Published OnlineFirst January 26, 2012; DOI: 10.1158/0008-5472.CAN-11-2055 Cancer Tumor and Stem Cell Biology Research BMK1 Kinase Suppresses Epithelial–Mesenchymal Transition through the Akt/GSK3b Signaling Pathway Runqiang Chen, Qingkai Yang, and Jiing-Dwan Lee Abstract Epithelial–mesenchymal transition (EMT) plays a crucial role in the development of cancer metastasis. The – jun mitogen-activated protein (MAP) kinases extracellular signal regulated kinase, c- -NH2-kinase, and p38 have been implicated in promoting EMT, but a role for the MAP kinase BMK1 has not been studied. Here, we report that BMK1 signaling suppresses EMT. BMK1 elevation augmented E-cadherin–mediated cell–cell adhesion, downregulated mesenchymal markers, and decreased cell motility. Conversely, BMK1 silencing attenuated E-cadherin–mediated cell–cell adhesion, upregulated mesenchymal markers, and stimulated cell motility. BMK1 depletion dramatically increased the accumulation of endogenous Snail in the nuclear compartment. Snail accumulation was mediated by Akt/GSK3b signaling, which was activated by a modulation in the expression of the mTOR inhibitor DEPTOR. In support of these observations, BMK1 depletion promoted metastasis in vivo. Together, our findings reveal a novel mechanism of EMT control via mTOR/Akt inhibition that suppresses cancer metastasis. Cancer Res; 72(6); 1–9. Ó2012 AACR. Introduction that control cancer progression. The majority of mitogenic/ – The process of epithelial–mesenchymal transition (EMT) oncogenic signal activated signaling pathways stimulate is critically involved in the progression of human diseases, EMT(2).Inparticular,3ofthe4mitogen-activatedprotein such as cancer metastasis and fibrosis (1). EMT involves (MAP) kinase pathways described to date (namely, Erk, JNK, profound phenotypic changes that include loss of cell–cell and p38; ref. 6) induce EMT (7). The function of the fourth adhesion, loss of cell polarity, and acquisition of migratory MAP kinase, BMK1, in EMT, however, remains unknown. and invasive properties (2). Loss of E-cadherin expression is Herein, we describe our investigations into the actions of a hallmark of EMT and has become an established biomark- BMK1 in EMT and in cancer metastasis. er of the process itself (3). One principal mechanism that actstoreduceE-cadherinexpressioninEMTistheactiva- Materials and Methods tion of its associated transcriptional repressors. The E- Cell culture and reagents cadherin repressors have been classified into 2 groups, based The human cell lines A549, MCF10A, DU145, T47D, and upon their interaction with the E-cadherin promoter (2). The mouse cell line 4T1 were purchased from the American Type first group directly binds to the E-cadherin promoter to Culture Collection (ATCC) and cultured no more than 6 physically inhibit transcription and is composed of the Snail, months after purchase for the experiments described herein. Zeb, E47, and KLF8 factors (4, 5). The second group indi- The cell lines were authenticated by ATCC and maintained as rectly binds to the E-cadherin promoter, interacting with recommended. LY294002, U0126, and SB203580 were pur- other direct binding factors, and includes Twist, Goosecoid, chased from Calbiochem. Sanguinarine was purchased from E2.2, and FoxC2. Gaining a thorough understanding of the Sigma-Aldrich. process by which epithelial cells transit to mesenchymal cells is necessary for elucidating the molecular mechanisms Plasmids, virus production, and infection of target cells The human plasmid pWZL-Neo-Myr-Flag-MAPK7, DEP- TOR short hairpin RNA (shRNA), and DEPTOR cDNA were Authors' Affiliation: Department of Immunology and Microbial Science, purchased from Addgene. The human shSnail plasmid was The Scripps Research Institute, La Jolla, California from OriGene. The procedures used to produce retroviruses Note: Supplementary data for this article are available at Cancer Research andtoinfecttargetcellshavebeendescribedpreviously(8). Online (http://cancerres.aacrjournals.org/). To produce recombinant lentiviruses, vectors encoding R. Chen and Q. Yang contributed equally to this work. shRNA against human BMK1, mouse BMK1, or control nontarget shRNAs were purchased from Open Biosystems; Corresponding Author: Jiing-Dwan Lee, Department of Immunology and fl Microbial Science, The Scripps Research Institute, 10550 N. Torrey Pines lentiviruses were generated by cotransfection of subcon u- Rd., IMM12, La Jolla, CA 92037. Phone: 858-784-8703; Fax: 858-784- ent human embryonic kidney (HEK) 293FT cells (Invitrogen) 8343; E-mail: [email protected] with one of the above expression plasmids and a packaging doi: 10.1158/0008-5472.CAN-11-2055 plasmid (pMDL, pREV, or pVSVG) using the GenJet DNA Ó2012 American Association for Cancer Research. in vitro transfection reagent (SignaGen