Spi-B–Mediated Silencing of Claudin-2 Promotes Early

Published OnlineFirst July 28, 2017; DOI: 10.1158/0008-5472.CAN-17-0020

Cancer Molecular and Cellular Pathobiology Research

Spi-B–Mediated Silencing of Claudin-2 Promotes Early Dissemination of Lung Cancer Cells from Primary Tumors Wei Du1, Xing Xu1, Qing Niu1, Xuexi Zhang1, Yiliang Wei1, Ziqiao Wang1,2, Wei Zhang1, Jun Yan3, Yongxin Ru4, Zheng Fu1,5, Xiaobo Li1, Yuan Jiang1,5, Zhenyi Ma1,5, Zhenfa Zhang6, Zhi Yao1,5, and Zhe Liu1,5

Abstract

Dissociation from epithelial sheets and invasion through the lymphatic metastasis, and short overall survival. Mechanistically, surrounding stroma are critical early events during epithelial Spi-B disrupted intercellular junctions and enhanced invasiveness cancer metastasis. Here we ﬁnd that a lymphocyte lineage–restrict- by reconﬁguring the chromatin structure of the tight junction gene ed transcription factor, Spi-B, is frequently expressed in human claudin-2 (CLDN2) and repressing its transcription. These data lung cancer tissues. The Spi-B–expressing cancer cells coexpressed suggest that Spi-B participates in mesenchymal invasion, linking vimentin but repressed E-cadherin and exhibited invasive behav- epithelial cancer metastasis with a lymphatic transcriptional ior. Increased Spi-B expression was associated with tumor grade, program. Cancer Res; 77(18); 4809–22. 2017 AACR.

Introduction Spi-B (encoded by SPIB), an Ets family transcription factor, is expressed exclusively in mature B cells, T-cell progenitors, and Every stage of cancer progression is accompanied with genetic plasmacytoid dendritic cells (4–6). B cells in SPIB / mice are and epigenetic dysregulation. Consequent aberrant activation defective in B-cell receptor (BCR) signaling and are unable to and/or silencing of a series of functional genes confer premalig- generate antibody responses to T-dependent antigens (7). Spi-B nant epithelial cells with multiple distinct properties including has been shown to regulate many genes that are important for unrestrained proliferation, resistance to cell death, evasion from BCR-mediated signaling including, Igu heavy chain, Ig light immune destruction, and progression to frank malignancy (1–3). chains (l and k), mb-1 (Iga), and the tyrosine kinases Btk (8). Epithelial cancer cells in their primary site are knit together by More recently, Spi-B has been detected in intestinal M cells, whose extensive intercellular junctions to form an epithelial cell sheet. To expression activates GP2 gene and endows M cells with antigen metastasize, individual or a small cluster cancer cells must first presentation capacity, thus playing essential role in controlling M- dissociate from their neighboring epithelial cells and invade the cell differentiation (9). surrounding stroma. The specific epigenetic mechanisms control- Many reports have linked Spi-B with hematopoietic tumor- ling this process are poorly understood. igenesis. SPIB is recurrently amplified and occasionally trans- located in the activated B-cell–like subtype of diffuse large B-cell lymphoma (ABC DLBCL). Its expression is required for 1 Department of Immunology, Biochemistry and Molecular Biology, Collaborative the survival of ABC DLBCL lines and contributes to apoptosis Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of resistance via the PI3K–AKT pathway (10–12). In addition, Medical Epigenetics, Tianjin Medical University, Tianjin Medical University Can- fi cer Institute and Hospital, Tianjin, China. 2School of Medical Laboratory, Tianjin gene expression pro ling analysis has detected Spi-B in some Medical University, Tianjin, China. 3Department of Pathology, Tianjin First Center malignant solid tumors including gastric cancer (13) and Hospital, Tianjin, China. 4State Key Laboratory of Experimental Hematology, colorectal cancer (14). IHC staining also detected Spi-B in Institute of Hematology and Blood Diseases Hospital, Chinese Academy of hepatocellular carcinoma (15, 16), suggesting that Spi-B may 5 Medical Sciences and Peking Union Medical College, Tianjin, China. Key Lab- be aberrantly expressed in some solid tumors. However, the oratory of Immune Microenvironment and Disease of the Ministry of Education, functional consequence of Spi-Bexpressionincarcinomasis Tianjin Medical University, Tianjin, China. 6Department of Lung Cancer Center, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. completely unknown. Here, we report that Spi-B is expressed in invasive cancer Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). cells in human primary lung cancer tissues. Expression of Spi-B in lung cancer cells downregulates claudin-2 thereby W. Du, X. Xu, and Q. Niu contributed equally to this article. disrupting intercellular junctions and enhancing invasive Corresponding Author: Zhe Liu, Tianjin Medical University, 22 Qixiangtai Road, behavior. These data identify an epigenetic process linking Heping District, Tianjin 300070, China. Phone: 86-22-8333-6533; E-mail: hematopoietic lineage gene control with local invasion in [email protected] metastatic carcinoma cells, and suggest that Spi-B may be an doi: 10.1158/0008-5472.CAN-17-0020 effective biomarker for both prognosis and treatment of lung 2017 American Association for Cancer Research. cancer.

