Background
Lung cancer is the leading cause of cancer-related mortality worldwide [
1]. The main cause of death is the invasion and metastasis of tumor. EMT is the early event of lung cancer metastasis which is characterized by a loss of cell-cell adhesion and polarity, down-regulation of epithelial markers, and acquisition of mesenchymal markers and phenotype [
2]. Thus, a better understanding of cellular and molecular mechanisms leading to metastatic dissemination of carcinoma cells is of utmost importance.
The human tumor suppressor gene DAL-1 was identified using Differential Display PCR as a gene whose expression was lacking in non-small cell lung cancer when compared with matched normal tissue [
3]. Frequent loss of DAL-1 in cervical cancer [
4], laryngeal squamous cell carcinoma [
5], breast cancer [
6] and esophageal squamous cell carcinoma [
7] suggested that DAL-1 could be a tumor suppressor [
8,
9]. It has also been reported that loss of DAL-1 expression and methylation of the DAL-1 promoter are involved in development and progression of NSCLC (Non small cell carcinoma), providing a possible indicator of poor prognosis. Further studies demonstrated DAL-1 contained FERM (protein 4.1-ezrin-radixin-moesin) and SABD (spectrin-actin-binding domain) domain [
10], enable it links to the transmembrane protein and cytoskeleton. Consequently, DAL-1 was considered as membrane associated protein contributing to cell shape, cell-cell and cell-matrix adhesion, and cell motility. DAL-1 also participates in the organization of cytoskeleton [
6,
11]. E-cadherin the key role in EMT and DAL-1 co-locate in the junction part of cells, therefore, loss of DAL-1 may associate with disruption of cell-cell adhesion, loss of polarity and cancer metastasis.
Herein, the effect of DAL-1 on EMT was identified, and its role on regulating EMT was illuminated in lung cancer cell lines. In this paper, we showed that expression of DAL-1 was downregulated in metastatic tumours and further identified DAL-1 as an EMT/metastasis suppressor.
Material and methods
Tissue samples and cell lines
The paraffin-embedded tissues of 190 primary lung cancers and 163 matched corresponding tumor adjacent tissues were obtained from patients who underwent primary surgical treatment without systemic chemotherapy or radiotherapy during the period 2005–2008. This project had the informed consents from all the patients and was approved by the First Affiliated Hospital of Guangzhou Medical University and informed consent was taken from all subjects. The average age of the patients at diagnosis was 60.34 years, ranging from 25 to 82 years. These cases included 77 squamous cell carcinomas (36 cases were well or moderately differentiated, 41 cases were poor differentiated), 85 adenocarcinomas (59 cases were well or moderately differentiated, 26 cases were poor differentiated), 11 adeno-squamous carcinomas (moderately differentiated), 6 small cell carcinomas (poor differentiated), 5 large-cell carcinomas (poor differentiated), 6 sarcomatoid carcinomas (poor differentiated). All samples were diagnosed and classified according to the World Health Organization grading system and the General Rules for Clinical.
The cell lines used in this study were purchased from American Type Culture Collection (ATCC).
Plasmids and stable cell lines
Plasmid pcDNA3-DAL-1 containing full-length DAL-1 coding region was kindly provided by Dr. Philip Washbourne (University of Oregon, USA). This plasmid was verified by DNA sequencing. The control vector pcDNA3 was purchased from Invitrogen (Grand Island, NY, USA). Lipofectamine® LTX & Plus Reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect. Transfected condition was pre-optimized using pEGFP-C1 (Clontec, Mountain View, CA, USA) to monitor the transfection efficiency under inverted fluorescent microscopy.
To obtain stable transfectants, cells seeded in six-well plates were transfected with 2.5 μg/well plasmids using 10 μl/well Lipofectamine®LTX Reagent and 2.5 μl/well Plus Reagent. After 6 h incubation in serum and antibiotic free condition, the medium was replaced with RPMI 1640 containing 10% FBS, and the cells were cultured for 48 h before submitting to a 2-week selection in medium containing G418 (600 μg/ml).
Immunohistochemical staining
5-μm sections of paraffin-embedded tissues were treated with pepsin or 30 min and blocked with BSA for 30 min at room temperature. Tissue sections were then incubated with antibodies to DAL-1, E-cadherin, Snail or vimentin overnight at 4°C. Isotype control antibody (Sigma-Aldrich) was used as a negative control. Each slide was washed three times in TBS and incubated with biotinylated anti–mouse IgG (Vector Laboratories) in a humid chamber for 30 min. The positive cells were detected using peroxidase-conjugated streptavidin (Vector Laboratories) followed by 3′3-diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories). The slides were counterstained with hematoxylin.
