Introduction
Gastric cancer (GC), a leading cause of cancer-related death, is the fifth most common type of cancer worldwide [
1]. In China, owing to the ageing and sociodemographic changes and substantial population, the morbidity of GC ranks the second [
2], with massive advanced GC. Despite of the use of chemotherapy and surgical resection, the long-term survival remains unsatisfactory on account of recurrence and metastasis. Currently, effective comprehensive treatment, which is able to heal the advanced GC, is not obtainable. Thus, it is of paramount importance to figure out the molecular mechanisms leading to invasion and metastasis of GC.
PODXL, an anti-adhesive transmembrane glycoprotein, is a member of CD34 family, which is initially revealed in podocytes of renal glomeruli. Subsequent study showed that PODXL can also be identified in vascular endothelia [
3], hematopoietic progenitors [
4] and neurons [
5]. Previous study showed that PODXL was involved in cell adhesion of kidney cells via affecting the transepithelial resistance and modifying the distribution of junctional proteins [
6]. Recent studies have suggested that the role of PODXL may be of pivotal importance in the development and progression of various cancers [
7‐
9]. It was first revealed that PODXL can be detected as a stem cell marker in malignant cells of testicular cancer [
7]. Thereafter, PODXL expression was reported to be associated with tumor aggression and adverse prognosis in several cancers, such as, urothelial bladder cancer [
8], colorectal cancer [
10], pancreatic cancer [
11], hepatocellular carcimoma [
9] and GC [
12].Emerging evidence revealed that the altered expression of PODXL was implicated with migration, epithelial-mesenchymal transition (EMT), invasion and metastasis of cancers [
11,
13,
14]. Lin et al. [
13] demonstrated that the level of PODXL was closely correlated with motility and invasiveness of breast cancer cells via activating the Rac1/Cdc42/cortactin signaling. Enforced expression of PODXL increased the formation and activation of invadopodia, resulting in the tumor invasion and metastasis. Additionally, it was found that the altered expression of PODXL lead to the molecular changes (e.g., vimentin, E-cadherin, collagen I) associated with TGF-β induced EMT of human lung adenocarcinoma cells [
14], demonstrating the significant role of PODXL in the EMT and metastasis of cancers. All of above clinical and experimental evidences are in favor of the oncogenic role of PODXL in the development of human cancers. However, little is known about the precise functions and underlying mechanisms of PODXL in GC EMT, invasion and metastasis. Moreover, the molecular mechanisms regulating PODXL remain unclear.
Krüppel-like factor 4 (KLF4), a conserved zinc finger transcription factor of Krüppel-like factor family, is a key regulator in maintaining the integrity of progression of cell cycle [
15]. Mounting evidences from both experimental and clinical data have suggested that KLF4 functioned as a tumor suppressor in cancers and played a critical role in tumor differentiation, EMT, invasion and metastasis [
16,
17]. Interestingly, the villin-positive gastric progenitor cells of transgenic mice with KLF4 inactivation contributed to the transformation of gastric mucosa and then lead to the tumorigenesis in the antrum in mice, which further verified the prominent role of KLF4 in the development of GC [
18]. Additionally, recent study indicated that miR-103 can enhance the proliferation and metastasis of GC by repressing KLF4 expression [
19]. While, the underlying mechanism that how KLF4 regulates the EMT and metastasis of GC is still unknown.
In our current study, we determined to explore the role of PODXL in the EMT and metastasis of GC and demonstrate the regulatory function of KLF4 in PODXL expression and function. Our study concerning KLF4/PODXL signaling pathway might be conducive to better understand the process of EMT, invasion and metastasis of GC, and provide an independent prognostic indicator and therapeutic target for GC.
