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Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene
© Sukka-Ganesh et al.; licensee BioMed Central Ltd. 2012
Received: 28 December 2011
Accepted: 28 June 2012
Published: 23 July 2012
Tumor formation is a complex process which involves constitutive activation of oncogenes and suppression of tumor suppressor genes. Receptor EphA2 and its ligand ephrin-A1 form an important cell communication system with its functional role in cell-cell interaction and tumor growth. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential. Claudins, the integrated tight junction (TJ) cell-cell adhesion proteins located on the apico-lateral portion of epithelial cells, functions in maintaining cell polarity. There is extensive evidence implicating Eph receptors and ephrins in malignancy, but the mechanisms how these molecular players affect TJ proteins and regulate tumor growth are not clear. In the present study we hypothesized that EphA2 signaling modulates claudin-2 gene expression via induction of cdx-2, a tumor suppressor gene in NSCLC cells.
The expression of EphA2, claudin-2 was determined in various NSCLC cell lines by using real-time quantitative polymerase chain reaction and Western blot analysis. The claudin-2 expression was also analyzed by immunofluorescence analysis. EphA2 and erk1/erk2 phosphorylation in ephrin-A1 activated cells was evaluated by Western blot analysis. The cell proliferation and tumor colony formation were determined by WST-1 and 3-D matrigel assays respectively.
NSCLC cells over expressed receptor EphA2 and claudin-2. Ephrin-A1 treatment significantly down regulated the claudin-2 and EphA2 expression in NSCLC cells. The transient transfection of cells with vector containing ephrin-A1 construct (pcDNA-EFNA1) decreased the expression of claudin-2, EphA2 when compared to empty vector. In addition ephrin-A1 activation increased cdx-2 expression in A549 cells. In contrast over-expression of EphA2 with plasmid pcDNA-EphA2 up regulated claudin-2 mRNA expression and decreased cdx-2 expression. The transient transfection of cells with vector containing cdx-2 construct (pcMV-cdx-2) decreased the expression of claudin-2 in A549 cells. Moreover, silencing the expression of receptor EphA2 by siRNA significantly reduced claudin-2 expression and decreased cell proliferation and tumor formation. Furthermore, silencing cdx-2 gene expression before ephrin-A1 treatment increased claudin-2 expression along with increased cell proliferation and tumor growth in A549 cells.
Our study suggests that EphA2 signaling up-regulates the expression of the TJ-protein claudin-2 that plays an important role in promoting cell proliferation and tumor growth in NSCLC cells. We conclude that receptor EphA2 activation by ephrin-A1 induces tumor suppressor gene cdx-2 expression which attenuates cell proliferation, tumor growth and thus may be a promising therapeutic target against NSCLC.
Tight junctions (TJ), the most apical cell-cell adhesion, owing to their cellular location are responsible for maintaining the cellular integrity. Any deregulation of the TJ characteristics could potentially lead to cellular transformation and acquisition of tumorogenesis potential . However, emerging details from many studies related with claudin and cancer have implicated claudin family members in a wide range of human cancers. The expression of claudins would decrease during tumorogenesis as tight junctions are lost during cellular transformation, but it is understood that claudins are expressed in a tissue specific manner [2–11]. Down-regulation of claudins in cancer seems to be well understood, but increased expression of claudin contributing to neoplastic progression is less clear . Aberrant tissue expression of certain claudins may contribute to neoplasia by directly altering TJ structure and function . Furthermore it is also postulated that claudins may affect cell-signalling pathways .
Cdx-2 is a transcriptional factor crucial to the normal proliferation and differentiation of intestinal epithelial cells , however little is known about the transcriptional program that controls genes involved in NSCLC tumor growth. In colorectal cancer reduced expression of cdx-2 has been reported in rodents and humans [14, 15]. In addition, cdx-2 null mice embryos failed to survive and heterozygote’s developed intestinal tumors. Furthermore the polyps developed in the colon do not express cdx-2 which indicates that loss of cdx-2 promotes tumorogenesis . Cdx-2 regulates claudin-2 functions by binding to its 5’ flanking region and affects the expression of downstream pathway genes . However, if receptor EphA2 activation with ephrin-A1 induced expression of cdx-2 plays any role in NSCLC tumor growth is not known.