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Materials and Methods were seeded into the top chamber and complete media was placed in the bottom chambers as a chemoattractant. The chambers were Cells incubated for 20 hours at 37 C with 5% CO . Experiments were Human bronchial epithelial cells (HBEC) are normal human 2 performed in triplicate. Migrated cells on the undersides of filter bronchial epithelium immortalized by hTERT and CDK4, and membrane were fixed in 4% formalin and stained with crystal were obtained from Dr. Jerry Shay (UT Southwestern Medical violet. The migrated cells were counted using light microscopy. Center, Dallas, TX) in 2008 at the passage 30. A549, H460, HCC827, H1155, H69, H526, H82, and Lewis lung carcinoma (LLC1) cells were obtained from ATCC within the past 10 years Soft agar 4 and maintained in ATCC recommended media supplemented Cells (1 10 ) were resuspended in DMEM containing 10% m FBS with 0.35% agarose and layered on top of 0.6% agarose in with 10% FBS, 100 U of penicillin/mL, and 100 g of strepto- mycin/mL. All experiments were performed within 1 month after DMEM on 6-well plates. Cells were cultured for 21 days at 37 C thawing early-passage cells. A549, H460, HCC827, H1155, H69, with 5% CO2. Experiments were performed in triplicate. Colonies H82, and H526 cells were authenticated in April 2017. DNA were stained, analyzed morphologically, and counted using light purified from above cell lines were tested by the short tandem microscopy. repeat analysis method using Promega PowerPlex 1.2 analysis system (Genewiz Inc.). Data were analyzed using GeneMapper4.0 3D Matrigel culture software and then compared with the ATCC databases for refer- These assays were optimized from previous publication (17). ence matching. Tumor cells were detached with 0.25% Trypsin-EDTA, centrifuged (1,000 rpm for 3 minutes), resuspended, and counted. Single cells 3 In vivo metastasis assay (2 10 per well performed in triplicate) were mixed into 0.4 mL LLC1 expressing empty vector or SPIB were selected by cell of RPMI1640 medium supplemented with 2% FBS and 5% – sorting for GFP expression (FACS Vantage, BD Biosciences). A chilled growth factor reduced Matrigel (BD Biosciences), and 5 m cultured in suspension in 24-well ultra-low attachment plate total of 10 cells of each group in 100- L saline were subcutaneously injected into 8-week-old C57BL/6 mice. Tumors in situ were (Corning) at 37 C for 14 days. Experiments were performed in excised 2 weeks after the inoculation of cells. Two weeks after triplicate. The organoids were categorized on the basis of their resection, the mice were sacrificed and metastatic nodules forma- morphology (18). tion in the lungs was analyzed. A total of 106 cells of LLC1 cells in 100-mL saline were injected Chromosome conformation capture into the tail vein of 8-week-old C57BL/6 mice. At one month Chromosome conformation capture (3C) was performed as following injection, the mice were sacrificed and metastatic described previously (19). A total of 106 cells were cross-linked, nodules formation in the lungs was analyzed. lysed, and nuclei were digested with DpnII. After ligation and LLC1-luc cell line was established using a lentivirus encoding subsequent DNA purification, the cross-linking frequencies the luciferase gene, and stable clones were isolated by puromycin between the anchor and test fragments were estimated by PCR selection. GFP-sorted LLC1-luc cells expressing empty vector, reactions relative to standards. Three PCR products together SPIB,orCLDN2 and SPIB were subcutaneously injected as above. containing from the CLDN2 gene promoter to all upstream The whole lungs were immediately grinded in liquid nitrogen and regulatory elements were amplified, mixed at equal molar ratios, total protein was used to detect tumor metastasis by assaying digested with DpnII, and ligated at high concentrations to generate luciferase activity. All animal procedures were approved by Ani- all possible ligation products. The cross-linking and ligation mal Care and Use Committee at Tianjin Medical University and efficiencies between different samples were normalized by setting conform to the legal mandates and national guidelines for the care the highest cross-linking frequency to 1.0. Primers used in this and maintenance of laboratory animals. study are provided in the Supplementary Table S1.

Immunohistochemical analysis Chromatin immunoprecipitation Lung cancer tissues were obtained from Tianjin Medical Uni- Chromatin immunoprecipitation (ChIP) was performed as versity Cancer Institute and Hospital in China. Samples were fixed described previously (19). Antibody against H3K4me3 was from in 4% paraformaldehyde at 4C overnight and embedded in Millipore; antibody against flag M2-agarose was from Sigma. paraffin. Paraffin blocks were cut into 5-mm sections, and were Results were quantified by real-time PCR with SYBR Green dye immunostained with antibodies against Spi-B (Abcam) and clau- using the ABI Prism 7900 system (Applied Biosystems). All PCR din-2 (Bioworld Technology), E-cadherin (BD Transduction lab- signals from immunoprecipitation samples were referenced to oratories), vimentin (Sigma), and processed following the stan- their respective inputs to normalize for differences in primer dard protocol for DAB staining. The use of all human lung cancer efficiencies. Primers used in this study are listed in Supplementary tissues and clinical data was approved by the Institutional Review Table S1. Board of Tianjin Medical University. Informed consent was provided in accordance with the Declaration of Helsinki. Samples Luciferase assay were deidentified prior to analysis. DNA fragments of CLDN2 promoter and cis-regulatory elements upstream were amplified from HUVEC DNA using primers Invasion assay listed in Supplementary Table S1. CLDN2 promoter was inserted Assays were performed in Transwell inserts with 8-mm pores into the XhoI and the HindIII site of the polylinker region pGL3- (BD Biosciences) coated with 20% growth factor–reduced Matri- basic. Cis-regulatory elements were inserted into the KpnI and XhoI gel. Tumor cells in serum-free medium (2 105 cells per well) site. Cell lines were transiently cotransfected either in triplicate or

