Stomatin-Like Protein 2 Regulates Survivin Expression in Non-Small

Yang et al. Cell Death and Disease (2018) 9:425 DOI 10.1038/s41419-018-0461-9 Cell Death & Disease

ARTICLE Open Access Stomatin-like protein 2 regulates survivin expression in non-small cell lung cancer cells through β-catenin signaling pathway Cheng-Ta Yang1,2,Jhy-MingLi1,Li-FuLi1,2,Yu-ShienKo2,3 and Jeng-Ting Chen4

Abstract The overexpression of stomatin-like protein-2 (SLP-2) is commonly observed in non-small cell lung cancer (NSCLC) cells. In the present study, we transfected a number of NSCLC cells with an SLP-2 shRNA-expressing vector (AdSLP2i) and examined its possible effects on cell growth and apoptosis. We found that suppression of SLP-2 expression inhibited cell growth, and that the apoptosis induced by SLP-2 suppression was correlated with decreased survivin protein expression. Moreover, the reduced survivin expression was found to be associated with reduced β-catenin nuclear localization and appeared not to be modulated through the AKT signaling pathway. By using immunoprecipitation and proteomics to analyze protein–protein interactions in A549 cells with SLP-2 overexpression, we found that annexin A2 interacted with SLP-2 and β-catenin directly. Our data further suggested that the knockdown of SLP-2 gene affected the SLP-2/Annexin A2/β-catenin cascade formation, reduced the translocation of cytoplasmic β-catenin into nucleus, and downregulated downstream target genes. The results presented in this study, together with our previous ﬁndings, suggest that SLP-2 promotes NSCLC cell proliferation by enhancing survivin expression mediated via β-catenin pathway. 1234567890():,; 1234567890():,;

Introduction found that the transfection of SLP-2 antisense into ESCC The stomatin gene superfamily consists of stomatin, cells suppressed cell growth and cell adhesion both – stomatin-like protein-1 (SLP-1), SLP-2, and SLP-31 3. in vitro and in vivo6. Furthermore, cell cycle analysis Because of its homology to stomatin, SLP-2 was predicted showed that the transfection of SLP-2 antisense led to S- to be cytoplasmically located and to link stomatin and/or phase arrest without apoptosis and the downregulation of other integral membrane proteins to the peripheral ﬁbronectin in ESCC cell lines5,6. cytoskeleton4. In addition, SLP-2 was found to be upre- The frequent overexpression of SLP-2 in NSCLC cells gulated by more than sixfold in esophageal squamous cell suggests a role in carcinogenesis. Here we explored its carcinoma (ESCC) tissues5 compared with their normal signaling network and therapeutic potential in this counterparts. Subsequently, the overexpression of SLP-2 malignancy. We constructed an adenoviral vector gene was also reported in human non-small cell lung expressing small hairpin RNA (shRNA) against SLP-2 cancer (NSCLC) cells, laryngeal carcinoma cells, and (AdSLP2i) to knockdown SLP-2 expression in NSCLC endometrial adenocarcinoma cells6. The same study also cells on a longer term basis. We found that the long-term suppression of SLP-2 expression in NSCLC cells resulted in cell apoptosis and, as such, we propose a possible Correspondence: Cheng-Ta Yang ([email protected]) 1Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, regulatory mechanism explaining how SPL-2 regulates Taiwan cell proliferation. 2Department of Respiratory Therapy, College of Medicine, Chang Gung University, Taoyuan, Taiwan Full list of author information is available at the end of the article Edited by A. Stephanou