The Eph family of receptor tyrosine kinases plays key role in the development of cancer. The Eph receptors and ephrins were originally discovered as neuronal guidance and vasculature formation proteins during embryonic development . Eph receptors and their ligands, ephrins are often dysregulated in malignant phenotypes including NSCLC [19–23]. However the precise role of these proteins in tumor growth is not well understood. Defining the role of EphA2 and ephrin-A1 in NSCLC is particularly important, as EphA2 receptor is highly expressed in NSCLC which contributes to tumor development. The aim of our study was to investigate the underlying mechanisms of tumor suppressor effect of ephrin-A1 in NSCLC. We report a novel mechanism of ephrin-A1 mediated attenuation of NSCLC tumor growth due to down regulation of claudin-2 and induction of tumor suppressor gene cdx-2. Thus providing the evidence that receptor EphA2 may be a promising therapeutic target for NSCLC.
NSCLC cell culture
A549 NSCLC cell line was obtained from American Type Culture Collection (Manassas, VA) and NCI-H2126, NCI-H838, NCI-H522, NCI-H23 NSCLC cell-lines were a kind gift from Dr. Frederic Kaye, MD, Division of Haemato-logy/Oncology, University of Florida, Gainesville, Florida. The NSCLC cells were resuspended in RPMI-1640 (Gibco Laboratories, Grand Island, NY) containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were plated in 100 mm Petri dishes (Corning Costar Corporation, MA) and incubated at 37°C in 5% CO2 and 95% air. The cell culture medium was changed on alternate days. When the cells were confluent they were trypsinized and seeded into 100 mm culture dishes or transwell chambers as required for different assays.
Construction of vectors containing ephrin-A1 (EFN-A1) and EphA2 and transient transfection of NSCLC cells
The gene transfer vector, pcDNA3.2/V5-DEST was used as an expression vector for the expression of ephrin-A1 (EFN-A1), and receptor EphA2, and pcDNA3.2/V5/CAT was used as a control vector (Invitrogen, Carlsbad, CA) as reported earlier . For the over expression of cdx-2 gene, pcMV6-XL5 was used as an expression vector for cdx-2 and control vector in A549 cells (Origene Technologies, Inc.; Rockville, MD). The cloned vectors were designated as pcDNA-EFN-A1, pcDNA-EphA2 and pcMV-cdx2 respectively. The control vectors were designated as Empty vector or pcMV-control. The NSCLC cells were transfected with vectors using lipofectamine-2000 reagent (Invitrogen, Carlsbad, CA). The transfected cells were used for further experiments.
Transfection of NSCLC cells
The siRNA targeting the receptor EphA2 and cdx-2 were designed using Oligoperfect design (Invitrogen, Carlsbad, CA). A549 cells were plated into 6-well plates or 35 mm plates as required for the experiments. The cells were allowed to adhere for 24 hours. The transfection of siRNA was performed using lipofectamine-2000 (Invitrogen) according to the manufacturer’s recommendation. The concentration of siRNA used was 100nM. After 4 hours of transfection, the culture medium with serum was added. The assays were carried out 48 hours post-transfection as reported earlier .
Total RNA isolation and quantitative real time PCR analysis
Primers Used in Quantitative Reverse Transcriptase-Polymerase Chain Reaction analysis
Forward Primer (5'-3')
Reverse Primer (5'-3')
Western blot analysis
NSCLC cells were cultured in 60 mm size cell culture dishes (Fisher Scientific, Pittsburgh, VA) to confluence and the cells were lysed in lysis buffer with the method reported earlier . Protein was estimated by BCA method (PIERCE, Rockford, IL) and equal amount of protein (20 μg/lane) were loaded. Proteins in the sample were separated in denaturing sodium dodecyl sulphate (SDS) polyacrylamide gels (Bio-Rad), and transferred electrophoretically onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). The blots were blocked with 5% Blotting Grade Blocker Non-fat Dry milk (Bio-Rad, Hercules, CA) for 1 hr on shaker at room temperature, and were overnight incubated at 4°C with respective antibodies - rabbit EphA2 antibody, rabbit cdx-2 antibody (Cell signaling, Beverly, CA) and rabbit claudin-2 antibody (Invitrogen, Grand Island, NY), at 1:1000 dilutions. After washing, they were incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG Ab) at a dilution of 1:1000 for 1 hr. Finally respective proteins were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). The Molecular mass (kDa) of the proteins was determined using the prestained protein marker (Bio-Rad).