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in duplicate with pRL-CMV Renilla luciferase reporter, which was testing (GraphPad Prism). Comparison of expression levels used for normalization (Promega). Cell lines were harvested and between Spi-B and claudin-2 was determined by using the Pearson assayed for luciferase activity using Dual-Luciferase reporter assay correlation test and the Spearman rank correlation test. A P value systems (Promega) following the manufacturers' instructions at of less than 0.05 was considered statistically significant for all tests. the time of 24–48 hours after transfection. Results EMSA Nuclear proteins from A549-expressing Spi-B cells were Spi-B expression is correlated with poor prognosis in human extracted using a NucBuster protein extraction kit (Novagen) lung cancer – according to the manufacturer's instructions. Double-stranded We studied Spi-B expression in 130 non small cell lung cancers oligonucleotides corresponding to the potential Spi-B–binding (NSCLC), including lung adenocarcinoma and squamous lung sites 50-GGT CCC CAA ACA TTC CTC CCT CAG TGA CAC CTT TC- carcinoma, in 14 small-cell lung cancers (SCLC), and in 10 tumor- 30and 50-CAA TTG GTA GTT CCT CCC ACT TTT CA-30 were end- adjacent normal lung tissues using IHC staining. Epithelial cells in labeled with biotin. Binding assays were performed in 10 mLof tumor-adjacent lung tissues did not express Spi-B. Lymphocytes in reaction mixture containing 2 mg of nuclear proteins, 10 mmol/L tumor-adjacent tissue expressed Spi-B (Fig. 1A, a), consistent with Tris, 55 mmol/L KCl, 1 mmol/L dithiothreitol, 5% glycerol, previous reports (6). Both NSCLC and SCLC expressed Spi-B (Fig. – 0.05% NP40, 2.5 mmol/L MgCl , 0.25 mmol/L EDTA, 1 mgof 1A, b d). Interestingly, we noticed strong nuclear staining of Spi-B 2 – poly (dIdC), and 1 nmol/L labeled probes at room temperature in cancer cells in the invasive strand at the tumor stromal border – for 30 minutes. For supershift assays, 2 mg of nuclear protein in NSCLC tissue sections (Fig. 1A, e). These Spi-B expressing extracts were incubated with Spi-B antibody for 30 minutes on ice invasive NSCLC cells lacked the epithelial marker E-cadherin (Fig. before incubating with oligonucleotide. Reactions were analyzed 1A, f), but coexpressed the mesenchymal marker vimentin (Fig. by electrophoresis on a 6.0% nondenaturing polyacrylamide gel 1A, g). These data indicate that Spi-B is expressed in cancer cells at 100 V for 1 hour. After transfer, the membrane was immediately with mesenchymal attributes. fi cross-linked for 1 minute on a UV-light crosslinker instrument Quanti cation of staining based on the intensity of Spi-B – equipped with 254 nm bulbs. A chemiluminescent detection nuclear staining and percentage of Spi-B positive tumor cells method utilizing a luminol/enhancer solution and a stable per- revealed higher expression of Spi-B in SCLCs than in NSCLCs oxide solution (Pierce Biotechnology) was used as described by (Fig. 1B). In addition, higher Spi-B staining intensity was associ- P ¼ the manufacturer and the membrane was exposed to X-ray films to ated with higher histologic grade of NSCLC (Fig. 1C, ANOVA, fi visualize the bands. 0.0018787). To assess the prognostic signi cance of Spi-B, we examined expression levels in resected NSCLC from subjects with known clinical outcomes. Subjects with stage II disease (n ¼ 73), Transmission electron microscopy whose tumors had low Spi-B staining intensity (staining scores Cells in three-dimensional basement membrane gels were fixed 4, n ¼ 47) had longer survival times than those whose tumors had in a fixative solution containing 2.5% glutaraldehyde. After being high staining intensity (staining scores > 4, n ¼ 26), with median washed several times in PBS, the cells were then postfixed with 2% survivals of 48 months (low Spi-B) versus 39.5 months (high Spi- osmium tetroxide for 1 hour at room temperature in darkness, B, P ¼ 0.001, Fig. 1D). Consistently, a highly significant negative dehydrated in ascending ethanol solutions and absolute acetone, correlation was found between Spi-B expression level and overall immersed in 50% Epon812 in acetone and finally embedded in survival time in 9 SCLC subjects with available survival data (Fig. Epon812. Ultrathin sections were mounted on copper slot grids 1E). Analysis of Spi-B staining intensity in another cohort of 62 and stained with uranyl acetate and lead citrate, and then primary NSCLC tissues (stage II), including 43 primary NSCLC observed under a HITACHI HT7700 electron microscope. tissues without lymphatic metastasis and 19 primary NSCLC tissues with different degrees of lymphatic metastasis, revealed fi Expression pro ling that increased Spi-B expression was associated with lymphatic fi The expression and functional pro les of genes were compared metastasis (Fig. 1F). These results indicate that the lymphocyte- SPIB between A549 cells expressing empty vector and using restricted protein, Spi-B, is ectopically expressed in lung cancers, Agilent SurePrint G3 Human Gene Expression 8 60 K v2 and its high expression level correlates with poor prognosis in fi Microarray (Agilent Technologies). Analysis of functional pro l- human lung cancer. ing of Spi-B–regulated genes was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Spi-B expression promotes lung metastasis in vivo based on the biological pathways from KEGG (Kyoto Encyclo- To test the functional consequences of Spi-B expression in lung pedia of genes and genomes) database. cancers in vivo, lentiviral constructs were introduced into mouse Lewis lung carcinoma (LLC1) cells, which lack detectable endog- Accession numbers enous Spi-B expression, and the metastatic capability was The Gene Expression Omnibus accession number for A549 cell assessed. Subcutaneous engraftment of Spi-B–expressing LLC1 expression profile data is GSE90645. cells formed comparable size of primary tumors but produced significantly more lung metastatic nodules, compared with vec- Statistical analysis tor-transduced cells (Fig. 2A and B), indicating a prometastatic Results were reported as mean SD unless otherwise noted. role of Spi-B. However, direct intravascular inoculation of cells SPSS 18.0 was used for statistical analysis. Correlation of the into the tail vein of syngeneic mice, which bypasses the local expression levels between Spi-B and survival rates were deter- invasion and vascular invasion steps, showed a modest increase mined with Kaplan–Meier analysis using Mantel–Cox log-rank in numbers of metastatic nodule in lung in mice bearing

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Figure 1. Spi-B is expressed in lung cancer cells and predicts poor prognosis. A, IHC staining with anti-Spi-B was performed on 10 tumor-adjacent normal lung tissues, 130 NSCLC, and 14 SCLC specimens. Representative fields show lack of Spi-B staining in normal lung epithelial cells but positive Spi-B staining in lymphocytes (arrows) in tumor adjacent tissue (a), positive Spi-B staining in human lung adenocarcinoma (b), squamous lung carcinoma (c), and SCLC (d). e–g, Series sections of human primary lung cancer tissues stained with Spi-B (e), E-cadherin (f) and vimentin (g). Scale bars, 50 mm. Arrows, invasive cancer cells. B, The frequency of cases with no (0), low (0.1–3.9), or high (4.0–8.0) Spi-B staining stratified by IHC-defined lung cancer subtype. C, The frequency of cases with no (0), low (0.1–3.9), or high (4.0–8.0) Spi-B staining stratified by tumor grade. D, Kaplan–Meier survival rates for 73 subjects with stage II NSCLC disease with low (staining scores 4, n ¼ 47, red line) versus high (staining scores > 4, n ¼ 26, blue line) Spi-B expression were compared. Median survivals were 48 months (low Spi-B) versus 39.5 months (high Spi-B; P ¼ 0.001). E, Semiquantitative scoring was performed and Spi-B scores were correlated with overall survival time of SCLC patients (r2 ¼ 0.874; P ¼ 0.0002). F, Spi-B expression in human primary lung cancer tissues stratified by lymphatic metastasis.