© The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Materials and methods Western blotting Cell lines The cells (1 × 106) were plated onto 10 cm dishes and The NSCLC cell lines A549 (ATCC CCL-185), H460 incubated for 16 h at 37 °C before infection. The cells were (ATCC HTB-177), H157 (ATCC CRL-5802), and H838 subsequently mock-infected or infected with either (ATCC CRL-5844), and the fetal lung fibroblasts cell line AdCtrl or AdSLP2i, and incubated for 24~96 h. Total cell WI-38 (ATCC CCL-75) were purchased from American lysates were prepared with lysis buffer (M-PER Mamma- Type Culture Collection (ATCC, Manassas, Virginia, lian Protein Extraction Reagent; Thermo Fisher Scientific, USA). Adult normal human lung fibroblasts cell line HLF Inc., Rockford, IL) and nuclear protein was then extracted were purchased from Clonetics (BioWhittaker, Inc., using a nuclear extraction kit (AY2002; Panomics). The Walkersville, MD, USA). primary antibodies used were mouse anti-SLP-2 monoclonal antibody (60052-1-lg; Protein Tech Group, Inc.), Adenoviral vectors rabbit anti-SLP-2 polyclonal antibody (sc-98709; Santa A 19nt SLP-2 targeting sequence, 5′-TCGACA Cruz Biotechnology, Santa Cruz, CA), mouse anti- ATGTAACTCTGCAAA-3′, designed by SABioscience survivin monoclonal antibody (2802; Cell Signaling shRNA (catalog number KH07204G) was selected. A ring Technology), rabbit anti-poly-(ADP-ribose) polymerase sequence of 6 base pairs (5′-ATCGAT-3′) existed (PARP) polyclonal antibody (9542; Cell Signaling Tech- between the sense and antisense strands. Using BLAST nology), mouse anti-active-β-catenin monoclonal anti- analysis, it was confirmed that the targeting sequence body (anti-ABC; 05-665; Millipore), mouse anti-β-catenin shared no homology with other coding sequences in the monoclonal antibody (P35222; Millipore), rabbit anti- human genome. As described previously7, we used pUC- AKT polyclonal antibody (9272; Cell Signaling Technol- U6 plasmid and the pAdTrack vector to generate ogy), mouse anti-phospho-AKT (Ser473) monoclonal recombinant adenovirus expressing shRNA against SLP-2 antibody (4051; Cell Signaling Technology), goat anti-γ (AdSLP2i). The recombinant adenovirus AdCtrl, which tubulin polyclonal antibody (sc-7396; Santa Cruz Bio- carries a green fluorescence protein (GFP) gene regulated technology), mouse anti-GAPDH monoclonal antibody by the cytomegalovirus (CMV) promoter was used as the (sc-47724; Santa Cruz Biotechnology), mouse anti- control in these experiments7. The adenovirus vectors annexin A2 monoclonal antibody (sc-47696; Santa Cruz were amplified by using 293 cells and titered by using Biotechnology), and rabbit anti-annexin A2 polyclonal Adeno-X Rapid Titer Kit (BD Biosciences, San Jose, CA) antibody (sc-9061; Santa Cruz Biotechnology), and in 293 cells. horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, or donkey anti-goat antibody was used as the Development of stable A549 cell lines with high SLP-2 secondary antibody (Santa Cruz Biotechnology). expression To make the SLP-2 expression plasmid under the Cell proliferation control of the CMV promoter (pCMVSLP2), a 1071 bp The cells (1 × 105/well) were plated in six-well culture fragment of the human SLP-2 gene was generated plates and incubated for 16 h at 37 °C before infection. (nucleotides 64–1134, GenBank Accession Number The cells were then mock-infected or infected with NM_013442) by PCR reaction using A549 cDNA as the either AdCtrl or AdSLP2i at varying doses (multiplicity template. The oligonucleotide primers used were as of infection [m.o.i.] of 10, 50, 100, and 200). On follows: forward primer 5′-GAA ATG CTG GCG CGC days 1, 2, 3, 4, 5, and 6 after infection, the cells GCG GCG CGG G-3′ and reverse primer 5′-CTA ACT were collected by trypsinization and suspended in CAT CTT GAC TCG ATC AAG C-3′. pcDNA3-SLP2 phosphate-buffered saline (PBS). Viable cells were plasmid was constructed by subcloning the fragment of determined by direct microscopic counting using trypan the entire SLP-2 encoding sequence from pJET1/blunt blue exclusion. All counts were done on triplicate plasmid into the pcDNA3 between the XhoI and samples. BamHI sites. The A549 cells were subsequently transfected with pcDNA3-SLP2 using a liposome-mediated Construction of survivin promoter plasmids transfection technique with Lipofectamine 2000 reagent To construct luciferase expression plasmid under the (Invitrogen, Grand Island, NY). Real-time PCR was used control of survivin promoter (pSurvivin-Luc), a 977 bp to screen the SLP-2 expression of the individual clones. fragment of the human survivin gene promoter was The stable cell lines with high SLP-2 expression, namely generated (nucleotides 1824–2800, GenBank Accession A549SLP2 cells, were used for further experiments. In Number U75285) by PCR reaction from the A549 geno- addition, A549EV was developed from A549 cells by mic DNA. The oligonucleotide primers used were as transfection with empty vector pcDNA3 for the control follows: forward primer 5′-ATA CGA GAT CTGG CCA studies. TAG AAC CA-3′ and reverse primer 5′-ATG TAA AGC