NSCLC cell proliferation
NSCLC cell proliferation was assessed by using an assay based on cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases (Roche, Indianapolis, IN) as reported earlier . With this assay, an increase in the number of viable cells results in an increase in the overall activity of the mitochondrial dehydrogenases in the sample. The augmentation in enzyme activity leads to an increase in the formazan dye formed. The formazan dye formed was quantified by using a plate reader at 450 nm. A549 cells were plated in 96-well microplate at a density of 0.5 X 105 cells per well. The cells were transfected with pcDNA-EFNA1, pcDNA-EphA2, siRNA for EphA2 or with scrambled siRNA (sc-siRNA), cdx-2-siRNA by using lipofectamine-2000 reagent, and a few wells were left untreated. The negative controls received serum-free media, and some of the wells were activated with recombinant ephrin-A1. Cells were allowed to incubate for 48 hours. Then, the WST-1 reagent was applied for 4 hours to measure cell proliferation. The cell proliferation was assessed in triplicate. The data are presented as a percentage of negative control proliferation with P values <0.05 were considered significant.
Claudin-2 expression in NSCLC cells was analyzed by using confocal laser-scanning microscopy (Zeiss LSM 510, Axiovert 100 M; Zeiss, Thornwood, NY), as reported previously . In brief, the cells were cultured to confluence on gelatinized glass cover slips and fixed in 5% paraformaldehyde (with 50 mM phosphate buffer) in 50% Tris wash buffer (TWB). The glass cover slips were rinsed 3 times and permeabilized with 1.2% Triton X-100 for 5 minutes, rinsed 3 times, incubated with 1% bovine serum albumin (BSA) in 100% TWB for 1 hour, then stained for the expression claudin-2 using primary antibody rabbit anti-claudin-2 at 1:150 dilution and secondary antibody goat anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate (FITC) (Zymed Laboratories, San Francisco, CA). 4, 6-diamino-2-phenylindole (DAPI) was used as a nuclear stain.
NSCLC tumor growth in matrigel
A 48-well culture plate was coated with 200 μl of matrigel per well and then allowed to polymerize for about 1–2 hours at 37°C. NSCLC cells at a density of ~1 x 103 cells per well were plated in 0.3 ml of 2% FBS containing RPMI-1640 as reported earlier . The cells were activated with ephrin-A1 or transfected with plasmid pcDNA-EphA2 or pcDNA-EFNA1 or siRNA for EphA2 or cdx-2 or empty vector by using lipofectamine-2000 and few wells were left untreated as controls, media were changed every three days. The number of colonies formed was recorded after 7 days of incubation. 4–6 randomly chosen fields (x10) from the sample were photographed.
The Sigma Stat 3.5 statistical software program was used to calculate statistical significance. Kruskal-Wallis One Way Analysis of Variance (ANOVA) was used to compare the experimental groups from the control groups. The post hoc test Holm-Sidak method was applied for pairwise comparisons. The differences at p < 0.05 were considered statistically significant.
NSCLC cells express receptor EphA2 and Caludin-2
Ephrin-A1 down regulated claudin-2 expression in NSCLC cells
In order to determine if over expression of the receptor EphA2 in A549 cells promotes claudin-2 expression, A549 cells were transiently transfected with pcDNA-EphA2 and the expression of claudin-2 was evaluated. A significant increase in the expression of claudin-2 was noted when compared to empty vector transfected cells (Figure 2B and C). To investigate whether this increase in claudin-2 expression was directly due to the increases in EphA2 receptor expression, NSCLC cells were transfected with EphA2-siRNA. In A549 cells, silencing the EphA2 receptor with siRNA significantly reduced the claudin-2 expression when compared to sc-siRNA transfected cells (Figure 2B and C). These results suggest that increased EphA2 expression modulated claudin-2 expression, which may play an important role in tumor growth in NSCLC.
Claudin-2 expression decreased in Ephrin-A1 treated A549 cells
In order to evaluate the cellular distribution of caludin-2, and morphological changes in activated cells, A549 cells were activated with ephrin-A1 and analyzed by immunofluorescence microscopy. Fluorescence immuno-staining analysis revealed that A549 cells showed punctuated expression of claudin-2 whereas, ephrin-A1 activation decreased claudin-2 expression (Figure 2E). In cells transfected with pcDNA-EFNA1 a marked decrease in claudin-2 expression was noticed. In addition, ephrin-A1 activated cells showed distorted cytoskeleton, and rounded morphology. However, in cells transfected with pcDNA-EphA2 a dense and higher expression of claudin-2 was noticed when compared to control and empty vector transfected cells. These data suggests that over expression of receptor EphA2 promotes the expression of claudin-2 in NSCLC cells. Ephrin-A1 activation or transfection of cells with plasmid containing the ephrin-A1 construct inhibits the expression of claudin-2 confirming its anti-tumor effects on NSCLC cells.