Dual-luciferase reporter assay
The E-cadherin promoter (643 bp, from-450 to +193) was cloned into pGL3-Enhancer vector (Promega). A549 cells were plated in 24-well plates at a density of 1 × 105 cells/well and grown overnight prior to transfection. The cells were transfected with either DAL-1 plasmid or control plasmid, and were co-transfected with pGL3-E-cadherin promoter plasmid and pGL3-TK plasmid. Forty-eight hours after transfection, the luciferase activities were analyzed by the Dual-luciferase system according to the manufacturer’s instruction (Promega). Three independent experiments were performed.
Chromatin immunoprecipitation analysis
ChIP analysis was carried out as described previously [
12]. Briefly, Protein-DNA complexes were immunoprecipatited with DAL-1 antibody, with human IgG as a control, respectively. ChIP DNA was isolated with the Qiaquick PCR Purification Kit (Qiagen). Subsequent qPCR using 1⁄50 fraction of ChIP-enriched DNA and 100 nM primers in a total volume of 20 μl was conducted to assess the amount of DNA that had been precipitated. Standard curves from 0.1-100 ng of sonicated genomic DNA were also amplified by qPCR as a reference. DNA from these samples was then subjected to PCR analysis. Primers sets for E-cadherin promoter were 5′-gca gac tgg aag ggc gg-3′ and 5′-ggc agg cag ccc agc-3′.
Co-immunoprecipitation and mass spectrometric assay and protein identification
Co-immunoprecipitation was performed using Pierce® Co-IP kit (Thermo scientific, USA) following the manufacturer’s instruction. A549 cell lines stable tranfected either DAL-1 or control were lysed in ice-cold IP Lysis/Wash Buffer. After centrifugation at 13,000 g at 4°C for 10 min, the supernatant was collected. Pre-clear lysate using the control agarose resin before co-immunoprecipitation by the resin coupled with goat anti-DAL-1 polyclonal antibody. The immunoprecipitate was isolated by the elution buffer and separated by 10% SDS-PAGE. Sliver staining was used to detect the protein on the gel. The protein bands were submitted for tryptic peptide mass fingerprinting for identification by matrix-assisted laser desorption/ionization time-of-flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Protein identification was performed by searching protein databases of Mascot (
http://www.matrixscience.com).
Cells were lysed on ice for 30 min with a lysis buffer (150 Mm NaCl, 25 mM Hepes, pH 7.4, 10 mM NaF, 5 mM MgCl2 1 mM EGTA, 1% Nonidet P-40, protease inhibitors mixture:10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF). Total lysates were precleared with protein G-Sepharose for 1 h at 4°C and then immunoprecipitated overnight with 2 μg of anti-Myc antibody (Santa Cruz Biotechnology). Protein G-Sepharose was then added, and the mixture was incubated for an additional 1 h at 4°C. The beads were washed three times with lysis buffer, followed by elution in Laemmli buffer. The proteins were subjected to SDS-PAGE and Western blotting according to standard procedures.
Migration and invasion assay
Cells were transfected with DAL-1, DAL-1 RNAi or negative control using Transfection Agent (Ambion, TX, USA) following manufacture’s protocol in 24-well plates. 24 h after transfection, Transwell migration assay and Matrigel invasion assay were performed separately using 24-well Transwell inserts with 8 μm pore size (Corning Costar Corp). For Transwell migration assay, 2 × 104 A549 or H460 cells suspended in 100 μl corresponding culture medium without fetal bovine serum (FBS) were loaded into the top chamber of transwell insert with non-coated membrane. For Matrigel invasion assay, 5 × 104 A549 or H460 cells were plated in 100 μl serum-free medium in the upper Matrigel-coated chamber instead. In both assays, the bottom chamber was containing 600 μl medium with 20% FBS. Cells were then allowed to migrated or invaded for 12 h at 37°C. The cells that migrated or invaded into the bottom chamber were fixed, stained with 1% nuclear fast red staining solution, visualized under phase contrast microscope and photographed. Total number of migrated or invaded cells was counted by IPP (Image-Pro Plus 6.0) software. All experiments were independently repeated at least three times.
Statistical analysis
Data were expressed as the mean ± SD for each group. The difference between groups was analyzed using a factorial model one-way analysis of variance. SPSS statistical software, version19.0 (Chicago, Illinois, USA) was used to carry out all analyses and P less than 0.05 was considered statistically significant. Each experiment was performed in triplicate.
Discussion
Tumor metastasis of is a complex multi-step and multi-stage process [
13,
14]. The mortality of lung cancer is often related to extensive metastasis [
15,
16]. During the metastasis cascade, carcinoma cells often activate EMT to underlie metastasis by promoting acquisition of migratory and invasive abilities [
17-
19]. Therefore, understanding the mechanisms inducing EMT is particularly important for developing strategies for clinical therapy [
20-
22]. DAL-1 was originally considered as a tumor growth suppressor gene based on its downregulation in lung adenocarcinoma [
23,
24]. Recent research reported that DAL-1 could suppress tumor metastasis [
25].