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TTC CAC CTC TGC CA-3′. After the digestion and Immunofluorescence microscopy purification of restriction enzymes, the fragment was The cells were infected with AdCtrl or AdSLP2i for 72 h inserted into the luciferase vector pGL2-basic (Promega, on coverslips and grown to 50–80% confluence, fixed for Madison, WI) between the XhoI and HindIII sites. For an 15 min at room temperature using 4% formaldehyde/PBS, empty experimental control plasmid (pGL2-basic-CD), and permeablized with 0.1% Triton X-100 in PBS. The we used a promoter-less pGL2-basic vector in which the cells were then blocked using 100% non-immune goat luciferase gene was replaced with the survivin promoter serum (50–062Z; Invitrogen) for 30 min and stained using gene. These plasmids were confirmed by sequence anti-active-β-catenin monoclonal antibody (anti-ABC; analysis. 05–665; Merck). Primary antibodies were detected by rhodamine (TRITC)-conjugated donkey anti-mouse IgG Transient transfection and reporter assays antibodies (Santa Cruz Biotechnology). Immuno- The luciferase reporter assay was used to normalize fluorescence microscopy was performed using a confocal transfection efficiency. The cells were grown in 12-well microscope with a 63 × NPL Fluotar objective. plates with the suggested medium until 60–80% con- fluent. The cells were then transiently transfected with Immunoprecipitation of SLP-2 containing proteins 1.6 μg of survivin promoter plasmid DNA or pGL2-basic Immunoprecipitation was performed using agarose- plasmid DNA using 2 μl of Lipofectamine 2000 conjugated rabbit polyclonal anti-SLP-2 antibody (Santa (Invitrogen) in 1 ml of Opti-Medium (Life Technology) Cruz Biotechnology). The cell lysate was prepared from per well for 4–6 h. Cell lysates were prepared 48 h SLP-2 overexpressed A549 cells with a modified RIPA after transfection. The light intensity was measured buffer (50 nM Tris-HCl, pH 7.4; 1% NP−40; 0.25% using a luminometer (MiniLumat LB 9506, Berthold) sodium deoxycholate; 150 nM NaCl; 1 mM EDTA; 1 mM and all measurements were done with duplicate phenylmethylsulfonyl fluorie; 1 μg/ml aprotinin, leu- samples. The luciferase ratio was defined as the peptin, pepstatin, 1 mM Na3VO4; 1 mM NaF), and the luciferase activity of the survivin promoter plasmids protein concentration was adjusted to 1 μg/μl with PBS. divided by the luciferase activity of pGL2-basic Ten milligrams of cell lysate was added with 25 μl of anti- plasmid. SLP-2 and incubated overnight. After microcentrifuga- tion, the supernatant was discarded and the precipitated RNA preparation and microarray assay beads were washed three times with modified RIPA buf- RNA was extracted from H838 cells that were infected fer, and the protein was dissociated from the beads with with AdCtrl or AdSLP2i using Trizol according to the SDS-polyacrylamide gel electrophoresis sample buffer. manufacturer’s protocol (Invitrogen). A microarray analysis was performed by Phalanx Biotech Group (Hsin- Mass spectrometric analysis of gel-fractionated proteins Chu, Taiwan). Briefly, total RNA (100 ng) was reverse The protein bands of interest excised from silver- transcribed to cDNA, amplified, labeled, and hybridized to stained gels were destained by 1% potassium ferricyanide the Human Genome U133 Plus 2.0 Array (Affymetrix, and 1.6% sodium thiosulfate, subjected to reduction and USA) according to the manufacturer’s instructions. After alkylation by 10 mM dithiothreitol/55 mM iodoacetamide normalization of the expression values, the data from the in 25 mM NH4HCO3, and then in-gel digested with AdSLP2i-transfected sample were compared with the trypsin (20 μg/mL in 25 mM NH4HCO3) at 37 °C for 16 h. AdCtrl-transfected sample. Data were considered sig- The reaction products were resolved with 0.1% Formic nificant when (1) the false discovery rate from the sig- acid (FA) for liquid chromatography tandem mass spec- nificance analysis of the microarray analysis for the trometry (LC-MS/MS) analysis. LC-MS/MS data acqui- comparison of knockdown and unknockdown expression sition was performed on a NanoLC U3000 (Dionex, values was < 0.05 and (2) the p-value of the comparison Sunnyvale, CA) equipped with micrOTOFq (Bruker Dal- between the knockdown and unknockdown expression tonik GmbH, Bremen, Germany), with micrOTOFcontrol values by Student’s t-test was < 0.05. In order to focus on software. Samples were loaded into a trapping column genes exhibiting a low degree of regulation, we restricted (Agilent Zorbax 300SB-C18 was from Agilent Technolo- the majority of the analysis to genes with changes in gies (Santa Clara, CA) at 5 μl/min 100% elution buffer A expression levels of at least twofold. (0.05% FA). After reducing the flow to 25 nl/min by using a splitter, peptides were separated by an analytical column Assays for Rac1 activation (capillary RP18 column, Synergy hydro-RP, 2.5 μm, Activation of Rho family small GTPases was detected 0.15 × 100 mm, packed in-house) with a gradient from 5% using an EZ-DetectRac1 Activation Kit (Pierce Bio- to 80% elution buffer B (0.05% FA in 100% acetonitrile) in technology) and executed according to the manufacturer’s 75 min. Peptide fragment spectra were acquired from one instructions. MS scan, followed by four MS/MS scans of the more

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(A) HLF Wi-38 A549 H460 H838 H157 SLP-2 39 KDa

