Abstract
Background
XB130 has been reported to be expressed by various types of cells such as thyroid cancer and esophageal cancer cells, and it promotes the proliferation and invasion of thyroid cancer cells. Our previous study demonstrated that XB130 is also expressed in gastric cancer (GC), and that its expression is associated with the prognosis, but the role of XB130 in GC has not been well characterized.
Methods
In this study, we investigated the influence of XB130 on gastric tumorigenesis and metastasis in vivo and in vitro using the MTT assay, clonogenic assay, BrdU incorporation assay, 3D culture, immunohistochemistry and immunofluorescence. Western blot analysis was also performed to identify the potential mechanisms involved.
Results
The proliferation, migration, and invasion of SGC7901 and MNK45 gastric adenocarcinoma cell lines were all significantly inhibited by knockdown of XB130 using small hairpin RNA. In a xenograft model, tumor growth was markedly inhibited after shXB130-transfected GC cells were implanted into nude mice. After XB130 knockdown, GC cells showed a more epithelial-like phenotype, suggesting an inhibition of the epithelial-mesenchymal transition (EMT) process. In addition, silencing of XB130 reduced the expression of p-Akt/Akt, upregulated expression of epithelial markers including E-cadherin, α-catenin and β-catenin, and downregulated mesenchymal markers including fibronectin and vimentin. Expression of oncoproteins related to tumor metastasis, such as MMP2, MMP9, and CD44, was also significantly reduced.
Conclusions
These findings indicate that XB130 enhances cell motility and invasiveness by modulating the EMT-like process, while silencing XB130 in GC suppresses tumorigenesis and metastasis, suggesting that it may be a potential therapeutic target.
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Results
Silencing XB130 inhibits proliferation of GC cell lines
Among the 5 common human GC cell lines, we found that XB130 expression was higher in SGC7901 (a poorly-differentiated cell line) and MKN45 (a well-differentiated cell line) than in the other cell lines (Additional file 1: Figure S1A). Accordingly, we chose these two cell lines for transfection with sh-XB130. The knockdown effect of sh-XB130 was confirmed by real-time PCR (Additional file 1: Figure S1B) and Western blotting (Additional file 1: Figure S1C). Compared with Scramble shRNA-transfected cells (Scramble cells), colony formation by sh-XB130-transfected cells (sh-XB130 cells) was markedly reduced in the plate colony forming assay (Figure 1A). In addition, the number of colonies that grew in soft agar was significantly reduced by transfection of sh-XB130 (p < 0.01) (Figure 1B). When the MTT assay was used to assess cell viability over a period of 7 days, we found that viability was significantly lower in sh-XB130 cells than in Scramble cells, indicating that cell viability was suppressed by knockdown of XB130 (Figure 1C). Cell cycle analysis revealed that sh-XB130 cells were arrested in G1 phase, accompanied by a significant reduction of cells in S phase (Figure 2A). The BrdU labeling assay showed that DNA synthesis was also strongly inhibited in sh-XB130 cells (Figure 2B). These results indicate that cell proliferation was remarkably inhibited by silencing of XB130.
Silencing XB130 inhibits GC cell motility and invasiveness and alters the phenotype of GC cells
To assess the effect of down-regulation of XB130 on cell motility, the wound healing assay and Transwell assay were performed. After knockdown of XB130, we found that fewer cells migrated to the center of the wound in the wound healing assay (Figure 3A) or migrated into the lower chamber in the Transwell assay (p < 0.01) (Figure 3B). In addition, sh-XB130 cells were relatively smooth spheroids with few projections, while Scramble cells and Control cells developed a multipolar invasive morphology in 3D culture (Figure 3C and Additional file 2: Figure S2A). We also investigated the cell structure by staining F-actin filaments. We found that XB130 was expressed in the F-actin filaments and XB130 knockdown resulted in GC cells adopting an epithelial-like morphology (Figure 3D). These findings indicate that the motility of GC cells was suppressed along with a decrease of invasive morphologic features after down-regulation of XB130.