As DAL-1 is associated with the cytoskeleton molecules and can regulate the cellular functions in adhesion and migration under physiological conditions [
26,
27], we speculated that DAL-1 might suppress metastasis via inhibiting EMT. Forced DAL-1 expression, however, abolished the metastatic capacity of a fibroblastoid, highly metastatic human lung cancer cell line through reversal to an epithelial phenotype. Loss of DAL-1 also predicted metastasis and impaired survival in a lung cancer tissue arrays from 190 patients, suggesting DAL-1 as an excellent biomarker for human metastatic lung cancer. Since DAL-1 abolishes metastatic capacity by re-inducing epithelial properties in dedifferentiated tumour cells, it might also be a candidate for gene therapy in metastatic cancer. DAL-1 overexpression altered the EMT markers expression, the expression of E-cadherin and β-catenin were upregulated, and expression of vimentin and N-cadherin were downregulated. Furthermore, inhibition of DAL-1 dramatically promoted the migration and invasion abilities of lung cancer cells. Our data together suggest that DAL-1 might contribute to the development of EMT.
Studies to unravel possible mechanisms, how loss of DAL-1 disturbs epithelial polarity with the outcome of EMT, are hindered by the enormous complexity of these processes. E-cadherin was observed to be upregulated after DAL-1 reexpression. Consistent with previous report, we confirmed that E-cadherin upregulation was sufficient to inhibit EMT in lung cancer cells [
28]. We demonstrated that DAL-1 might increase the expression of E-cadherin via transcriptional activating the E-cadherin promoter. Our result of the dual-luciferase reporter assay and ChIP analysis identified that DAL-1 could bind E-cadherin promoter directly and increase expression of E-cadherin as a transcriptional factor. Recent study identified a putative NSL (nuclear localization signal) in exon 12 of DAL-1 [
29]. Interestingly, a casein kinase II site, SAAE, is 26 amino acids upstream of the NLS in exon 12. It has been reported that proteins containing an NLS often have casein kinase II site 10–30 amino acids from the NLS [
30]. The conformation of DAL-1 would be changed if the kinase site was phosphorylated, and the NLS would be exposed to be identified, leading DAL-1 into nuclear to act a role as transcriptional factor.
DAL-1 as an important component of membrane-associated cytoskeleton was originally detected in cell-cell junction part. DAL-1 contains some specific domains, including SABD and FERM domains [
31,
32] which lead DAL-1 function as a scaffold protein binding to the cell membrane and connecting to the cytoskeleton [
27,
33,
34]. This kind of structure is essential for the maintainance of cell polarity and cell-cell adhesion [
35,
36]. Loss of DAL-1 may cause impairing of this structure and result in EMT development.
DAL-1 associated proteins were investigated by co-immunoprecipitation assay. E-cadherin, β-tubulin, P4HA1, 14-3-3ε and HSPA5 had been found in the DAL-1 binding complex. Previous research showed that DAL-1 interacted with 14-3-3ε and contributed to cell apoptosis [
37,
38]. So we performed endogenous co-immunoprecipitation experiment to conform the direct interaction between DAL-1 and P4HA1, E-cadherin or HSPA5. P4HA1 as a Prolyl hydroxylase family member could upregulate E-cadherin expression via induce HIF degradation [
39,
40]. The results showed that only HSPA5 was the combined protein to DAL-1. Molecular chaperone HSPA5 was a key survival factor in development and cancer [
41]. HSPA5 may also be important for tumor metastasis because it is elevated in metastatic cancer cell lines, lymph node metastasis, and knockdown of HSPA5 inhibits tumor cell invasion in vitro and growth and metastasis in xenograft models [
42,
43]. The notable component of our work described here was that HSPA5 interacted with DAL-1 directly. HSPA5 knockdown could impact on EMT markers. Whether DAL-1 suppresses EMT via inhibiting HSPA5, further investigations will explore this mechanism.
Conclusions
In this study, we have demonstrated that the endogenous expression of DAL-1 was negatively correlated with the tumor pathological grading of clinical lung cancer samples. Further in vitro experiments have showed that DAL-1 reduced migration and invasion, transcriptional activated E-cadherin promoter. Moreover, HSPA5 as a DAL-1 co-precipitated protein contributed in EMT process. Based on our observations and results reported by other groups, we have proposed DAL-1 could attenuate EMT and be important for tumor metastasis in the early transformation process of lung cancer. The experimental data and conclusions in the present study furnish valuable information regarding the biological functions of DAL-1 and the possible mechanism of the migration and invasion of lung tumor. Thus, further studies will be focused on the molecular network involved in the DAL-1 regulation on EMT.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XLC and XYG performed the experiments and drafted the manuscript. HYZ, XBX, HYW and JL participated in the experiments. THC, SHL and ZL contributed to final data analysis. YJZ contributed to the design of this study, and edited the manuscript. All authors read and approved the final manuscript.