γ-Tubulin 50 KDa

(B) A549 H460 H838 H157

Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i

SLP-2 39 KDa 39 KDa 39 KDa 39 KDa

GAPDH 37 KDa 37 KDa 37 KDa 37 KDa

Fig. 1 The expression of SLP-2 after AdSLP2i transfection in various cell lines. a SLP-2 expression levels in normal and NSCLC cells were analyzed by western blotting using antibodies against SLP-2 and γ-tubulin. The primary antibodies used are shown on the left of the panel. The cell lines are shown on the top of the panel. b Western blot assays for the expressions of SLP-2 protein in A549, H460, H838, and H157 NSCLC cells on day 3 after treatment with mock infection (Mock), or with either AdCtrl or AdSLP2i at m.o.i. of 100 abundant parent ions. Each precursor was analyzed twice vector, AdSLP2i, containing a U6 promoter to introduce and then excluded in the following minute. LC-MS/MS ectopic SLP-2 shRNA expression to knockdown SLP-2 data were calibrated and processed with DataAnalysis expression in NSCLC cells. We infected A549, H460, v.4.0 (Bruker Daltonics) for database search. H838, and H157 NSCLC cells with AdSLP2i or AdCtrl at The emerging spectra identified via LC-MS/MS were m.o.i. of 100 or conducted mock infection. After a 3-day used in searches of the uniprot_sprot201006 database period, the SLP-2 expression in the AdSLP2i-treated cells (selected for Homo sapiens, 20 367 entries) assuming the was markedly decreased compared with those in the digestion enzyme trypsin. The MASCOT search engine AdCtrl- or mock-infected cells (Fig. 1b), indicating that (http://www.matrixscience.com; v.2.2 Matrix Science) was AdSLP2i effectively inhibited SLP-2 expression in the used with two missing cleavage site with charge states NSCLC cell lines. + + from 1 to 3 , MS mass tolerance was set to be 50 p.p.m. In order to find an optimal dose for the subsequent for LC-MS/MS and MS/MS tolerance was set to be 0.1 Da studies, we treated A549, H460, H838, and H157 cells for both fix modification: Carbamidomethyl (C) and with AdSLP2i or AdCtrl at 0, 10, 50, 100, or 200 m.o.i. As variable modifications: protein Acetyl (Protein N-term), shown in Fig. 2, in the H460 and H383 cells there were no Gln- > pyro-Glu (N-term Q), Oxidation (M). Protein significant differences in survival ratio (i.e., the ratios of identification was performed using Mowse scores survival cell number in infected cells to the mock-infected (p < 0.05) and MudPIT algorithm of MASCOT search cells) between AdSLP2i and AdCtrl transfection at m.o.i. engine. The dat files were produced by Mascot of 10 examined on day 5 nor in H157 cells at m.o.i. of 10 Daemon and subjected to Scaffold (v.2.06.02; and 50 on day 3. Of the other NSCLC cell lines examined, http://www.proteomesoftware.com) searching which the numbers of viable cells in the AdSLP2i-infected group assumed 99% protein probability, 95% peptide probability were significantly lower than those in the AdCtrl-infected with two peptides minimum for protein and peptide groups at all m.o.i. tested (p < 0.05 in all comparisons). identification. The cytotoxicity of AdSLP2i transfection was increased in a dose-dependent manner (Fig. 2); however, different Statistical analyses NSCLC cell lines exhibited different sensitivities. At day 5, Results were expressed as means ± SE. Statistical com- the ratios of viable cell numbers in A549, H460, H838, parisons were made with a two-sided t-test. A p-value < and H157 cells with AdCtrl treatment at m.o.i. of 100 to 0.05 was accepted as significant. those subjected to mock infection were not < 50% (50.0 ± 1.36%, 59.5 ± 1.86%, 57.6 ± 0.63%, and 65.6 ± 0.00%, Results respectively) and the ratios of viable cell numbers in the AdSLP2i induced apoptosis in NSCLC cells AdSLP2i-infected cells treated at m.o.i. of 100 to those The levels of SLP-2 expression in normal and NSCLC subjected to mock infection treatments were not > 50% cell lines were evaluated by western blotting with anti- (0.31 ± 0.05%, 26.4 ± 0.89%, 8.22 ± 0.63%, and 46.12 ± SLP-2-specific antibody. The expressions of SLP-2 in the 0.91%, respectively). Therefore, we chose the m.o.i. of 100 NSCLC cell lines were much higher than those in normal to study the effect of AdSLP2i infection on the cell growth HLF cell line (Fig. 1a). We then constructed an adenoviral in the NSCLC cell lines.

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Fig. 2 Cytotoxicity of AdSLP2i transfection on NSCLC cells. A549, H460, H838, and H157 cells were treated with varying doses of AdSLP2i or AdCtrl. Culture medium alone was used for mock infection. Triplet cultures were performed for each treatment and viable cells were counted on day 3 and day 5. The results are expressed as a percentage of the mock-infected cells (survival ratio). Points, mean; bars, SD