Spi-B–expressing LLC1 cells, but this did not reach statistical tently, enriched Spi-B–expressing lung cancer cells were observed signiﬁcance (P ¼ 0.0849; Fig. 2C and D). Thus, Spi-B expression in lymphatic metastases compared with their corresponding pri- may function at early events of metastasis, without markedly mary tumor (Fig. 2E). Quantitative analysis revealed signiﬁcantly affecting the later metastatic cascades including circulation, higher Spi-B staining intensity in lymphatic metastases than in extravasation, and colonization. primary lung cancer tissues (Fig. 2F). Altogether, these data indicate We next examined Spi-B expression in 27 primary human lung that Spi-B expression promotes lung cancer metastasis via increas- cancer tissues and their matched lymphatic metastases. Consis- ing early dissemination of cancer cells from their primary sites.

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Figure 2. Spi-B promotes lung metastasis. A, LLC1 cells expressing empty vector or SPIB were subcutaneously injected into 8-week-old female C57BL/6 mice. Two weeks later, the primary tumors were removed. Another two weeks later, the mice were sacrificed and the lung metastases were analyzed. Left, images of primary tumors. Middle, representative images of lungs. Black arrow, metastatic nodules. Right, quantitation of lung metastatic nodules. B, Representative hematoxylin and eosin stain of lungs from mice subcutaneously injected with vector or SPIB-expressing LLC1 cells. Scale bars, 50 mm. C, LLC1 cells expressing empty vector or SPIB were injected into the tail vein of 8-week-old female C57BL/6 mice. One month later, the mice were sacrificed and the lung metastases were analyzed. Left, representative gross lung pictures. Black arrow, metastatic nodules. Right, quantitation of lung metastatic nodules. D, Representative hematoxylin and eosin stain of lungs from mice injected via tail vain with vector or SPIB-expressing LLC1 cells. Scale bars, 50 mm. E and F, IHC staining with anti-Spi-B was performed on 27 primary human lung cancer tissues and their matched lymph node metastases. E, Representative pictures of Spi-B staining in human primary lung cancer tissues and lymphatic metastases. Scale bars, 50 mm. F, Quantified Spi-B expression in primary human lung cancer tissues and lymph node metastases.

Spi-B disrupts intercellular junctions and increases invasive mesenchymal marker vimentin or the expression of EMT-related capacity in vitro transcription factors (Snail1, Snail2, ZEB1, and Twist1; Supple- We next investigated the effects of Spi-B expression in estab- mentary Fig. S1C), indicating that Spi-B alone is not sufﬁcient to lished lung cancer cell lines in vitro. We examined Spi-B expression induce the EMT program; other oncogenic mutations may be in various lung epithelial cell lines, including 1 immortalized required. Indeed, knockdown of Spi-B in H1155, a NSCLC line human bronchial epithelial cell line (HBEC), 4 NSCLC cell lines, that expresses endogenous Spi-B and have mesenchymal pheno- and 3 SCLC cell lines. Spi-B mRNA was detected in 1 NSCLC cell type, resulted in upregulation of E-cadherin and downregulation line (H1155) and 3 SCLC cell lines (H526, H69, H82; Fig. 3A), of vimentin, Snail1, ZEB1, and Twist1 (Supplementary Fig. S1B consistent with the immunostaining results showing that more and S1C), fostering epithelial characteristics of H1155. These data cases of SCLC expressed Spi-B than NSCLC. Given that Spi-B is suggest that Spi-B might be essential in maintaining the mesen- primarily expressed in cancer cells with mesenchymal attributes in chymal phenotype of lung cancer cells although it is not able to human primary NSCLC tissues, we tested whether Spi-B can induce the EMT program in epithelial cells. induce the epithelial–mesenchymal transition (EMT) program. Then, we transduced lentiviral constructs into A549 and H460 Transient expression of Spi-B in HBECs caused elongated mor- cells, two NSCLC lines that do not express endogenous Spi-B, and phology in two-dimensional culture and inhibited expression of analyzed the resultant biological effect. Spi-B expression did not the epithelial marker E-cadherin in HBECs (Supplementary Fig. alter the number of colonies formed in soft agar (Fig. 3B and C). S1A and S1B); however, Spi-B did not alter expression of the Interestingly, Spi-B expression caused signiﬁcant morphologic

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Figure 4. Changes of gene expression profiling upon Spi-B expression. A, Functional profiling of genes differentially expressed between control A549 cells and SPIB- expressing A549 cells. Double-headed arrows, 315 genes upregulated and 278 genes downregulated by Spi-B. Representative Spi-B–induced (red) and Spi-B–repressed genes (green) are listed vertically (left) and under each molecular pathway (right). B, Quantitative RT-PCR was performed to confirm the transcriptional change of indicated genes identified in the microarray. RNA was purified from control or SPIB-expressing A549 cells and control or Spi-B shRNA–expressing H1155 cells. Relative expression is shown as fold differences relative to GAPDH. C, Representative zymograms showed gelatinolytic activity of MMP9 in control or Spi-B–expressing A549 cells.

change of the clones in soft agar. Whereas control cells formed exhibited fewer cell–cell interactions (Fig. 3B and C). In addition, tightly organized spheres, 43% of Spi-B–expressing A549 cells and while control cells developed into round or oval cell aggregates 38% of Spi-B–expressing H460 cells appeared dispersed and with strong cell–cell adhesion in three-dimensional basement