Laboratories). www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst January 26, 2012; DOI: 10.1158/0008-5472.CAN-11-2055 Chen et al. Infectious lentiviruses were collected 48 hours after trans- Luciferase assay fection and centrifuged to remove cell debris. A549 cells The luciferase assay was done as described previously (10). were transduced with the viruses expressing shRNA against The pG5E1bLuc and internal control pRL-TK plasmids were humanBMK1,DEPTOR,Snail,ornontargetshRNAs.4T1 cotransfected into cells along with a construct encoding the cells were transduced with lentiviruses expressing shRNA GAL4-binding domain fused to MEF2C. that targeted mouse BMK1 or nontarget shRNAs. Immunoflourescence Antibodies and immunoblotting Cellsweregrownonglasscoverslipsina6-wellplateand Cells were lysed in radioimmunoprecipitation assay buffer washed 3 times with PBS before fixing with 2% formaldehyde and blots were carried out as described previously (9). in PBS (pH 7.4) for 15 minutes at room temperature. The Antibodies used were as follows: anti–glyceraldehyde-3- cells were then washed 3 times with PBS and permeabilized phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich); with 0.2% Triton X-100 plus 1% normal goat serum (NGS) in anti-Snail, anti-GSK3b,anti-GSK3b (Ser 9), anti-Akt, anti- PBS for 5 minutes on ice. Cells were again washed 3 times p-Akt (Ser 473), anti-p-BMK1 (T218/Y220), anti-p-Akt (Thr with 1% NGS in PBS. Coverslips were incubated with respec- 308), anti-ZO-1, and anti–E-cadherin (Cell Signaling); anti- tive primary antibodies at 1:100 dilutions overnight at 4oC. DEPTOR (Novus Biologicals); anti-vimentin V9 (NeoMar- After 3 washes with 1% NGS in PBS, incubation with fluo- kers); anti–N-cadherin (BD Transduction); anti-twist1, rescein-conjugated secondary antibodies at 1:50 dilutions anti-Zeb1, and anti-Zeb2 (Santa Cruz Biotechnology); and was carried out. Cells were then washed 4 times with PBS, anti-BMK1 (10). All immunoblotting experiments were mounted with medium containing 40,6-diamidino-2-pheny- repeated at least 3 times. The statistics for immunoblotting lindole (DAPI; Vector Laboratories), and analyzed using experiements in Figs. 1 to 5 were presented in Supplemen- fluorescence microscopy. tary Fig. S1 and S2 (statistics for immunoblotting in Fig. 1C,Fig.2BandE,andFig.3A–C and G are in Supplementary Cell motility assays Fig. S1A–S1G, respectively. Statistics for immunoblotting Cells were harvested with Trypsin/EDTA, washed twice in Fig. 3E and F, Fig. 4A–DandF,andFig.5AandCare with Dulbecco's modified Eagle's medium, and resuspended in Supplementary Fig. S2A–S2I, respectively). in serum-free medium at a density of 106 cells/mL. Lower EV BMK1 A C EV BMK1 D 1.0 2.2 6 E-Cadherin 1.0 2.3 markers ZO-1 Epithelial 4 1.0 0.6 EV BMK1 B N-Cadherin 1.0 0.4 Vimentin 2 E-Cadherin markers Mesenchymal Mesenchymal 1.0 7.3 Relative luciferase activity BMK1 0 1.0 1.0 EV BMK1 ZO-1 GAPDH E 200 150 100 * N-Cadherin per field 50 Migratory cells 0 EV BMK1 Vimentin EV BMK1 Figure 1. BMK1 enhances cell epithelial properties. A, phase-contrast microscopic images of A549 cells carrying control vector (EV) or expression vector encoding BMK1. Scale bar, 50 mm. B, fluorescence microscopic staining of E-cadherin, ZO-1, N-cadherin, and vimentin (green) is indicated in the EV and BMK1 A549 cells. Nuclear DNA was stained with DAPI (blue). Scale bar, 20 mm. C, expression levels of epithelial markers, E-cadherin and ZO-1, as well as mesenchymal markers, N-cadherin and vimentin, were examined by immunoblotting of EV and BMK1 A549 cells. GAPDH was used as a loading control. The relative expression levels of indicated proteins in BMK1 cells was determined by setting the expression level, measuring by densitometry, in EV cells at a value of 1.0. D, MEF2C transactivation activity was determined in EV and BMK1 A549 cells using luciferase assay. E, motility of EV and BMK1 A549 cells was analyzed. Each bar represents the mean Æ SD of samples measured in triplicate. Ã, P < 0.05. Scale bar, 50 mm. OF2 Cancer Res; 72(6) March 15, 2012 Cancer Research Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst January 26, 2012; DOI: 10.1158/0008-5472.CAN-11-2055 BMK1 Suppresses EMT Du145 MCF10A A C E shCont shCont shBMK1 shCont shBMK1 shBMK1 1.0 0.2 1.0 0.4 800 * E-Cadherin 600 1.0 0.3 1.0 0.3 * ZO-1 400 1.0 4.2 1.0 6.1 Snail 200 3.61.0 1.0 3.1 * N-Cadherin Migratory cells per field Migratory 0 shContshBMK1 shCont shBMK1 shCont shBMK1 1.0 3.0 1.0 3.3 1 h 3 h 5 h Vimentin 1.0 1.0 1.0 1.0 GAPDH B D shCont shBMK1 1,500 shCont shBMK1 1,200 * 1.0 0.3 0 h 900 E-Cadherin 600 300 * 1.0 0.2 0 Migratory cells per field per cells Migratory markers ZO-1 Epithelial 30 h F 1.0 3.3 N-Cadherin 1.0 3.4 100 Vimentin markers 80 * Mesenchymal Mesenchymal 1.0 0.2 60 BMK1 40 20 1.0 1.0 Ratio of migration of initial width)(% 0 GAPDH Figure 2.