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The A549, H460, H838, and H157 cells were activity of survivin promoter in the AdCtrl-treated mock infected or infected with AdCtrl or AdSLP2i at group was 4.33-folds higher than that in mock- m.o.i. of 100. The numbers of viable cells were infected group, and the activity of survivin promoter counted from days 1 to 6 after transfection. The growth in AdSLP2i-treated group was 7.53% of that in AdCtrl- rates of the AdSLP2i-infected cells were substantively treated cells. These results indicated that AdSLP2i acted suppressed by SLP-2 gene silencing (Supplemental Fig. 1). on the suppression of survivin gene transcription and From day 3 to day 6, the numbers of viable cells in the thus decrease survivin protein levels. AdSLP2i-infected groups were significantly lower than those in the AdCtrl-infected groups in all the NSCLC cell Investigation of the links between survivin and SLP-2 lines examined (p < 0.05 in all comparisons). The results Several signaling pathways have been suggested to be indicated that knockdown of SLP-2 expression by involved in the transcriptional control of survivin gene, AdSLP2i treatment effectively inhibited NSCLC cell including the Wnt and AKT signaling pathways8. The growth. survivin gene is also an important target gene in the TCF- To assess whether SLP-2 inhibition induces apoptosis, 4/β-catenin signaling pathway9. In this regard, we found A549, H460, H838, and H157 cells were transfected with that in the cytosolic and nuclei of the AdSLP2i- AdSLP2i or AdCtrl at m.o.i. of 100, or were subjected to transfected cells, β-catenin was decreased (Fig. 4a). Pre- mock transfection. On day 5 after the infection, cells were vious study has demonstrated that cyclin-dependent collected and cell cycle analysis was performed7. The kinase 2-mediated β-catenin phosphorylation at residues results showed that there was a marked increase in the Ser23, Ser37, Thr41, and Ser45 promotes rapid degradation subG1 population of cells in the AdSLP2i-treated of cytosolic β-catenin10. We further utilized the antibody group (Fig. 3a). To confirm the presence of the recognizing active form of β-catenin with depho- apoptotic process, all the NSCLC cell lines were treated sphorylation on Ser37 or Thr41 (05-665, Anti-ABC, with either AdSLP2i or AdCtrl at m.o.i. of 100 and were Merck) and immunofluorescence to confirm the change examined for the presence of PARP cleavage by western of β-catenin localization after AdSLP2i or AdCtrl-trans- blot analysis. From day 3 onwards, the degradation of fection (Fig. 4b). The results indicated that the nuclear PARP was clearly detected in the AdSLP2i-treated cells content of the active form of β-catenin was consistently but not in the mock- nor AdCtrl-infected cells (Fig. 3b). and obviously reduced in the AdSLP2i-transfected cells. The results indicated that SLP-2 inhibition by AdSLP2i These results suggested that survivin down-expression transfection induced programmed cell death, as was evi- was probably regulated through the TCF-4/β-catenin denced by the increased subG1 cells and PARP protein signaling pathway. cleavage. We further examined AKT expression in NSCLC cell lines after either mock transfection or transfection with Microarray hybridization to identify gene(s) involved in AdSLP2i or AdCtrl at m.o.i. of 100. Three days after SLP-2-related apoptosis AdSLP2i transfection, phospho-AKT (pAKT) was found A cDNA microarray (Phalanx Biotech Group) was to be up-regulated in the AdSLP2i-transfected cells used to survey the probable factors involved in H838 cell compared with the cells in the other two experimental apoptosis induced by SLP-2 inhibition. Marked down- groups (Fig. 5a). regulation of c-myc and survivin protein levels were To confirm that survivin, pAKT, and β-catenin were observed on day 3 after transfection with AdSLP2i at m. regulated by SLP-2 inhibition, we generated a SLP-2 o.i. of 100 compared with cells transfected with AdCtrl. overexpression cell line, A549SLP2, to study this issue. Western blot analysis showed that in AdSLP2i- The A549SLP2 cell line was established by the stable transfected human lung cancer cells, including A549, transfection of CMV promoter-controlled SLP-2 expres- H460, H838, and H157 cells, the survivin protein sion vector. The expression of SLP-2 in the A549SLP2 expression levels were all suppressed (Supplemental cells was markedly higher than that in A549EV cells. We Fig. 2A). To explore how SLP-2 regulates survivin found that the nuclear β-catenin and survivin were up- expression, we cloned the survivin promoter gene and regulated and that pAKT was downregulated in the subcloned it into the upstream of the luciferase reporter A549SLP2 cells compared to the A549EV cells (Supple- gene in the pGL2-Basic vector (pGL2, Promega) to mental Fig. 3A-D). These results further established that generate a new plasmid, pSurvivin-Luc. pSurvivin-Luc the nuclear translocation of β-catenin affected survivin was then transfected into A549 cells; 4 h after that expression and that it was regulated by SLP-2 expression transfection, the cells were either mock transfected or status. transfected with AdCtrl or AdSLP2i at m.o.i. of 100. A previous report indicated Rho-GTPases regulate After incubation for 48 h, the cells were collected for cytoskeleton by becoming inactive when GDP-bound or luciferase assay. As shown in Supplemental Fig. 2B, the by becoming active when GTP-bound11. Another study

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A. A549 H460 H838 H157

Mock

AdCtrl

AdSLP2i

B. A549 H838 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i 116 KDa 116 KDa PARP PARP 89 KDa 89 KDa GAPDH 37 KDa GAPDH 37 KDa

H460 H157 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i 116 KDa 116 KDa PARP PARP 89 KDa 89 KDa GAPDH 37 KDa GAPDH 37 KDa

Fig. 3 Cell apoptosis induced by knockdown of SLP-2 in NSCLC cells. a A549, H460, H838, and H157 cells were treated with mock infection or with either AdSLP2i or AdCtrl at m.o.i. of 100. On day 5 post infection, cell cycle analysis showed a marked increase in the subG1 population of the AdSLP2i-treated cells. b A549, H460, H838, and H157 cells were treated with either AdSLP2i or AdCtrl at m.o.i. of 100. On day 3 post infection, degradation of poly-(ADP-ribose) polymerase (PARP) was clearly detected by western blotting analysis in all four AdSLP2i-treated cell lines

