Background
Cervical cancer is the second most common cancer in women worldwide and it has been steadily increasing in young women [
1]. Human papillomavirus (HPV) infection causes virtually all cases of cervical cancer. Recent years the great advancement of prophylactic vaccines which aim directly at different types of HPV has been achieved and results in a substantial reduction in the incidence of cervical cancer [
2]. Notwithstanding, many issues related to the radical therapy of existing cervical cancer remain unresolved. One major problem is the cervical cancer metastasis, which is the leading cause of mortality.
Cancer metastasis is composed of a complex series of phenotypic and biochemical changes such as angiogenesis, lymphangiogenesis, gene expression, motility or cell shape. These alterations are regulated by several sets of growth factors and their cognate receptors [
3]. Vascular endothelial growth factor C (VEGF-C), a dimeric glycoprotein belonging to VEGF family of cytokines, is found to be implicated in the most aggressive tumors. By binding to its receptor Flt-4, VEGF-C promotes angiogenesis and/or lymphangiogenesis, thus accelerates cancer metastasis to lymph nodes and distant organs [
4,
5]. Correspondingly, clinical studies have verified that VEGF-C expression is closely related to invasion phenotype and affects the patient's survival in cervical carcinomas [
6‐
8]. Previously, it was reported that the Flt-4 expression was restricted in the endothelial cells of lymphatic vessels. Recent years the Flt-4 has been shown to be expressed in a variety of human malignancies, including cervical cancer [
9,
10]. These observations hint the possibility that as the specific ligand to Flt-4, VEGF-C may be implicated in cervical cancer progression by direct impacts on tumor cells.
Cell migration is critical to cancer cell invasion and metastasis. The first step is represented by dynamic filamentous actin cytoskeletal remodeling, which allows the formation of protrusions that adhere to the extra-cellular matrix and generate intra-cellular forces for cell movement [
11,
12]. Indeed, actin remodeling is involved in cancer transformation and metastasis [
13]. Our previous work indicated that actin remodeling is the primary step, not only for cancer cell metastasis [
14], but also for endothelial and neuron cell migration [
15,
16]. These events are mediated by moesin, an actin-binding protein belonging to the ezrin/radixin/moesin (ERM) family. Indeed, moesin gene expression is shown to be strongly associated with metastatic phenotypes of cervical cancer [
17]. There is also evidence showing that VEGF-C enhances cervical cancer cell motility [
10], but the underlying mechanisms remain largely unknown. In the present study, we purpose to investigate the effects of VEGF-C on actin cytoskeleton remodeling and on cervical cancer cell migration and to characterize the role of moesin and the signaling cascade implicated in these actions.
Methods
Cell cultures and treatments
An established cervical carcinoma cell line (SiHa) was used for this study. SiHa cells were incubated in RPMI 1640 medium containing 10% fetal calf serum (FCS), L-glutamine and penicillin streptomycin under a 5% CO2 atmosphere at 37°C. An inhibitor was always to be added 1 h before starting the treatments. Recombinant human VEGF-C wild type (2179-VC-025) and the fusion protein of human IgG with the extracellular ligand-binding domains of Flt-4 (Flt-4/IgG) (349-F4-050) were purchased from R&D Systems (Minneapolis, MN). The selective inhibitor of the Rho-associated protein kinase Y-27632 was from Sigma-Aldrich (Saint-Louis, MO).
Immunoblottings
Cell lysates were separated by SDS-PAGE. Antibodies used were: moesin (clone 38, Transduction Laboratories, Lexington, KY), Thr558-P-moesin (sc-12895, Santa Cruz Biotechnology, Santa Cruz, CA), RhoA (sc-418, Santa Cruz), ROCK-2 (sc-5561, Santa Cruz), β-actin (sc-1615, Santa Cruz). Primary and secondary Abs were incubated with the membranes with standard technique. Immunodetection was accomplished using enhanced chemiluminescence. Chemiluminescence was acquired with a quantitative digital imaging system (Quantity One, BioRad, Hercules, CA) allowing to check for saturation. Densitometry values were adjusted to β-actin intensity and normalized to expression level from the control sample.