Ephrin-A1 treatment increased cdx-2 expression in NSCLC cells
Silencing cdx-2 expression blocked ephrin-A1 mediated inhibition of claudin-2 expression in NSCLC cells
Silencing cdx-2 expression blocked Ephrin-A1 mediated inhibition of cell proliferation in NSCLC cells
Ephrin-A1 activation inhibited tumor growth and silencing cdx-2 expression blunted Ephrin-A1 mediated suppression of tumor growth in NSCLC cells in vitro
The major finding of our present study is that receptor EphA2 is over expressed in NSCLC cell lines which promotes tumor growth. In addition we also found that EphA2 promotes tight junction protein claudin-2 expression in A549 cells. However, the expression of ephrin-A1 was found to dysregulated and A549 cells showed minimal levels. There is accumulating evidence that activation of receptor EphA2 with its ligand ephrin-A1 attenuates tumorogenic potential of malignant cells [24, 27, 28]. The molecular mechanisms responsible for tumor suppressive effects of ephrin-A1 are still elusive. In the present study, we report that proliferating NSCLC cells showed enhanced expression of EphA2, and claudin-2. The activation of receptor EphA2 with ephrin-A1 inhibited the expression of EphA2 and claudin-2. To further examine the effect of ephrin-A1 on NSCLC we transfected the cells with vector expressing ephrinA1 construct, pcDNA-EFNA1. We found that forced expression of ephrin-A1 down regulated the receptor EphA2 and inhibited cell proliferation and tumor growth in 3D matrigel. In addition the activation of receptor EphA2 with ephrin-A1 induced phosphorylation of EphA2 and inhibited the downstream singling MAP kinase pathway Erk1/Erk2. Furthermore, the activation of EphA2 receptor with ephrinA1 induced cdx-2, a tumor suppressor gene in A549 cells. These data suggests that ephrin-A1 activation/transfection could effectively bind and activate endogenous EphA2 in NSCLC and led to internalization and degradation of EphA2. In order to understand if receptor EphA2 signaling modulates TJ protein claudin-2 we transfected the A549 cells with EphA2 expressing vector, pcDNA-EphA2. The expression of claudin-2 was higher in A549 cells transfected with pcDNA-EphA2 as compared to empty vector transfected cells or control cells. In addition, over expression of receptor EphA2 significantly enhanced tumor growth. Whereas silencing the expression of receptor EphA2 by siRNA, decreased the expression of claudin-2 and interestingly a significant up-regulation of cdx-2 was noticed in NSCLC cells as compared to sc-siRNA transfected A549 cells. However, silencing cdx-2 gene with siRNA and subsequent activation with ephrin-A1 or transfection with pcDNA-EFNA1 failed to inhibit tumor growth in A549 cells. Collectively these data suggests that an ephrin-A1 mediated anti-oncongenic effect is due to downregulation of EphA2, claudin-2 expression and induction of cdx-2 gene in NSCLC.
EphA2 is an oncoprotein which promotes cell survival, abnormal cell growth and invasion in a number of malignancies, including NSCLC [18, 20, 21, 29]. In malignant cells such as A549, due to dysregulated cell division and abnormal growth the cell-cell contacts are loose which hinders the interaction between neighbouring cells. The loss of contact among the adjacent cells results in accumulation of high levels of intracellular EphA2 and claudin-2 an integral component of tight junction. Tight junctions are the apical cell-cell adhesions that regulate paracellular permeability and are critical for cell polarity. Alteration in tight junction protein claudin-2, can cause the defects in normal regulation of growth factor receptor activation due to a differential distribution of the receptor and their respective ligands, which can be observed with respect to receptor EphA2 and its ligand Ephrin-A1 . In this study, we attempted to understand the underlying mechanisms by which EphA2 over expression leads to enhanced or irregular claudin-2 expression via cdx-2 modulation and promote tumor growth in NSCLC cells. Several studies reported that receptor EphA2 is over expressed in a number of malignancies [19, 20, 30]. Previously we have reported that EphA2 is over expressed in malignant mesothelioma cells (MMC) and posttranslational silencing of EphA2 significantly suppresses the proliferation and haptotactic migration of MMC [22, 24]. In addition, EphA2 receptor activation in MMC by its ligand ephrin-A1 inhibited the RAS MAP kinase signaling pathways [23, 24]. Our study in A549 cells revealed that receptor EphA2 signaling up regulates the TJ protein claudin-2. In turn, the over expression of claudin-2 along with EphA2 promotes A549 cell proliferation and tumor growth. It was reported that EGF signaling induced claudin-2 expression which promoted colonization of mammary tumor cells . The up regulated levels of claudin-2 caused leaky cellular barriers in MDCK1 cells . The junctional claudin-2 forms the selective cation channels that are sufficient to transform the functional “tight” junction into a “leaky” one . The leaky barriers may contribute to increase uptake of nutrients and growth factor which promote exaggerated tumor colonization. In the present study we noted exaggerated tumor colonies formation when EphA2 was over expressed in A549 cells. It is plausible that over expression of receptor EphA2 promotes claudin-2 which in turn enhances tumor colonization of A549 cells.