Silencing XB130 reduces tumor growth in nude mice
To determine the influence of XB130 on tumor growth in vivo, a xenograft nude mouse model was used. Colonies of sh-XB130 cells, Scamble shRNA cells, and Control SGC7901 cells were prepared. After subcutaneous injection into nude mice, all three types of cells formed tumors (Figure 4A). However, tumor growth was much slower after injection of sh-XB130 cells than after injection of Control or Scramble cells (Figure 4B). After 3 weeks, tumor volume was significantly smaller in the sh-XB130 group than in the Control and Scramble groups (p < 0.01, Figure 4B and C). These findings indicate that GC tumor growth was inhibited by downregulation of XB130.
XB130 activates the PI3K/Akt pathway and alters both EMT markers and metastasis-associated proteins in GC
To explore the mechanisms underlying the above-mentioned changes induced by silencing of XB130, we postulated that its downregulation might influence the expression of EMT markers and metastasis-associated proteins via the PI3K/Akt pathway. We found that knockdown of XB130 decreased the phosphorylation of Akt in xenograft GC tissues (Figure 4D) and in GC cell lines (Figure 5A). Immunofluorescence, immunohistochemistry, and Western blotting were combined to assess the expression of EMT markers. In contrast to the Scramble group, silencing of XB130 in xenograft GC tissues and cultured GC cell lines led to higher expression of the epithelial marker E-cadherin and lower expression of the mesenchymal marker vimentin (Figure 4D, Figure 5B, and Additional file 2: Figure S2B). Western blotting also showed that silencing of XB130 substantially increased the expression of epithelial markers (E-cadherin, α-catenin and β-catenin), while causing a significant decrease in the expression of mesenchymal markers (fibronectin and vimentin) and metastasis-associated proteins (MMP2, MMP9 and CD44) (Figure 5C and D).
Discussion
The present study has provided the first evidence concerning the role of XB130 in GC, showing that (1) XB130 contributes to GC cell proliferation and invasiveness, (2) XB130 is involved in phosphorylation of Akt and EMT-like changes, and (3) XB130 could be a potential therapeutic target in patients with GC.
XB130 was initially cloned as a homologue of actin filament-associated protein (AFAP-110) [1], which has been suggested to have a role in mechanotransduction, stress fiber stabilization, focal adhesion formation, and podosome dynamics [15–17]. Regarding the functional properties of XB130, similar to AFAP-110, it is associated with regulation of the cytoskeleton, exhibits a high affinity for lamellipodial F-actin, and influences the motility and invasiveness of thyroid tumor cells [5]. Consequently, knockdown of XB130 in thyroid cancer cells decreases the wound closure rate, inhibits cell invasion, reduces lamellipodial persistence, and slows cell spreading [5]. Consistent with that report, we found that silencing of XB130 decreased the motility of GC cells along with significant inhibition of the transition from epithelial-like to fibroblast-like morphology, indicating that XB130 affects the motility and invasiveness of these tumor cells by interfering with an EMT-like process.
The EMT is a highly conserved process that has been well characterized in embryogenesis. In epithelial tumors, epithelial-like cancer cells undergo a phenotypic change to become mesenchymal-like cells which is similar to fibroblasts [18, 19]. These changes lead to loss of polarity for epithelial cells and resulted in promotion of tumor cell metastasis [6]. However, tumor cells seldom exhibit a complete change from an epithelial to mesenchymal phenotype (as occurs in the embryological setting), but rather show more plastic and dynamic changes that are better classified as “EMT-like” or as a partial EMT [20, 21]. Such EMT-like changes have been reported to be important in the metastasis of epithelial tumors [22].