Kinase assays
SiHa cells were harvested in 20 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 0.5% IGEPAL and 0.1 mg/mL PMSF. Equal amounts of cell lysates were immunoprecipitated with 40 μg Rhotekin RBD agarose (14-383, upstate) vs. GTP-RhoA or an Ab vs. ROCK-2 (sc-5561, Santa Cruz). The IPs were washed three times with buffer containing 20 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 0.1% IGEPAL and 0.1 mg/mL PMSF. For ROCK-2 activity assay, two additional washes were performed in kinase assay buffer (20 mM MOPS, 25 mM b-glycerophosphate, 5 mM EGTA, 1 mM DTT) and the samples were therefore resuspended in this buffer. 5 mg of de-phosphorylated myelin basic protein (Upstate) together with 500 mM ATP and 75 mM MgCl2 were added to each sample and the reaction was started at 30°C for 20 min. The reaction was stopped on ice and the samples were resuspended in Laemmli Buffer. The samples were separated with SDS-PAGE and Western blot analysis was performed using antibodies recognizing RhoA (sc-418, Santa Cruz) or Thr98-P-myelin basic protein (05-429, Upstate).
Cell immunofluorescence
SiHa cervical cancer cells were grown on coverslips and exposed to treatments. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X for 5 min. Blocking was performed with 3% normal serum for 20 min. After washing the nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) (Sigma) and actin was stained with Texas Red-phalloidin (Sigma). The coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence was observed by an Olympus BX41 microscope and images were captured by DP70 Olympus digital camera with high-resolution.
Transfection experiments
Each plasmid (15 μg) was transfected into SiHa cervical cancer cells using the Lipofectamine (Invitrogen) according to the manufacturer's instructions. Plasmids containing RhoA T19 and RhoA G14V were transfected. These constructs were obtained from the Guthrie cDNA Resource Center
http://www.cdna.org. As control, parallel cells were transfected with empty pcDNA3.1+ plasmid (mock-transfected). After transfection for 24 h, cells (60-70% confluent) were treated with VEGF-C (100 ng/mL) for 48 h and cellular extracts were prepared according to the experiments to be performed.
Moesin on-target plus siRNA (J-011772-06), ROCK-2 siGENOME SMARTpool siRNA (L-004610-00) and scrambled control siRNA (D-001810-01) were obtained from Dharmacon. SiHa cells (40% confluent) were serum-starved for 1 h followed by incubation with 100 nM target siRNA or control siRNA (scrambled siRNA) for 6 h in serum-free media. The serum-containing media was then added (10% serum final concentration) for 42 h before experiments and/or functional assays were conducted. Target protein silencing was assessed through protein analysis up to 48 h after transfection.
Cell migration assays
Cell migration was assayed with razor scrape assays as we previously described [
14]. Briefly, a razor blade was pressed through the confluent SiHa cervical cancer cell monolayer into the plastic plate to mark the starting line. Cells were swept away on one side of that line. Cells were washed, and 2.0 mL of RPMI 1640 containing steroid-deprived FBS and gelatin (1 mg/mL) were added. Migration was monitored for 48 hours. Fresh medium and treatment were replaced every 12 h. Cells were digitally imaged and migration distance was measured by phase-contrast microscopy.
Cell invasion assays
As we previously described [
14], cell invasion were assayed following the standard method by using the BD BioCoat™ Growth Factor Reduced (GFR) Matrigel™ Invasion Chamber (BD Bioscience, USA). In brief, after rehydrating the GFR Matrigel inserts, the test substance was added to the wells. An equal number of Control Inserts (no GFR Matrigel coating) were prepared as control. 0.5 mL of SiHa cell suspension (2.5 × 10
4 cells/mL) was added to the inside of the inserts. The chambers were incubated for 48 h at 37°C, 5% CO
2 atmosphere. After incubation, the non-invading cells were removed from the upper surface of the membrane using cotton tipped swabs. Then the cells on the lower surface of the membrane were stained with Diff-Quick stain. The invading cells were observed under the microscope at 100 × magnification. Cells were counted in the central field of triplicate membranes. The invasion index was calculated as the % invasion test cell/% invasion control cell.