found that Rac1 is a member of the Rho family that can Immunoprecipitation and proteomics to analyze controll the nuclear localization of β-catenin via the Wnt protein–protein interactions in cells with SLP-2 signal pathway12. In this study, we sought to investigate overexpression whether the Rac1-GTPase could control the nuclear Our data showed that the knockdown of SLP-2 gene localization of β-catenin to mediate survivin expression inhibited survivin expression via the TCF-4/β-catenin after AdSLP2i treatment. Our data indicated that Rac1- signaling pathway, but not via PI3K/Akt-Rac1 signaling. GTPase was active in A549 and H157 cells but inactive in By then using LC-MS/MS and the database, we detected H460 and H838 cells after cells of each type were treated the interaction between SLP-2 and annexin A2 in with AdSLP2i (Fig. 5b). These results indicated that Rac1- A549SLP2 cells (Fig. 6a, b). We further used GTPase did not participate the in TCF/β-catenin signaling immunoprecipitation and western blotting to conﬁrm pathway. if there were interactions among SLP-2, annexin A2, and

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A549 H838

(A) Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i

Active β-catenin 92 KDa Active β-catenin 92 KDa Cytosol Cytosol Tublin 55 KDa Tublin 55 KDa Active β-catenin 92 KDa Active β-catenin 92 KDa Nuclear Nuclear Lamin A/C 65 KDa Lamin A/C 65 KDa

H157 H460 Mock AdCtrlAdSLP2i Mock AdCtrl AdSLP2i Active β-catenin 92 KDa Active β-catenin 92 KDa Cytosol Cytosol Tublin 55 KDa Tublin 55 KDa Active β-catenin 92 KDa Active β-catenin 92 KDa Nuclear Nuclear Lamin A/C 65 KDa Lamin A/C 65 KDa

(B) A549 H460 H838 H157 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2iMock AdCtrl AdSLP2i

GFP

Active β-catenin

DAPI

Merge

Fig. 4 Western blotting and immunoﬂuorescence analysis for the nuclear localization of β-catenin by SLP-2 inhibition in NSCLC cells. a Western blot analyses for the expressions of nuclear active β-catenin in A549, H460, H838, and H157 cells treated with either AdSLP2i or AdCtrl at m.o. i. of 100. b Immunoﬂuorescence analyses for the nuclear localization of β-catenin in A549, H460, H838, and H157 cells treated with either AdSLP2i or AdCtrl at m.o.i. of 100. On day 3 post infection, the nuclear localization of β-catenin was clearly reduced in all four AdSLP2i-treated cell lines

β-catenin. Our results indicated that annexin A2 reduced in H460, A549, and H838 cells transfected with interacted with SLP-2 and β-catenin directly AdSLP2i at various m.o.i. on day 3 and day 5 compared (Supplemental Fig. 4A). In addition, we found that with the cells in the AdCtrl group (Fig. 2). However, cell the expression of annexin A2 was not changed by death induced by AdSLP2i transfection was not apparent the knockdown or overexpression of SLP-2 gene in H157 cells nor was cell growth suppression induced by (Supplemental Fig. 4B). AdSLP2i transfection apparent in H460 and H157 cells. These data might suggest that the inhibition of SLP-2 Discussion expression reduced cell proliferation, and that this sup- The SLP-2 is a novel oncogene that is over-expressed in pressive effect varied in different cell types. Moreover, the human ESCC cells, NSCLC cells, laryngeal carcinoma suppression of SLP-2 expression resulted in PARP clea- cells, and endometrial adenocarcinoma cells6. In the vage and cell apoptosis, and both the microarray analysis present study, we ﬁrst analyzed the expression of SLP-2 in and western blotting showed a correlation between the normal and NSCLC cells using western blotting and, as decrease of survivin expression and cell apoptosis. The expected, the expression of SLP-2 in the NSCLC cells was results observed in this study, together with our previous much higher than that in the normal cells. We then report7, suggest that the following regulatory circuit is designed an adenovirus vector, designated as AdSLP2i, involved in the regulation of NSCLC cell growth: SLP-2 harboring SLP-2 shRNA under the control of a U6 pro- repression leads to reduced survivin activity, which in moter in order to silence the expression of SLP-2 in turn results in PARP cleavage and then culminates in cell NSCLC cells. The cell survival was found to be greatly apoptosis.

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(A) A549 H838 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i pAkt pAkt 60 KDa 60 KDa Akt Akt 60 KDa 60 KDa

H460 H157 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i pAkt pAkt 60 KDa 60 KDa Akt 60 KDa Akt 60 KDa

(B) A549 H838 A549SLP2 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i

GTP -Rac1 21 KDa GTP-Rac1 21 KDa

Total Rac1 21 KDa Total Rac1 21 KDa

GTP-Rac1/Total Rac1 Ratio 48.75% 55.05% 58.08% 92.60% GTP-Rac1/Total Rac1 Ratio 41.15% 62.88% 50.12%

H460 H157 Mock AdCtrl AdSLP2i Mock AdCtrl AdSLP2i GTP-Rac1 21 KDa GTP-Rac1 21 KDa Total Rac1 21 KDa Total Rac1 21 KDa GTP-Rac1/Total Rac1 Ratio 26.43% 33.51% 26.53% GTP-Rac1/Total Rac1 Ratio 59.68% 23.27% 58.21%