Figure 3. Spi-B disrupts intercellular junctions and promotes invasiveness and migration. A, RT-PCR shows Spi-B transcription in HBECs and various lung cancer cell lines. B and C, Spi-B–expressing cancer cells and control cells were allowed to grow in soft agar for 3 weeks, and colonies were counted (top). Representative images of the clones are shown at the bottom. The bar chart shows the percentage of mass (blue) and loose colonies (red). Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 100 mm. D and E, Phase-contrast micrographs of control and SPIB-expressing cancer cells cultured on Matrigel for 8 days. The bar chart shows the percentage of mass (blue) and grape-like (red) colonies. Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 25 mm. F and G, TEM sections show intercellular junctions of control and SPIB-expressing cancer cells cultured on Matrigel for 8 days. The bar chart shows the number of tight junctions per field. Error bars, means SD for eight fields in a representative experiment. Scale bars, 0.2 mm. H and I, Control and SPIB-expressing cancer cells were subjected to an invasion assay. Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 100 mm. J, H526 cells expressing control or shRNA against Spi-B were subjected for invasion assay. The efficiency of Spi-B knockdown is shown in top panel. Error bars, means SD for a representative experiment performed in triplicate. K, Images of wound-healing assay showed the motility of control and SPIB-expressing A549 cells.

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Figure 5. Spi-B negatively correlates with claudin-2 in human lung cancer tissues. A, Immunoblots show expression of claudin-2, Spi-B, and actin. B, IHC for Spi-B and claudin-2 was performed on 39 human primary lung cancer specimens (stage I–II). Representative fields show strong Spi-B staining in peripheral lung cancer cells. Cancer cells in the center field of the tumor mass are Spi-B negative. Antibodies to claudin-2 stain the cancer cells in the center field of the tumor mass; peripheral cancer cells are negative for claudin-2 staining. C, Semiquantitative scoring was performed and Spi-B scores were correlated with claudin-2 scores (r2 ¼ 0.236; P ¼ 0.00054).

membrane gels, a major portion of Spi-B–expressing cancer cells different molecular pathways are shown in Supplementary Tables developed into a grape-like morphology with poorly adhesive S2 and S3. colonies (Fig. 3D and E). Transmission electron microscopy We used quantitative RT-PCR to confirm Spi-B–dependent (TEM) showed impaired formation of tight and adherence junc- differential transcription of 9 genes identified in the microarray tions in Spi-B–expressing cells compared with control cells in analysis, including the downregulation of tight junction genes three-dimensional basement membrane gels (Fig. 3F and G). CLDN2, CLDN14, CGN, and CRB3, and the upregulation of PGF, Consistently, knockdown of Spi-B in H526 cells resulted in tighter FOS, RAC2, and TNFSF10, which promotes cancer metastasis, and cell aggregates with strong cell adhesion in two-dimensional upregulation of MMP9, which degrades extracellular matrix and culture (Supplementary Fig. S2A). These data indicate that Spi- enhances tumor invasion (Fig. 4B). The upregulation of secreted B expression disrupts intercellular junctions. active MMP9 by Spi-B was further confirmed by gelatin zymo- In addition, Spi-B overexpression caused enhanced invasive- graphy (Fig. 4C). Consistently, knockdown of Spi-B in H1155 ness of A549 and H460 cells through Matrigel (Fig. 3H and I). cells caused reciprocal changes in expression levels of these genes Conversely, knock down of Spi-B in H526 cells caused reduced compared with expression of Spi-B in A549 cells (Fig. 4B). These invasion (Fig. 3J). Spi-B overexpression also increased motility of data indicate that Spi-B regulates tight junctions, proteases, and A549 cells on collagen-coated plates in scratching assays (Fig. 3K). metastasis-related genes. Transient expression of Spi-B had no effect on cell proliferation or adherence to fibronectin, a major extracellular matrix component, Expression of Spi-B is negatively correlated with claudin-2 in as evaluated by bromodeoxyuridine (BrdUrd) incorporation human primary lung cancer tissues analysis and replating assay (Supplementary Fig. S2B and S2C). Epithelial cells interact with neighboring cells through various Altogether, these data indicate that the major function of Spi-B in intercellular junctions such as tight, gap, and Adherens junctions. epithelial cancer cells is to disrupt the intercellular junctions and Tight junctions are associated with cell polarity and permeability promote invasion. (20). Given that overexpression of Spi-B in epithelial cancer cells resulted in the disruption of cell–cell adhesion and the loss of Spi-B downregulates tight junction genes polarity, we focused on the tight junction genes that were down- To better understand the molecular mechanisms of Spi-B– regulated by Spi-B expression. Claudin-2, a key component within mediated promotion of metastasis, we expressed Spi-B in A549 tight junctions and whose downregulation was associated with cells and used a microarray platform to analyze the resultant breast cancer metastasis (21–23), was primarily expressed in expression profile. Genes for which expression was substantively bronchial epithelium in lung tumor-adjacent tissue (Supplemen- altered in the stable Spi-B-expressing cell lines were functionally tary Fig. S3A) and detected in some epithelial cells of alveoli in classified by gene ontology. Through analyses of enrichment lung tumor-adjacent tissue (Supplementary Fig. S3B). Downre- within each category, two groups of genes that significantly gulation of claudin-2 by Spi-B was confirmed by immunoblot at changed expression levels (2-fold in either direction) after Spi- the protein level (Fig. 5A). B overexpression were identified. Tree 1 included 278 down- To define the correlation between Spi-B and claudin-2 in regulated genes and tree 2 contained 315 upregulated genes on individual primary lung cancer tissues, we used IHC to study the Spi-B overexpression (Fig. 4A). Functional profiling of these expression levels of Spi-B and claudin-2 in serial sections of downregulated genes revealed that the major proportion of the NSCLC samples. The representative pictures are shown in Fig. genes was associated with regulation of cell interactions including 5B. Claudin-2 was expressed in cancer cells that located in the regulation of actin cytoskeleton and tight junctions, which is central part of the lung cancer mass. These cancer cells did not consistent with the morphologic changes observed in Matrigel express Spi-B. The invasive cancer cells at the tumor-stromal and soft agar. The upregulated genes are involved in the BCR border expressed significant levels of Spi-B but lacked claudin-2. signaling pathway, which is also consistent with the physiologic When intratumoral staining was quantified, a highly significant role of Spi-B. Individual Spi-B–regulated genes and subgroups of negative correlation was found between Spi-B and claudin-2