Patients, specimens, and immunohistochemical staining
Tissues were from cervical cancer patients hospitalized in the first hospital of Sun Yat-sen University (Guangzhou, China) with informed patient consent, including 24 patients with cervical intra-epithelial neoplasia (CIN), 22 patients with squamous carcinoma at stage I, and 20 patients with squamous carcinoma at stage II without radiotherapy or chemotherapy. The histological diagnosis was made according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Lymph node status was evaluated. Tissues from the normal cervix of 20 patients suffering from hysteromyoma who were undergone hysterectomy were taken as the control. All tissues were stored at -80°C until use.
Immunohistochemical staining was performed as we previously described [
18]. Histological sections of 4 μm were mounted on silanized slides and allowed to dry for 1 h at room temperature (RT), followed by 1 h incubation in an oven at 60°C. Briefly, after deparaffination and rehydration, epitope retrieval was performed by immersing slides in DAKO Epitope Retrival Solution (0.01 M citrate buffer, pH 6.0) in a water bath at 98°C for 40 minutes followed by a 20 minutes cool-down period at room temperature (RT). The working dilution for the anti-human moesin monoclonal antibody (clone 38, Transduction Laboratories, Lexington, KY) was 1:50. The sections were incubated with primary antibodies at RT for 1 hour. Expression of moesin was evaluated according to the ratio of positive cells per specimen and staining intensity as described previously [
19]. The ratio of positive cells per specimen was evaluated quantitatively and scored 0 for staining of ≤ 1%, 1 for staining of 2 to 25%, 2 for staining of 26 to 50%, 3 for staining of 51 to 75%, and 4 for staining ≥ 75% of the cells examined. Intensity was graded as follows: 0, no signal; 1, weak; 2, moderate; and 3, strong staining. A total score of 0 to 12 was finally calculated and graded as negative (-; score: 0-1), weak (+; 2-4), moderate (++; 5-8), and strong (+++; 9-12).
Statistical analysis
Values are expressed as mean ± SD. Statistical differences between mean values were determined by ANOVA, followed by the Fisher's protected least significance difference (PLSD). To compare the differences of groups for immunohistochemistry, Kruskal Wallis Test and Chi-Square Test were used to calculate the P value. All differences were considered significant at P < 0.05.
Discussion
Cervical cancer is one of the most frequent types of tumor worldwide and its metastasis is the leading cause of death in patients with cervical cancer. However, the current understanding on the molecular mechanisms of cervical cancer metastasis is unclear. In this study, we demonstrated that VEGF-C accelerated cervical cancer metastasis by directly driving cancer cell migration and invasion. These processes were closely related to the effects of VEGF-C on moesin expression and activation through RhoA/ROCK-2 signaling pathway. To our knowledge, this is the first time reporting that moesin is the target protein of VEGF-C and responsible for cervical cancer metastasis.
Tumor metastasis is a complex process. A number of cellular alterations occur as cancer cell spread to lymph nodes and distant organs, including cellular transformation and tumor growth, angiogenesis and lymphangiogenesis. VEGF-C, the dimeric glycoprotein belonging to VEGF family of cytokines, plays critical role in a most of aggressive tumors. Indeed, the elevated level of serum VEGF-C has been found in patients with breast cancer [
22], lung cancer [
23] and cervical cancer [
8] and it appears to be a unique marker for an early diagnosis of cancer metastasis. Moreover, increased VEGF-C mRNA expression in tumor tissues correlates positively with lymphatic metastasis and poor prognosis [
24‐
26].
VEGF-C is mainly produced by tumor and stromal cells and it induces lymphangiogenesis that promotes the growth and metastasis of neoplasms. These biological effects are predominantly elicited by the activation of VEGF-C specific receptor Flt-4 [
27,
28]. It has been proposed that Flt-4 is restricted to be expressed on the lymphatic endothelium and tumor blood vessels [
29]. However, recent studies have indicated that Flt-4 is also expressed in a variety of human malignancies [
28], indicating that VEGF-C may affect cancer development and progression by direct effects on tumor cells. In agreement, we found that VEGF-C/Flt-4 axis drove cervical cancer cell horizontal migration and three-dimensional invasion into matrices, which are consistent with limited reports that VEGF-C directly stimulated the motility of other types of cancer cells [
10,
30,
31], confirming that in addition to the regulatory actions on lymphangiogenesis, VEGF-C can promote tumor cell metastasis by directly triggering cell migration and invasion in an autocrine fashion.
