inhibition by HLJ1 of invasion ©2010 American Association for Cancer Research. and metastasis of lung cancer. Our results show that HLJ1 1656 Cancer Res; 70(4) February 15, 2010 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst February 9, 2010; DOI: 10.1158/0008-5472.CAN-09-2453 HLJ1 Switches the Double-Faced Role of Nucleophosmin downregulates the activities of signal transducer and activa- and then resolved by SDS-PAGE, followed by Western blot. tor of transcription 3 (STAT3) and matrix metalloproteinase- All experiments were performed in triplicate. For oligomeri- 2 (MMP-2) by blocking NPM1 oligomerization and regulating zation assay, we separately incubated recombinant His- the NPM1–AP-2α complex. These findings offer a new insight NPM1 alone and His-NPM1 plus His-HLJ1 in binding buffer into tumor progression and an opportunity to discover overnight at 4°C. After slowly mixing with native sample dye, potential genes that might act as targets in MTT. the protein complexes were incubated for 5 min at room temperature and then resolved by native-PAGE, followed by Western blot. Materials and Methods Real-time PCR. The primer sets used in SYBR-Green real- time PCR were listed as follows: HLJ1 forward primer, 5′- Cell culture. Human lung adenocarcinoma cell lines CL1-0 CCAGCAGACATTGTTTTTATCATT-3′ and reverse primer, and CL1-5, numbered in ascending order of invasive compe- 5′-CCATCCAGTGTTGGTACATTAATT-3′; NPM1 forward tence (4, 19), and their derivative transfectants were cultured primer, 5′-AGGTGGTTCTCTTCCCAAAGT-3′ and reverse in RPMI 1640 (Invitrogen) with 10% fetal bovine serum and primer, 5′-CACTGCCAGATCTTGAATAGC-3′.TheTBP was 1% penicillin-streptomycin (Invitrogen). used as an internal control (20). All experiments were per- Coimmunoprecipitation and Western blot analysis. The formed in triplicate. The relative expression level of HLJ1 preparations of whole-cell lysates, cytoplasmic and nuclear and NPM1 against that of TBP was calculated by the equation −Δ − − extracts, and Western blot analysis (20) and the details of CT = [CTHLJ1 or NPM1 CTTBP]. The RNA relative expres- −ΔΔ coimmunoprecipitation have been described previously sion was calculated as 2 CT × K, wherein K = constant. (21). The primary antibodies used for Western blot analysis Mammalian two-hybrid assay. The mammalian two- were monoclonal mouse anti-HLJ1 antibody (prepared in- hybrid assay was performed using the BD Matchmaker house), monoclonal mouse anti-NPM1 antibody (Zymed Lab- Mammalian Assay kit (Clontech) according to the manufac- oratories), polyclonal rabbit anti-AP-2α antibody (Santa Cruz turer's instructions. The recombinant pM-HLJ1 construct Biotechnology), polyclonal rabbit anti-STAT3 antibody (Up- was cotransfected into CL1-0 cells with the pVP16-NPM1 state Biotechnology, Inc.), monoclonal mouse anti-phospho- construct along with the reporter plasmids (pG5SEAP) STAT3 (Tyr705) antibody (Upstate Biotechnology), polyclonal and the β-galactosidase reporter at a defined molar ratio rabbit anti-MMP-2 (Santa Cruz Biotechnology), polyclonal (5:5:1:1). All experiments were performed in triplicate. rabbit anti-TATA-box binding protein (TBP) antibody (Santa The secreted alkaline phosphatase (SEAP) activity was Cruz Biotechnology), polyclonal rabbit anti–enhanced green measured by using BD Great EscAPe SEAP chemilumines- fluorescent protein (EGFP) antibody (Clontech), monoclonal cence detection kit (Clontech). The intensity of SEAP was mouse anti-β-tubulin (Upstate Biotechnology), and mono- normalized to β-galactosidase activity to calculate the clonal mouse anti-α-tubulin (Upstate Biotechnology). transfection efficiency. Chemiluminescent β-galactosidase Construction of expression vectors. The full-length cDNA activity was detected by a Galacto-Light plus system of NPM1 was constructed in-frame into pVP16 vector (Clon- (Applied Biosystems). tech), pcDNA3 expression vector (Invitrogen), or pEGFP-C3 Immunofluorescence staining. To identify the cellular vector (Clontech). To identify the protein-protein interacting localization of NPM1 cells were seeded onto eight-well cham- regions of HLJ1 with NPM1, one set of constructs was gener- ber slides. Cells were fixed and immunostained by anti-NPM1 ated for a pull-down assay. All primers used in this study are antibody and rhodamine-labeled antimouse secondary anti- listed in Supplementary Table S1. To overexpress HLJ1 in body. Nuclei were demarcated with 4′,6-diamidino-2-pheny- cells, the coding region of HLJ1 cDNA was cloned into the lindole (DAPI)