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expression (Fig. 5C). Therefore, endogenous Spi-B represses clau- A549 cells (Fig. 7B), indicating that P1 is the active promoter in din-2 in human lung cancer tissues. A549 cells. To further study whether the CLDN2 gene is regulated directly by Spi-B, we examined the in vivo binding status of Spi-B in Spi-B promotes metastasis by repressing claudin-2 genomic loci of CLDN2 using ChIP. Cis-regulatory elements are To assess whether Spi-B promotes cancer metastasis through identified by DNase I hypersensitive sites, as reported in the claudin-2 repression, we first tested the cellular effect of claudin-2 ENCODE database, and potential Spi-B–binding consensus depletion. The efficiency of claudin-2 knockdown is shown in Fig. sites were analyzed for Spi-B association (Fig. 7C). The ChIP 6A. Claudin-2 depletion in A549 cells exhibited similar cellular analysis revealed the occupancy of endogenous Spi-B in H526 effects as Spi-B overexpression, including decreased cell–cell cells, as well as the transfected Spi-B in A549 cells at three adhesion in soft agar, the loss of polarity in 3D Matrigel, and regions (sites 2, 4, and 8) upstream of the P1 (Fig. 7C). We enhanced invasion through Matrigel (Fig. 6B–D). termed these three Spi-B occupied cis-regulatory elements as S1 We next reexpressed claudin-2 in Spi-B–overexpressing A549 (26,001 to 24,893), S2 (21,765 to 21,450), and S3 cells (Fig. 6E). As expected, reexpression of claudin-2 completely (7,479 to 7,201), which are 25 kb, 21 kb, and 7.5 kb restored the formation of round cell aggregates with strong cell– upstream of the active promoter, respectively. cell adhesion in soft agar (Fig. 6F), and decreased the proportion Long-range communication requires physical interaction (24, of grape-like organoids in three-dimensional basement mem- 25). Assuming that S1, S2, or S3 controls CLDN2 gene transcrip- brane gels (Fig. 6G). In addition, reexpression of claudin-2 in tion, physical interactions between these three cis-regulatory ele- Spi-B–transfected A549 cells partially abolished Spi-B–induced ments with the promoter are expected in active or silenced invasion through Matrigel (Fig. 6H). However, reexpression of CLDN2. We therefore performed a chromosome conformation other Spi-B–downregulated tight junction proteins, claudin-14 capture (3C) assay to explore the chromatin configuration of the and Crumbs 3, was not able to restore these changes in cellular CLDN2 gene in A549 and H526 cells, focusing on DNA fragments behavior caused by Spi-B overexpression (Supplementary Fig. carrying the S1, S2, S3, and the P1. Briefly, cross-linked chromatin S4A–S4C). Therefore, the changes in cellular behavior caused by was digested with Dpn II, diluted, religated, and long-range enforced Spi-B expression are primarily due to repression of association frequencies were assessed with PCR. Indeed, physical claudin-2. interactions between the S3 and the P1 were detected in the active To determine whether Spi-B promotes metastasis in vivo CLDN2 gene in A549 cells when using P1 as an anchor fragment through claudin-2 repression, we reexpressed claudin-2 in Spi- (Fig. 7D). In addition to the S3–P1 interaction, the S1–P1 inter- B–expressing luciferase-labeled LLC1 cells and subcutaneously action was also detected in the silenced CLDN2 gene in H526 cells injected the cells into C57BL/6 mice as described above. Two (Fig. 7D). The S2, however, did not interact with the promoter weeks after removal of the primary tumors, the whole lung tissues either in A549 or H526 cells (Fig. 7D). Consistently, the H526- were collected for protein extraction and to perform luciferase specificS1–P1 interaction was also detected when using the S1 as assays to quantify metastases. Reexpression of claudin-2 reduced the anchor (Fig. 7E). These results suggest that the physical the numbers of both visible lung metastatic nodules and the interaction between the Spi-B–occupied cis-regulatory element metastatic luciferase-expressing LLC1 cells to baseline levels (Fig. S1 and the promoter may be associated with CLDN2 gene silenc- 6I and J). Therefore, we identified claudin-2, a tight junction ing. Then, we overexpressed Spi-B in A549 cells and evaluated the protein, as the main target of Spi-B in promoting metastasis of effect of Spi-B on chromatin configurations of CLDN2. Expect- lung cancer cells. These results offer an explanation for the edly, transient expression of Spi-B caused juxtaposition of the S1 association of Spi-B with lymphatic metastasis and overall short to the P1 but had no effect on the interaction between the S3 and survival in human lung cancer patients. the P1 (Fig. 7F). Thus, aberrant activation of Spi-B in epithelial Thus, we propose a model whereby ectopic expression of Spi-B cancer cells establishes a physical interaction between a cis-regu- by some cancer cells at the tumor-stromal border disrupts inter- latory element, S1, with the active promoter of CLDN2. cellular junctions by silencing CLDN2 transcription, facilitating To investigate the functional consequence of juxtaposition of the dissociation of cancer cells from the primary epithelial carci- S1 to the P1 on CLDN2 transcription, we performed luciferase noma cell sheet on one hand, and promoting the dissociated reporter gene assays, a commonly used method for promoter and cancer cells to invade through the surrounding stroma by increas- cis-regulatory element (enhancer or silencer) characterization (26, ing MMP9 expression on the other hand. Therefore, Spi-B–expres- 27). We cloned the S1 or the S3 upstream of the P1 flanking the sing cancer cells have higher metastatic capability (Fig. 6K). luciferase gene and transfected the constructs into HEK293 cells that normally express Spi-B (Fig. 7G). While the S3 has no effect Spi-B establishes a long-range silencer–promoter interaction on the promoter activity, ectopic placement of the S1 upstream of and represses CLDN2 transcription the P1 completely reduced the promoter activity to the baseline We next explored the mechanism by which Spi-B silences level (Fig. 7H), indicating that the S1 functions as a silencer of the claudin-2 expression. Claudin-2 is transcribed in A549 cells but P1. Two Spi-B–binding consensuses were identified within the S1. not in H1155, H526, H69 and H82 cells (Fig. 7A). This expression EMSA with super shift assay showed direct binding of Spi-B to pattern for claudin-2 is opposite from that which we found for these two sequences in vitro (Fig. 7I). Cotransfection of SPIB Spi-B. CLDN2 contains three alternative promoters generating shRNA or mutation of two Spi-B–binding sites within the S1 three differential transcript variants with distinct 50 untranslated partially restored promoter activity (Fig. 7J), suggesting that in regions, yet encoding identical proteins. The ChIP analysis to scan addition to establishing a silencer–promoter interaction Spi-B 10 regions along CLDN2 for distribution of H3K4Me3, which binding to the silencer represses promoter activity. marks active promoters, and H3K9acetyl, which associates with These data support a model whereby CLDN2 transcription the promoter and early coding region of active genes, revealed requires decolocalization of an upstream silencer, S1, with the enrichment of these two modified histones in P1 (Promoter1) in promoter. Spi-B associates with multiple upstream sites including