While a lot is known on the molecular mechanisms of VEGF-C on lymphangiogenesis [
32], little information is available on VEGF-C's direct impact on tumor cell motility. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and is essential for most types of cell migration [
33]. For example, our previous studies have demonstrated that actin cytoskeleton remodelling is the initial process for breast cancer metastasis [
14,
34,
35]. In this work, we indicated that VEGF-C provoked actin fibers rearrangement in cervical cancer cells and increased the formation of specialized membrane structures, which may interact with the extra-cellular matrix and with nearby cells, thus allowing the tumor cells to achieve locomotion. In analogy to our study, it's also reported by other groups that VEGF or VEGF-C induced actin reorganization and cell shape change in vascular endothelial cells, leading to the sprouts of endothelial cells [
36,
37].
Moesin, a member of the ERM family, is an actin-binding protein that plays a imperial role in cell motility by linking the actin cytoskeleton to a variety of membrane-anchoring proteins [
38,
39]. When activated through phosphorylation of Thr
558, moesin induces actin de-polymerization and re-assembly toward the cell membrane edge, being responsible for the formation of cortical actin complexes. Our data showed that VEGF-C up-regulated moesin expression and phosphorylation and the silencing of moesin abolished VEGF-C's impact on actin rearrangement, indicating that moesin is the target protein for VEGF-C and the key mediator for actin cytoskeleton remodelling. This may be strong evidence to understand the mechanism that high moesin expression correlates with lymph node metastasis. Actually, high expression of ERM proteins has been identified as the prognostic markers in clinical cancers [
40] and the expression pattern of moesin can be regarded as an independent prognostic factor in patients with oral squamous cell carcinoma [
41]. Therefore, moesin expression may serve as a potential marker for cervical cancer metastasis, which need be further clinically investigated.
As previously reported [
21,
42,
43], RhoA is a crucial mediator conveying the upstream signaling evoked by various factors to its downstream target ROCK-2, which is a known activator of ERM proteins. In this study, we showed that VEGF-C increased RhoA/ROCK-2 expression and activities. Moreover, the use of RhoA dominant negative construct or the inhibitor of ROCK-2 blocked VEGF-C-enhanced moesin expression and phosphorylation, implying that RhoA/ROCK-2 represents the link between VEGF-C and the activation of moesin. Indeed, the significance of RhoA/ROCK-2 in VEGF signalling has been displayed in endothelial cells, where RhoA/ROCK-2 mediates VEGF functions on microvascular permeability [
44], endothelial migration and angiogenesis [
45]. More importantly, the overexpression of RhoA is often observed in clinical cancers [
46] and it has been repeatedly identified as a gene associated with metastasis [
47,
48]. These findings may partly be elucidated by our data that RhoA/ROCK-2 was able to activate moesin and finally led to enhanced cell motility. However, it should be pointed out that our data is mainly obtained from
in vitro experiments. Whether the linkage between VEGF-C, RhoA/ROCK-2 and moesin exists and functions
in vivo as we expected remains obscure. Our future studies will be aimed to further define this issue and one possible way is to suppress these proteins expression respectively with effective approaches, such as siRNA gene therapy in cervical cancer mice model and then their relationship and functions in cancer metastasis will be elucidated.
VEGF-C-enhanced migration and invasion of SiHa cell is markedly inhibited by moesin or ROCK-2 specific siRNAs, suggesting that RhoA/ROCK-2/moesin cascade plays an important role in these processes. Notwithstanding, this is not the exclusive way that VEGF-C promotes cancer cell motility. For example, it has been shown that VEGF or VEGF-C promoted cancer cell metastasis by up-regulation of integrin αvβ6 expression or of the neural cell adhesion molecule contactin-1 through divergent cellular signalings [
10,
31]. Moreover, other members of ERM family, such as ezrin, function similarly as moesin in cancer cells. Therefore, further efforts should focus on the exact function of these proteins involving cervical cancer metastasis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MH designed and carried out the experiments, analyzed the data, drafted and revised the manuscript; YC, WL, QSL, JXL and JHH carried out the experiments; XDF designed the experiments, analyzed the data, drafted and revised the manuscript. All authors read and approved the final manuscript.