Fig. 5 GTP-Rac1 and pAKT did not participate in TCF/β-catenin signaling pathway after SLP-2 inhibition in NSCLC cells. a Western blot analyses for the expressions of pAkt in A549, H460, H838, and H157 cells treated with either AdSLP2i or AdCtrl at m.o.i. of 100. b Downregulation of SLP-2 increased the GTP-Rac1 proteins in A549 and H157 cells, but decreased the GTP-Rac1 proteins in H838 and H460 cells as determined by pull- down assay. Total cell lysates were subjected to the pull-down assay for GTP-Rac1 activities. Total cell lysates were analyzed for Rac1 expression as a loading control. The ratio of GTP-Rac1/total Rac1 in cells was analyzed by densitometry of the blot

The expression of survivin have been reported to be protein reduced pAKT expression but increased nuclear regulated by several signal pathways including TCF-4/β- β-catenin expression (Supplemental Fig. 3A-D). These catenin and PI3K/AKT-Rac18,9,11,12. The TCF-4/β-cate- results strongly suggest that β-catenin directly regulates nin pathway has an important role in the Wnt signaling survivin expression through the TCF-4/β-catenin signal- pathway. The activation of Wnt signaling enhances the ing pathway rather than through the PI3k/AKT signaling translocation of β-catenin into cell nuclei, where it binds pathway. to transcription factors of the TCF/LEF family. The β- Wu et al.12 reported that Rac1-GTPase activation catenin-TCF/LEF complex then induces transcription of enhances the nuclear localization of β-catenin in the Wnt downstream target genes in cancer, such as c-Myc, cyclin signaling pathway and that the knockdown of the D1, and Tcf-113, and subsequently lead to the transacti- expression of Rho family small GTPases stimulates AKT1 vation of survivin promoter14. upregulation, leading to enhanced cell migration and The PI3K/AKT signaling pathway has been shown to invasion in human bronchial cells17. Our data showed that regulate the cell survival, cell cycle progression, and cel- the knockdown of SLP-2 upregulated pAKT expression lular growth of many types of cancer cells. The activation and downregulated nuclear β-catenin. In the present of PI3K/AKT was found to promote β-catenin phos- study, we assessed Rac1-GTPase activity by western phorylation15, whereas the transfection of CA-AKT DNA blotting after AdSLP2i transfection. We found that Rac1- into cells has been shown to increase the expression of GTPase was upregulated in A549 and H157 cells but was both pAKT and survivin16. The results of the present downregulated in H460 and H838 cells (Fig. 5a–d). These study, however, showed that pAKT was upregulated but results indicated that β-catenin translocation into nuclei that nuclear β-catenin was downregulated after AdSLP2i was probably not regulated by Rac1-GTPase activation. transfection (Fig. 4a, b). In contrast, the increase of SLP-2 Further analysis by immune-precipitation and proteomics

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Fig. 6 Representative MS/MS spectra from micrOTOFq used to confirm the identification of STOML2 interactome. Tryptic peptides of protein bands in the SDS-gel were analyzed by LC-MS/MS using micrOTOFq. The amino acid sequences of peptides identified by MS/MS analysis and matched to the amino acid sequences of proteins are underlined. The representative MS/MS spectra of two selected peptides (indicated in blood red) for each protein are also denoted below the amino acid sequences. The assignments of the fragmented ions observed to specific amino acid residues were performed using Scadffold 2 search engine, and the search results are shown below the MS/MS spectra

showed direct interactions among SLP-2, β-catenin, and gene was reported to inhibit cell division and proliferation annexin A2, suggesting a role of annexin A2 in this reg- by blocking DNA synthesis27,28, reducing the levels of p11 ulatory cascade. and c-Myc, and causing cell cycle arrest29. Moreover, the Overexpression of annexin A2 has been found in many downregulation of annexin A2 has been shown to reduce cancers, such as NSCLC cancer18, Lewis lung cancer19, the activation of β-catenin and the expression of cyclin gliomas20, gastric cancer21, brain tumors22,23, acute pro- D130,31. Our data suggested that SLP-2/Annexin A2/β- myelocytic leukemia24, colorectal carcinoma25, and pan- catenin cascade formation might prevent the phosphor- creatic cancer26. Relatedly, the knockdown of annexin A2 ylation and subsequent degradation of β-catenin, so the