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4818 Cancer Res; 77(18) September 15, 2017 Cancer Research

Spi-B and Lung Cancer Metastasis

those within the S1, establishing silencer–promoter interactions, lung cancer cell lines downregulates E-cadherin expression and and blocking CLDN2 transcription (Fig. 7K). increases cell motility and invasive capability in vitro. Depletion of Spi-B in lung cancer cell lines that express endogenous Spi-B and have mesenchymal attributes promotes MET. These findings Discussion highlight the role of Spi-B in EMT program induction. Because For many years, the hypothesis that pathologic processes can be Spi-B is not able to induce expression of vimentin or EMT-TFs in achieved by co-opting a series of physiologic processes has been HBECs, and it has been documented that transformation of discussed. In this study, we identified Spi-B, a lymphocyte-restrict- HBECs requires series oncogenic mutations and/or genetic ed transcription factor, as a master modulator of invasion of lung manipulation accompanied with microenvironmental induction cancer cells. Physiologically, expression of Spi-B starts in pre-B (36–38), it is possible that Spi-B may promote EMT through lymphocytes and reach a peak in immature and mature B lym- disruption of intercellular junctions, but this effect requires other phocytes (6, 28), which parallels the process of expansion of oncogenic mutations or specific microenvironment. developmentally mature B lymphocytes from bone marrow to Second, Spi-B markedly enhance expression of MMP9, a key peripheral circulation. Loss of Spi-B can cause an increased pre-B protease that is highly expressed in lymphocytes for matrix population within the bone marrow environment with dimin- proteolysis to facilitate their departure from bone marrow (39– ished recirculating B lymphocytes (29). Therefore, Spi-B is critical 41), conferring carcinoma cells the ability to remodel the sur- for the departure of developmentally mature B lymphocytes from rounding matrix. Multiple steps of the invasion process require their developmental niche. Similarly, ectopic expression of Spi-B protease-mediated matrix remodeling. For example, protease is in lung cancer cells promotes their dissemination from the pri- required for carcinoma cells to breach the basement membrane mary sites. From this point of view, Spi-B may activate function- that confine the tumor to a local position, to liberate growth factor ally similar programs in lung carcinoma. molecules that are tethered to the basement membrane or stroma, Spi-B is first identified as a transcriptional activator, directly and to invade the stroma. Epithelial cells are unable to produce activating c-Rel (29), P2Y10 (30), and p50 (31). Our results show proteases. Carcinoma cells that retain epithelial markers follow that Spi-B can also act as a transcriptional repressor. Multiple behind stromal fibroblasts that remodel the extracellular matrix to transcription factors including Miz-1 and GATA3 have been found invade (42). Mesenchymal cells are capable of matrix remodeling to act as both a transcriptional activator and repressor. It was (42), which is consistent with our result that Spi-B is expressed in reported that whether they function as a transcriptional activator cancer cells with mesenchymal attributes and upregulates MMP9. or a repressor depends on their interactions with other transcrip- Knockdown of MMP9 or overexpression of its inhibitor tissue tional regulators (32, 33). Spi-B expression endows lung cancer inhibitor of metalloproteinases 1 (TIMP1) abrogated Spi-B– cells with two properties that are normally required for expansion induced invasion through Matrigel in vitro (Supplementary Fig. of developmentally mature B lymphocytes. First, Spi-B down- S5A and S5B). In addition to degrade matrix, MMP9 is involved in regulates proteins that constitute intercellular junction com- TGFb activation (43), angiogenesis by increasing VEGF bioavail- plexes, resulting in disruption of tight and adherens junctions. ability (44) and is critical for both the invasion of the primary It has been documented that disruption of tight and adherens tumor and the formation of the metastatic niche (42, 45–47). junctions impairs epithelial polarity and differentiation, inducing Invasion is a fundamental step in tumor progression and a EMT and metastatic behavior (34, 35). Consistent with that, Spi-B driving force for metastasis. Solid tumors invade the adjacent is mainly expressed in the invasive cancer cells at the tumor– tissue by single cancer cells or a cluster of connected tumor cells stromal border in human primary lung cancer tissues. These Spi- (47, 48). Two models have been proposed for individual cancer B–expressing cancer cells coexpress the mesenchymal marker cell invasion: mesenchymal invasion or amoeboid invasion. vimentin but lack the epithelial marker E-cadherin, and thus, Mesenchymal invasion is characterized by an elongated morphol- exhibit mesenchymal attributes. Enforced expression of Spi-B in ogy that requires extracellular proteolysis localized at cellular