We demonstrate that activation of receptor EphA2 with ephrin-A1 induced cdx-2 expression and inhibited tumor formation. The over expression of cdx-2 by vector pcMV-cdx-2 resulted in downregulation of claudin-2 and attenuation of cell proliferation and tumor growth on matrigels. In addition silencing cdx-2 expression using siRNA and activation with eprhin-A1 resulted in up regulation of claudin-2 in A549 cells. Cdx-2, a tumor suppressor gene is homeobox transcriptional factor that is known to control apical-basolateral polarity in mouse enterocytes and human colonic epithelial cells . Cdx-2 regulates epithelial cell polarity and morphogenesis through control of apical protein transport. At the transcriptional level, transcriptional factors such as cdx-2 can bind to the promoter regions of various claudin genes and affect their expression [1, 10]. In addition, certain characteristics of claudin-2 and cdx-2 show similarity that both are critical for epithelial cell polarity [1, 34]. Increased cdx-2 expression was used as a marker for progression in gastric carcinogenesis , while some of the gastric cancers studies showed aberrant expression of cdx-2 in intestinal metaplasia which is a subset of gastric adenocarcinoma . The down-regulation of cdx-2 mechanism was related to the induction of ulcer-associated cell lineage (UACL) . In addition loss of cdx-2 immunoreactivity was implicated as diagnostic feature in poorly differentiated colorectal adenocarcinoma . Whereas, the reduced expression of cdx-1 and cdx-2 genes were associated with the development of enterocolitis in intestinal mucosa . Furthermore, activation of Ras oncogene was associated with down regulation of the cdx-2 in colon cancer cells . All these studies confirm that expression of cdx-2 gene though disease specific and tissue specific, the expression of cdx2 was directly associated with tumor growth. The plausible mechanisms for the reduced cdx-2 expression in carcinogenesis, could be that homeodomain proteins signifies roles in directing the cells to specified cell-phenotype during organogenesis in early stages of development. However, a reduction of cdx-2 gene expression in the late stages such as in invasive tumors may be attributed to over expression oncogenic proteins which may lead to deviate from normal epithelial phenotype to the neoplastic phenotype .
It has been shown that, caudal-related homeobox gene cdx-2 is positively involved in the regulation of the human claudin-2 promoter activity . The EphA2 signaling caused reduced expression of transcription factor cdx-2 that hinder its binding to claudin promoter and thus cause irregular expression of claudin-2 which is reported to be increased in NSCLC cells in the present study. It is conceivable that due to the disrupted TJ or claudin-2 there is a disruption in epithelial cell polarity leading to leakage of large solutes passing across epithelial barriers to the other cells. Thus, the TJ disruption in premalignant neoplastic tissue can increase the proba-bility that it will develop into a complete carcinoma because of the continuous stimulation of cell division followed by disrupted natural barriers between growth factors and their receptors. The novel finding of our present study is that receptor EphA2 mediated the enhanced induction of functionally altered claudin-2 via down-regulation of tumor suppressor gene expression cdx-2 in NSCLC cells. It is possible that activation of receptor EphA2 with ephrin-A1 downregulated claudin-2 and induced the expression of cdx-2 suggesting oncogenic protein EphA2 play a major role in regulating cdx-2 expression in NSCLC. Whereas, the forced expression of ephrin-A1 induced tumor suppressive signals via downregulation and degradation EphA2 and inhibited the oncogenic singling pathway in NSCLC. However, this needs to be further investigated.
In conclusion, we present the first evidence that EphA2 signaling promotes the expression of claudin-2 in NSCLC cells. Activation of NSCLC with ligand ephrin-A1 suppressed the caludin-2 expression via the induction of transcriptional factor cdx-2. These studies suggest that targeting EphA2 by using ephrin-A1 may be a promising approach for the therapeutic inventions against NSCLC.
This work was supported by NIR grant # 09KN-09; RC1 (#09KW-08) from James & Esther King, Florida Department of Health (Nasreen, N); VA Merit Review (Mohammed, KA).
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