In most cases, downregulation of E-cadherin seems to be the final common pathway of the EMT. Epithelial cells undergoing the EMT tend to develop a spindle-shaped or fibroblast-like morphology, and display increased or new expression of mesenchymal markers, including vimentin and fibronectin [23]. E-cadherin is a cell adhesion molecule that is anchored to the actin cytoskeleton via a complex consisting of α-catenin and β-catenin [24], and it is thought to be the key molecule in the establishment of cell-cell adhesion at adherens junctions. Fibronectin and vimentin are generally considered to be typical mesenchymal markers have been reported to contribute to invasion and distant metastasis of GC [25–27]. In the present study, expression of E-cadherin was significantly increased by XB130 knockdown in vivo and in vitro, while vimentin expression was partially inhibited, suggesting that XB130 has a role in enhancing EMT-like changes of GC.
The PI3K/Akt signaling pathway has been reported to be influenced by XB130 [10, 11], and phosphorylation of Akt promotes EMT-like changes through repression of Snail-mediated cadherin 1[7]. MMP2 and MMP9 are members of the matrix metalloproteinase family, which bind to zinc and act on the extracellular matrix (ECM) to degrade type IV collagen in the basement membrane. After basement membrane integrity is lost, metastasis occurs and the survival rate decreases dramatically in GC patients [28–30]. CD44 is recognized as a marker of cancer stem cells, which are a small population of stem-like cells residing in tumor tissues that can cause tumor formation, recurrence, and metastasis [31]. As a transmembrane glycoprotein expressed on the cell surface, CD44 and its variants can bind to the ECM and are involved in making connections between cells and the matrix [31]. All of these extracellular factors contribute to EMT-like changes in tumor cells. In the present study, we found that phosphorylation of Akt, expression of matrix metalloptoteinases, and expression of cancer stem cell markers were all significantly suppressed by XB130 knockdown, further confirming that XB130 may enhance the EMT-like process and promote the motility and invasiveness of GC cells.
As an adaptor protein, XB130 promoted GC cell proliferation and migration, while knockdown of XB130 contributed to reduced growth of xenograft tumors, suggesting that XB130 is an oncoprotein in GC. It may seem paradoxical that our previous study demonstrated a positive correlation between expression of XB130 and the prognosis [4]. In fact, such discrepancy is not uncommon for oncogenes. Several oncogenes are known to be downregulated in tumors and their low expression predicts a poor prognosis. Clinical studies have shown that low expression of the oncoproteins Bcl-2 and Bcl-B is associated with a poor outcome of GC [32–34]. A similar “discrepancy” has also been noted for some tumor suppressor genes. For example, it has been reported that overexpression of the tumor suppressor gene p53 is significantly correlated with unfavorable clinicopathologic parameters and lower overall survival [35]. Moreover, a correlation between gene expression and the prognosis is not necessarily indicative of a causal relationship. Compensatory mechanisms may downregulate some oncogenes and upregulate some tumor suppressor genes.
In addition, clinical prognosis is influenced by various factors including gene expression and medical interventions. Currently, fluoropyrimidine derivative-based and platinum-based combination regimens are accepted as conventional first-line treatment for GC [36]. In our previous study [4], 80% of patients were treated with 5-fluorouracil (5-FU), and XB130-negative patients had a lower survival rate when they received 5-FU. In addition, sensitivity studies showed that XB130 knockdown reduces the sensitivity of GC cells to 5-FU [4]. These results indicate that tumors with high levels of XB130 expression show greater sensitivity to 5-FU, leading to an improved survival rate, which may be an explanation for the better prognosis of patients with high expression of XB130 [4]. Taken together, these findings suggest that XB130 may be a potential target for the treatment of GC.
Conclusions
In summary, the present study showed that XB130 is an oncogene that promotes tumor growth and metastasis, probably through its role in an EMT-like process. XB130 seems to be an important regulator of the metastasis of gastric cancer and the potential target for treatment of this cancer.