Figure 6. Spi-B disrupts intercellular junctions and promotes invasion by silencing claudin-2 expression. A, Immunoblots show the efficiency of claudin-2 knockdown. B, A549 cells were transduced with control or shRNAs against claudin-2 as indicated. The cells were grown in soft agar for 3 weeks. The representative images of the clones are shown in the left. The bar chart shows the percentage of mass (blue) and loose colonies (red). Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 100 mm. C, A549 cells were transduced with control or shRNAs against claudin-2 as indicated and plated in Matrigel. Phase contrast of acini is shown on left and quantified on right. The bar chart shows the percentage of mass (blue) and grape-like (red) colonies. Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 25 mm. D, A549 cells were transduced with control or shRNAs against claudin-2 as indicated and subjected to an invasion assay. Error bars, means SD for a representative experiment performed in triplicate. E, A549 cells were transduced with Spi-B or Spi-B and claudin-2 as indicated. Immunoblots show expression of Spi-B, claudin-2, and actin. F, A549 cells were transduced with Spi-B or Spi-B and claudin-2 as indicated. The cells were grown in soft agar for 3 weeks. The representative images of the clones are shown on the left and the percentages of clones with differential morphology are quantified on the right. The bar chart shows the percentage of mass (blue) and loose colonies (red). Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 100 mm. G, A549 cells were transduced with Spi-B or Spi-B and claudin-2 as indicated and plated in Matrigel. Phase contrast of acini is shown on left and quantified on right. The bar chart shows the percentage of mass (blue) and grape-like (red) colonies. Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 25 mm. H, A549 cells were transduced with Spi-B or Spi-B and claudin-2 as indicated and subjected to an invasion assay. Error bars, means SD for a representative experiment performed in triplicate. Scale bars, 100 mm. I and J, LLC1-Luc cells were transduced with Spi-B or Spi-B and claudin-2 as indicated. The cells were subcutaneously injected into C57BL/6 mice. Two weeks later, the primary tumors were removed and the lung metastases were analyzed. I, The images of the primary tumors (left), the lung (middle), and lung metastatic nodules (right). The proteins were extracted from the whole lung tissue and subjected to luciferase assay. The luciferase activity of each lung is shown in J. , P < 0.05. K, Schematic showing the role of Spi-B in epithelial cancer metastasis. Spi-B is activated in the cancer cells at the tumor–stromal border. Spi-B expression downregulates tight junction protein claudin-2, facilitating dissociation of cancer cells from the epithelial cell sheet on one hand and promoting local invasion through upregulation of MMP9 on the other hand.

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Du et al.

Figure 7. Spi-B establishes S1-CLDN2 promoter interactions. A, RT-PCR shows transcription of CLDN2 and GAPDH. B, Quantitative ChIP shows distribution of H3K4me1 and H3K9ac histone modiﬁcations in A549 cells. Location of regions assessed by ChIP is shown in schematic. Error bars, SD of three independent chromatin preparations. C, Quantitative ChIP was performed showing association of Spi-B in H526 cells and Spi-B-Flag in transfected A549 cells with regions 2–4. Schematic showing DNase I hypersensitive sites (vertical lines) and Spi-B–binding consensus (red stars). Bar graphs show fold enrichment of Spi-B binding. D and E, 3C was used to calculate cross-linking frequency between chromatin segments to assess proximity in A549 (claudin-2–expressing, solid circles) and H526 (claudin-2-nonexpressing, open circles) cells. Vertical lines, DpnII restriction sites; arrows, PCR primer sites and direction. Anchor symbols mark anchoring primer for each dataset. Cross-linking frequency between different segments and P1 (D), S1 (E) is shown. Top panels show representative PCR products. Mean SD of three independent chromatin preparations is shown. F, Cross-linking frequency is shown using the P1 as anchor in A549 cells transiently expressing Spi-B (open circles) or vector control (solid circles). Mean SD of three independent experiments is shown. G, RT-PCR shows transcription of SPIB and GAPDH. H, Luciferase reporter activity is shown with ectopic placement of S1 or S3 adjacent to the P1. Error bars, means SD for a representative experiment performed in triplicate. I, EMSA shows mobility shift of probe with S1 sequence containing the two Spi-B consensus sites, with supershift following anti-Spi-B treatment. J, Cotransfection of Spi-B shRNA or double mutation of two Spi-B–binding consensus within S1 partially abrogated S1 activity in luciferase assay. Error bars, means SD for a representative experiment performed in triplicate. K, Schematic showing the mechanism by which Spi-B represses claudin-2 transcription. Spi-B associates with silencer S1 and establishes physical interaction between S1 and P1, thereby repressing P1 activity.

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Spi-B and Lung Cancer Metastasis

protrusions, whereas amoeboid invasion is characterized by a Administrative, technical, or material support (i.e., reporting or organizing rounded morphology and proteolysis independent (49). These data, constructing databases): Y. Ru two invasion modes are both important for carcinoma metastasis Study supervision: Z. Ma, Z. Yao, Z. Liu and interconvert into each other to adapt to the altered environ- Acknowledgments ment (50). Our results lead us to suggest that Spi-B participates in The authors thank Baocun Sun at Tianjin Medical University for advice. the mesenchymal invasion of lung cancers. The significant association of Spi-B expression in human lung cancers with invasive behavior substantiates its clinical significance in both prognosis Grant Support and therapy. This work was supported by grants (91519331, 31371295 to Z. Liu; 81572271, 81372307 to Z. Ma; 81572882 to Z. Yao; 81502538 to W. Du; fl 81402121 to Y. Jiang) from the National Natural Science Foundation of China, Disclosure of Potential Con icts of Interest grant 2014CB910100 to Z. Liu from the Ministry of Science and Technology of fl No potential con icts of interest were disclosed. China, grant 15JCZDJC34800 from Tianjin Municipal Science and Technology Commission to Z. Liu, grant 2016M591397 to X. Li from the China Postdoctoral Authors' Contributions Science Foundation, and grant 20140602 to W. Du from Tianjin Municipal University Science and Technology Foundation. Conception and design: Z. Liu The costs of publication of this article were defrayed in part by the Development of methodology: Y. Ru payment of page charges. This article must therefore be hereby marked Acquisition of data (provided animals, acquired and managed patients, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate provided facilities, etc.): W. Du, X. Xu, Q. Niu, X. Zhang, Z. Wang, W. Zhang, this fact. J. Yan, Z. Fu, Y. Jiang, Z. Zhang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Wei, X. Li Received January 3, 2017; revised June 6, 2017; accepted July 19, 2017; Writing, review, and/or revision of the manuscript: Z. Liu published OnlineFirst July 28, 2017.

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4822 Cancer Res; 77(18) September 15, 2017 Cancer Research

Spi-B−Mediated Silencing of Claudin-2 Promotes Early Dissemination of Lung Cancer Cells from Primary Tumors

Wei Du, Xing Xu, Qing Niu, et al.

Cancer Res 2017;77:4809-4822. Published OnlineFirst July 28, 2017.

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