Methods
Patients and specimens
32 paired fresh GC and surrounding normal tissues were collected after radical gastrectomy in the Shanghai General Hospital and were stored at − 80 °C refrigerator for RNA extraction. 57 paired GC and surrounding normal tissues were obtained from patients diagnosed with GC at General Surgery Department of Shanghai General Hospital from 2013 to 2014. All specimens, preparing to construct tissue microarray (TMA), were paraffin-embedded, then validated by hematoxylin and eosin (H&E) staining and finally examined by two independent pathologists. The final TMA covered 54 GC tissues and 57 adjacent normal tissues. All patients enrolled had yet to receive radiotherapy and chemotherapy before surgery. Clinicopathological characteristics, diagnosed and confirmed by two independent pathologists, were presented in Table
1. All written informed consents were obtained before enrolling in the study. And the study was ratified by the Ethical Committee for Clinical Research of Shanghai General Hospital.
Table 1
Correlation between PODXL expression and clinicopathological parameters in GC (n = 54)
Age | | | | | 0.821339 |
< 65 | 26 | 11 | 15 |
≥ 65 | 28 | 11 | 17 | |
Gender | | | | | 0.753143 |
Male | 38 | 16 | 22 |
Female | 16 | 6 | 10 | |
T stagea | | | | | 0.025968* |
T2 | 5 | 4 | 1 |
T3 | 21 | 11 | 10 | |
T4 | 28 | 7 | 21 | |
N stage | | | | | 0.020323* |
N0 | 11 | 7 | 4 | |
N1 | 12 | 8 | 4 |
N2 | 13 | 3 | 10 | |
N3 | 18 | 4 | 14 | |
UICC stageb | | | | | 0.015122* |
I&II | 17 | 11 | 6 | |
III&IV | 37 | 11 | 26 | |
Nerve invasion | | | | | 0.117946 |
Yes | 29 | 9 | 20 | |
No | 25 | 13 | 12 | |
Vessel invasion | | | | | 0.510348 |
Yes | 29 | 13 | 16 | |
No | 25 | 9 | 16 | |
Differentiation | | | | | 0.000867*** |
Well | 3 | 3 | 0 | |
Moderate | 19 | 12 | 7 | |
poor | 32 | 7 | 25 | |
Tumor size | ≤ 3 | 23 | 8 | 15 | 0.442764 |
> 3 | 31 | 14 | 17 | |
Cell lines
The human gastric cancer cells, including AGS, SGC7901, MGC803, BGC823, MKN45, MKN28, HGC27, were obtained from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). All cells were cultured in 1640 medium mingled with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin–streptomycin. All the cells were maintained at 37 °C filled with 5% CO2.
The Oncomine and the Kaplan–Meier Plotter
RNA extraction and quantitative real-time PCR(qRT-PCR)
Based on the protocol previously described [
20], the total RNA of GC cell lines, tissues and adjacent normal tissues, was extracted and then transcribed into complementary DNA, respectively. The ΔΔCt method was performed to evaluate relative targeted genes levels. The specific primers used in study were the same as that depicted previously [
20]. Each result was carried out in triplicate.
Transient transfection and lentiviral transduction
The pcDNA3.1-KLF4 plasmid and negative control vector were synthesized from Obio Technology Co.,Ltd (Shanghai, China). The oligonucleotides were performed employing Lipofectamine™ 2000 (Invitrogen, USA) as described previously [
21]. The short hairpin RNA (shRNA) targeting PODXL was bought form Genechem Co., Ltd. (Shanghai, China). Sense: TATCAGTGAGATCAATTTC, Antisense: GAAATTGATCTCACTGATA. For lentivirus transfections, GC cells in logarithmic growth phase, cultured in 6-well plates, were transfected with lenti-shPODXL-virus or lenti-control virus. Thereafter, the GC cells with lentiviral transduction were purified with puromycin (Invitrogen, USA).