Abbreviations
- GC:
-
Gastric cancer
- EMT:
-
Epithelial-mesenchymal transition
- PI3K:
-
Phosphatidylinositol 3-kinase
- FAK:
-
Focal adhesion kinase
- shRNA:
-
Short hairpin RNA
- MMP:
-
Matrix metalloproteinase
- ECM:
-
Extracellular matrix
- 5-FU:
-
5- fluorouracil
- MTT:
-
Methyl thiazolyl tetrazolium
- PBS:
-
Phosphate-buffered saline
- PI:
-
Propidium iodide.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (31271564, to WL), the Wu Jie** Medical Foundation, Bei**g, China (845553317108091001, to WL), and the Science and Technology Program Planning Project of Guangdong Province (2011B031800281, to MS).
Agents and companies
Cells: Foleibao Biotechnology Development Company, Shanghai, China
Fetal bovine serum: Thermo Scientific HyClone, South Logan, UT, USA
Trizol kit, 4′,6-diamidino-2-phenylindole, and RPMI-1640 medium: Life Technologies, Carlsbad, CA, USA
Protease inhibitor cocktail: Roche, Basel, CH
MMLV-RT reverse transcriptase: ProSpec, East Brunswick, NJ, USA
Nitrocellulose membranes: Millipore, Bedford, MA, USA
Western Lightning Plus-ECL: Perkin Elmer, Waltham, MA, USA
Crystal violet, agar, and propidium iodide: Sigma Chemical Company, Louis, MO, USA
Microplate spectrophotometer and RNase A: Thermo Scientific, Pittsburgh, PA, USA
Flow cytometry: Beckman-coulter, Fullerton, CA, USA
Flowjo software: TreeStar, Ashland, Oregon, USA
Microscope: Olympus, Tokyo, Japan
Matrigel: BD Biosciences, San Jose, CA, USA
Nude mice: Sun Yat-Sen University, Guangzhou, China
Dako Envision System: Dako, Glostrup, Denmark
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The authors declare that they have no competing interests.
Authors’ contributions
WL, QZ, and MS contributed to conception and design of the study and revised the manuscript. LS, MZ, and WL performed experiments and were responsible for data collection, analysis, and interpretation of the results. LW, LL, and YW were responsible for conducting the data analysis. LS and WL drafted the manuscript and figures, and YL interpreted the data and critically revised the manuscript. All authors read and approved the final manuscript.
Min Shi, Dayong Zheng contributed equally to this work.
Electronic supplementary material
12967_2013_1699_MOESM1_ESM.tiff
Additional file 1: Figure S1: Baseline expression and silencing effect of XB130 in gastric cancer (GC) cell lines. (A) Baseline expression of XB130 in various GC cell lines. Knocking down effect of sh-XB130 in SGC7901 and MKN45 cell lines was evaluated. Real-time PCR (B) and Western blot (C) revealed that sh-XB130 effectively suppressed the expression of XB130 in both cell lines. (TIFF 2 MB)
12967_2013_1699_MOESM2_ESM.tiff
Additional file 2: Figure S2: Silencing XB130 changed the morphology of gastric cancer cells and altered the EMT-like associated proteins by cultured cells. (A) Matrigel 3D culture was performed to assess cell morphology. Aggressive protrusion-positive structures were found in the Scramble and Control group. In contrast, the invasiveness of sh-XB130 transfected cells was much lower and the cells were round spheroids with no or few protrusions. Pictures in upper line showed the view of 2–3 cells in each group under 100× magnification and the others were graphed under 200× magnification. (500 cells were incubated in each well). (B) E-cadherin and vimentin immunofluorescence staining. E-cadherin was significantly upregulated and vimentin was downregulated by XB130 knockdown. Scale bar = 100 μm. Experiments were repeated 3 times. (TIFF 5 MB)
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Shi, M., Zheng, D., Sun, L. et al. XB130 promotes proliferation and invasion of gastric cancer cells. J Transl Med 12, 1 (2014). https://doi.org/10.1186/1479-5876-12-1
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DOI: https://doi.org/10.1186/1479-5876-12-1