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inhibition of SLP-2 expression might reduce the accu- 6. Zhang, L. et al. Stomatin-like protein 2 is overexpressed in cancer and involved mulation and translocation of cytoplasmic β-catenin into in regulating cell growth and cell adhesion in human esophageal squamous cell carcinoma. Clin. Cancer Res. 12,1639–1646 (2006). nuclei, and thus downregulated downstream target genes 7.Yang,C.T.etal.Adenovirus-mediated transfer of siRNA against survivin such as survivin and cyclin D1. enhances the radiosensitivity of human non-small cell lung cancer cells. In summary, the molecular mechanisms of SLP-2 reg- Cancer Gene. Ther. 17,120–130 (2010). 8. Zhang, M., Yang, J. & Li, F. Transcriptional and post-transcriptional controls of ulation in cancer remain unclear. Our results suggested survivin in cancer cells: novel approaches for cancer treatment. J. Exp. Clin. that SLP-2 promotes NSCLC cell proliferation by Cancer Res. 25,391–402 (2006). enhancing survivin expression mediated through the 9. Li, F. Role of survivin and its splice variants in tumorigenesis. Br. J. Cancer 92, β 212–216 (2005). TCF4/ -catenin pathway (Supplemental Fig. 5). We also 10. Park, C. S. et al. Modulation of beta-catenin phosphorylation/degradation by suggest that the β-catenin pathway, but not the PI3K/ cyclin-dependent kinase 2. J. Biol. Chem. 279, 19592–19599 (2004). AKT pathway, is modulated by SLP-2 in NSCLC cells. 11. Hall, A. Rho GTPases and the actin cytoskeleton. Science 279,509–514 (1998). 12. Wu, X. et al. Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling. Cell 133,340–353 (2008). Acknowledgements 13. Eastman,Q.&Grosschedl,R.RegulationofLEF-1/TCFtranscriptionfactorsby We thank Professor Jau-Song Yu at Chang Gung University for technical Wnt and other signals. Curr. Opin. Cell. Biol. 11,233–240 (1999). support in conducting the proteomic experiments and Professor Jan-Ken Chen 14. Fang, Z. H. et al. Transcriptional regulation of survivin by c-Myc in BCR/ABL- at Chang Gung University for providing the inspiration for this study. The study transformed cells: implications in anti-leukaemic strategy. J.Cell.Mol.Med.13 was supported by the National Science Council, R.O.C. [96-2320-B-182-033 (to (8B), 2039–2052 (2009). CTY)] and Chang Gung Research Project [G680471 (to CTY)]. 15. Fang, D. et al. Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J. Biol. Chem. 282,11221–11229 (2007). ’ Authors contributions 16. Belyanskaya, L. L. et al. Cisplatin activates Akt in small cell lung cancer cells and C.T.Y. conceived and designed the study and wrote the article. The attenuates apoptosis by survivin upregulation. Int. J. Cancer 117,755–763 experiments were performed by J.M.L. and Y.S.K. The data were coordinated (2005). and analyzed by L.F.L. and C.T.Y. All the authors contributed to, read, and 17. Bousquet, E. et al. Loss of RhoB expression promotes migration and invasion fi approved the nal manuscript. of human bronchial cells via activation of AKT1. Cancer Res. 69,6092–6099 (2009). 18. Wang, C. Y. et al. Annexin A2 silencing induces G2 arrest of non-small cell lung Author details cancer cells through p53-dependent and -independent mechanisms. J. Biol. 1Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Chem. 287,32512–32524 (2012). Taiwan. 2Department of Respiratory Therapy, College of Medicine, Chang Gung 19. Sharma, M. R., Rothman, V., Tuszynski, G. P. & Sharma, M. C. Antibody-directed University, Taoyuan, Taiwan. 3Division of Cardiology, Chang Gung Memorial targeting of angiostatin’s receptor annexin II inhibits Lewis lung carcinoma Hospital, Taipei, Taiwan. 4Department of Surgery, Chang Gung Memorial tumor growth via blocking of plasminogen activation: possible biochemical Hospital, Taoyuan, Taiwan mechanism of angiostatin’s action. Exp. Mol. Pathol. 81, 136–145 (2006). 20. Mai,J.,Finley,R.L.Jr,Waisman,D.M.&Sloane,B.F.HumanprocathepsinB Conflict of interest interacts with the annexin II tetramer on the surface of tumor cells. J. Biol. The authors declare that they have no conflict of interest. Chem. 275,12806–12812 (2000). 21. Emoto, K. et al. Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma. Anticancer Res. 21(2B), 1339–1345 (2001). Publisher's note 22. Nygaard, S. J. et al. Expression of annexin II in glioma cell lines and in brain – Springer Nature remains neutral with regard to jurisdictional claims in tumor biopsies. J. Neurooncol. 38,11 18 (1998). published maps and institutional affiliations. 23. Roseman, B. J., Bollen, A., Hsu, J., Lamborn, K. & Israel, M. A. Annexin II marks astrocytic brain tumors of high histologic grade. Oncol. Res. 6,561–567 (1994). 24. Menell, J. S. et al. Annexin II and bleeding in acute promyelocytic leukemia. N. Supplementary Information accompanies this paper at https://doi.org/ Eng. J. Med. 340,994–1004 (1999). 10.1038/s41419-018-0461-9. 25. Emoto, K. et al. Annexin II overexpression correlates with stromal tenascin-C overexpression: a prognostic marker in colorectal carcinoma. Cancer 92, Received: 13 November 2017 Revised: 26 February 2018 Accepted: 28 1419–1426 (2001). February 2018 26. Esposito, I. et al. Tenascin C and annexin II expression in the process of pancreatic carcinogenesis. J. Pathol. 208,673–685 (2006). 27. Bao, H. et al. Overexpression of Annexin II affects the proliferation, apoptosis, invasion and production of proangiogenic factors in multiple myeloma. Int. J. References Hematol. 90,177–185 (2009). 1. Seidel,G.&Prohaska,R.MolecularcloningofhSLP-1,anovelhumanbrain- 28. Chiang, Y., Rizzino, A., Sibenaller, Z. A., Wold, M. S. & Vishwanatha, J. K. 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