Nude mice xenograft models
To establish subcutaneous tumor, about 7 × 10
6 cells were subcutaneously injected into a nude mouse. 28 days after injection, all mice were sacrificed, and then the xenograft tissues were excised. Then the tumor weight and volume were measured. The following formula was used to calculated tumor volume: volume = 0.5 × width
2 × length. For liver metastatic assay, 5 × 10
6 cells were injected via ileocolic vein as we did previously [
21]. The mice were killed and the livers were then removed after 21 days’ inoculation or when they were moribund. The tissues were then paraffin-embedded and the slides finally validated by hematoxylin and eosin (H&E) staining. All mice were used in line with the guidelines of Institutional Animal Care of Shanghai General Hospital, and all efforts were employed to weaken animal suffering.
Immunohistochemistry (IHC)
After deparaffinization in xylene, the microarray of GC were rehydrated in the gradient ethanol. Then the microarray, prepared for antigen retrieval, was boiled in 10 mM sodium citrate buffer (PH 6.0) for 4 min. After endogenous peroxidase activity of the slide was blockaded by hydrogen peroxide for 10 min, the slides were covered with primary antibody: PODXL (1:200, Santa cruz, CA, USA), KLF4 (1:200, Santa cruz, CA, USA) at 4 °C overnight. Next, incubated with secondary antibody for half an hour at room temperature, the microarrays were then soaked with diaminobenzidine and Mayer’s hematoxylin. Finally, the microarray was dehydrated in the gradient ethanol and covered with cover slips. The scores of GC specimens were undertaken by two independent pathologist as reported previously [
20].
Chromatin immunoprecipitation (ChIP)
SimpleChIP enzymatic Chromatin IP kit(magnetic beads) (Cell Signaling Technology, USA) was used for ChIP assay. 293T cells (4*106), transfected with vector or KLF4, were cross-linked, quenched with glycine and then lysed by sonication. Next, immunoprecipitation was performed with the antibody against KLF4 (R&D, #AF3640), and IgG was applied as a normal control. Finally, precipitated DNA was amplified by PCR and examined for the presence of PODXL promoter sequence. And the primers were used as follows: forward: 5′-TCTTGCCACCAGGACACCTA-3′, reverse: 5′-TACCTCTTCCCAGACCCAAT-3′.
Statistical analysis
SPSS 21 statistical software (SPSS Inc., USA) was carried out to analyse all data. The significance of paired and unpaired continuous variables was calculated by Student’s t test and Mann–Whitney U test. The significances between targeted genes expression and clinicopathological features were determined appropriately by Fisher’s exact test and the χ2 test. Kaplan–Meier method and log-rank test were performed for survival curves and differences, respectively. The HR with 95% confidence interval and significance between individual factors and overall survival (OS), disease-free survival (DFS) were performed via Cox proportional hazards regression. P value < 0.05 was considered to be significant.
Discussion
Owing to the high economic burden and low public awareness, the morbidity of advanced GC in China is extremely high, which leads to the great mass of cancer-related deaths [
1]. Despite of improvement of surgery and chemotherapy for GC, the recurrence and metastasis of advanced GC is still at its peak [
22]. Thus, there is an urgent need to figure out the molecular mechanisms of invasion, metastasis and EMT of GC. In our current study, we explored the pivotal roles of KLF4 and PODXL in development and progression of GC and their underlying mechanisms. We found that KLF4 and PODXL constitute a novel signaling pathway, which is irreplaceable in invasion, metastasis and EMT of GC cells. Our findings may have presented an unique perspective on the pathogenesis and aggressive biology of GC.
PODXL, located on chromosome 7q32-q33, is a transmembrane sialomucins of CD34 family [
23]. Initially, PODXL was identified in podocytes, vascular endothelia and neurons and PODXL was essential for development of cells [
3,
5,
24]. Subsequently, many lines of evidence supported that PODXL occupied a significant role in the development and progression of cancers via acting as an oncogene [
7,
25]. First, PODXL was reported to be overexpressed in various cancers [
8,
10,
26,
27]. It was demonstrated by Kaprio et al. that PODXL was evaluated to be overexpressed in the vast majority of colorectal cancers [
10]. Based on the recent study, the level of PODXL in GC tissues was significantly higher in comparison with normal ones [
27]. Furthermore, available articles manifested that PODXL may be an independent prognostic marker in cancers [
8,
12,
28]. Boman et al. found that PODXL overexpression was significantly correlated with a low 5-year OS in urothelial bladder cancer and PODXL was an independent prognostic indicator for patients with urothelial bladder cancer [
8]. Additionally, mounting evidence illustrated that PODXL level was significantly correlated with clinical features, such as advanced tumor stage, high-grade stage and so forth [
10,
11,
29]. Forse et al. revealed that elevated PODXL expression was correlated with high histological grade, large tumor size and so on [
29]. In our present study, we demonstrated that PODXL was an oncogene from different angles. In addition, elevated PODXL expression was positively correlated with advanced tumor stage, high-grade stage and lymph node metastasis, which was consistent with the roles of PODXL in other cancers. Taken together, we manifested from molecular and clinical evidence that PODXL could promote GC invasion and metastasis.
The process of invasion and metastasis is complicated and the detailed molecular basis remains to be explored [
30]. Emerging evidence suggested that PODXL was involved in the process of invasion, metastasis and EMT in various cancers [
13,
14,
23]. Enforced PODXL expression in breast and prostate cancer cell lines promoted the migrated and invasive abilities by increasing expression of MMP-1 and MMP-9, and activating MAPK and PI3K signaling [
31]. Moreover, silencing of PODXL level in human lung adenocarcinoma cells decreased the spreading and migration abilities and resulted in the phenotype changes of cells by affecting EMT-associated protein, such as vimentin and E-cadherin. This findings manifested that PODXL was implicated in cell migration and invasion via EMT process [
14]. Additionally, recent study also revealed that the deletion of PODXL in breast cancer cells led to inhibition of primary tumor growth and metastasis in vivo [
23]. However, little was known concerning the roles of PODXL in the invasion and metastasis of GC cells. In the present study, we found that the migration and invasion ability was attenuated when silencing PODXL expression in GC cell lines. Further assays revealed that silencing PODXL level in GC cell lines led to reduction of MMP-2 expression, which could give rise to the decrease of migration and invasion of GC cells. Additionally, we observed that knockdown of PODXL in GC cell lines contributed to the acquisition of epithelial phenotype by upregulation of epithelial marker (β-catenin) and downregulation of mesenchymal marker (N-cadherin). Furthermore, we proved that silencing PODXL in GC cell lines led to the inhibition of tumor growth and metastasis in nude mice, consistent with above study. Thus, we demonstrated first that PODXL can play a critical role in migration, invasion and EMT of GC.
In view of the significant role of PODXL in GC migration, invasion and EMT, we further explored the underlying molecular mechanisms regulating PODXL overexpression in GC. Our current study verified that KLF4, a critical transcriptional factor that had been reported to be involved in the development of different cancers, was the upstream target of PODXL. Supporting evidences were provided from diverse angles: First, the correlation between KLF4 and PODXL expression in GC microarray was negative. Second, the mRNA level of KLF4 was negatively associated with that of PODXL in GC tissues. Additionally, elevated expression of KLF4 directly contributed to decrease of PODXL. Moreover, further bioinformatics and luciferase reporter assay revealed that KLF4 could directly bind to the 5′UTR of PODXL and then negatively regulate the level of PODXL. Therefore, we concluded that there was a novel KLF4/PODXL signaling pathway contributing to GC invasion and metastasis from clinical and mechanistic evidence. Interestingly, our previous and other studies illustrated that PODXL can also be regulated by a great many of miRNAs [
20,
32,
33]. And it was necessary to figure out possibility of the cross-talks between miRNAs and KLF4 in regulating PODXL expression, contributing to aggression of GC.
To sum up, our study explored the vital role of PODXL in EMT, progression and metastasis of GC and the molecular mechanisms between KLF4 and PODXL. Collectively, our current study not only manifested that PODXL may serve as an independent prognostic indicator for GC, but also revealed a novel KLF4/PODXL signaling pathway in GC, which may be a promising molecular target for the treatment of GC.