Metastatic squamous cell carcinoma of the head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7), which can activate signaling pathways to promote invasion and survival of SCCHN cells. We hypothesized that the RhoA/Rho-associated kinase (ROCK) pathway is involved in the CCR7-induced invasion and migration of metastatic SCCHN cells. Thus, using migration, matrigel invasion and scrape wound-healing assays, we elucidated the role of RhoA in mediating CCR7-associated cellular mobility. Pull-down assays and western blotting were used to measure RhoA and its downstream expression. Immunohistochemical staining and analysis were useful in identifying the correlation between CCR7 and RhoA expression and clinicopathological factors. The results showed that inhibition of RhoA/ROCK reduced the tumor cell migration and invasiveness induced by CCL19. Activated RhoA, proline-rich tyrosine kinase-2 (Pyk2) and cofilin induced by CCL19 were elevated, and increased RhoA, Pyk2 and cofilin activity was eliminated by CCR7mAb, RhoA/ROCK and Pyk2 inhibitors, indicating involvement of the RhoA/ROCK-Pyk2-cofilin cascade. In summary, CCR7 via RhoA/ROCK-Pyk2 cofilin pathway promotes invasion and migration of metastatic SCCHN cells.
Squamous cell carcinoma of the head and neck (SCCHN) accounts for over 90% of all head and neck cancer (
Chemokines induce cytoskeleton rearrangement, firm adhesion to endothelial cells, and directional migration by binding to G-protein-coupled receptors (
Attention has focused on the involvement of Rho family GTPases and their downstream effectors in chemokine-elicited migration (
In the present study we examined whether RhoA was activated by CCR7, as well as the role and the molecular mechanisms of the RhoA pathway in CCR7 regulating SCCHN metastasis. The results showed that CCL19 induced activation of the Rho/ROCK-Pyk2-cofilin pathway, whereas MLC was not involved in the process. Since chemical inhibitors of this signal transduction pathway are currently in use clinically (
The PCI-37B metastatic SCCHN cell line, which strongly expresses CCR7, was a kind gift from the University of Pittsburgh (
SCCHN tissue specimens were obtained from 75 patients by biopsy prior to chemotherapy or radiotherapy at the Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University. The term ‘metastatic’ in the present study refers to patients with positive lymph nodes that were recognized at initial presentation or later based on histopathological diagnosis following neck dissection. The classification of SCCHN, including primary tumors (T), regional lymph nodes (N), distant metastasis (M) and stage grouping, was determined according to the regulations of the Union for International Cancer Control (UICC) for Head and Neck Cancer [tumor-node-metastasis (TNM) classification, 1997]. Ten samples of normal tissues adjacent to the benign tumor were chosen as controls. The study protocol was approved by the Medical Ethics Committee of the Affiliated Stomatological Hospital of China Medical University and performed according to the declaration of Helsinki. All the specimens were obtained with the consent of the patients prior to surgery and in accordance with the Health Insurance Portability. Written informed consent was obtained from all individuals.
The CCR7 chemokine ligand, CCL19 (MIP-3β) was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-CCR7 antibody was purchased from BD Biosciences (San Jose, CA, USA). C3 exoenzyme (Rho inhibitor) was purchased from Cytoskeleton Inc. (Denver, CO, USA) and Y-27632 (ROCK inhibitor) was purchased from Sigma (Santa Clara, CA, USA). Tyrphostin A9 (Pyk2 inhibitor) was purchased from Calbiochem (San Diego, CA, USA). Anti-RhoA, anti-phospho-Pyk2 (Tyr402), anti-phospho-cofilin (Ser3) and anti-phospho-myosin light chain (Ser19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Bio-Rad protein assay dye reagent was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Enhanced chemiluminescence was purchased from Amersham Pharmacia Biotechnology (Piscataway, NJ, USA).
Immunohistochemical staining used conventional horseradish peroxidase immunohistochemical staining methods. Briefly, 5-μm sections of the specimens were deparaffinized and hydrated with 0.6% H2O2 in methanol to inhibit endogenous peroxidase. Antigen retrieval was performed and slides were incubated with normal blocking serum for 10 min. Sections were incubated with primary antibodies (1:100 dilution): CCR7-specific monoclonal antibody, and rabbit anti-RhoA polyclonal antibody overnight at 4°C. Immunodetection was performed using peroxidase-labeled secondary antibody (R&D Systems) and diaminobenzidine for visualization. The sections were counterstained with hematoxylin (Sigma). Negative controls included omission of the primary antibody. Cell morphology was analyzed by microscopy (Nikon Eclipse 80i; Tokyo, Japan) at ×100–400 magnification. According to the percentage of positive tumor cells, the cells were scored as negative (−), <10% or no staining; weak positive (+), 11–50%; positive (++), 51–75%; or strongly positive (+++), >75%.
Chemotaxis in response to chemokines was measured by counting cells migrating through a polycarbonate filter (8-μm pore size) in 24-well Transwell chambers in triplicate in DMEM with 0.5% (w/v) BSA (Invitrogen). Cell suspensions (2×105 cells/200 μl) were placed in the top chamber of the filter. Aliquots of chemokines were added to wells. After 24 h, the cells in each lower well were counted under a light microscope in at least five different fields (original magnification, ×200). Means ± SD were recorded for each condition, and an index was calculated based on the control involving random migration.
Cell invasion was quantified
Migration of PCI-37B cells was measured using an
RhoA-GTP was measured with recombinant purified glutathione-S-transferase rhotekin Rho-binding domain (GST-TRBD) bound to glutathione beads and purified as previously described (
Cells were harvested in a lysis buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaF, 1 mM NaV3O4, 1% Triton X-100 and 1X protease inhibitor of protein tyrosine phosphatases). Lysates were sonicated for 3 sec and centrifuged at 4°C at 12,000 rpm for 30 min. The supernatant was collected for protein quantification using the Bio-Rad protein assay dye reagent. Protein (50 mg) was size-fractionated through a 10% SDS-PAGE gel and transferred onto nitrocellulose filters which were blocked (1% non-fat dry milk, 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5) and incubated with primary antibody (1:1,000 dilution). Nitrocellulose filters were incubated with horseradish peroxidase-conjugated secondary antibodies and bands were visualized with enhanced chemiluminescence and quantified by scanning densitometry using ImageJ software.
Experiments were run in triplicate and repeated at least three times. Numerical data were expressed as means ± standard deviation (SD). Correlations were analyzed using the Spearman’s and χ2 tests. Statistical differences between two groups were evaluated using an unpaired Student’s t-test. P<0.05 was considered to indicate a statistically significant result. Statistical analyses were performed with SPSS 13.0 software.
Immunohistochemistry was utilized to examine CCR7 and RhoA expression in SCCHN tumor tissues, metastatic and normal lymph nodes and oral mucosal tissues. CCR7 and RhoA were evident in the cell membrane and cytoplasm, and were mainly expressed in the stromal surroundings of tumors and metastatic lymph node cells. Stained cells were few or absent in normal lymph nodes and oral mucosal tissues (
Since CCR7 regulates actin organization (
Using chemotaxis and matrigel invasion assay we analyzed the role of RhoA in PCI-37B migration and invasiveness in response to CCL19. The presence of CCL19 in the lower chamber stimulated a >1.5-fold increase in chemotaxis and invasion of PCI-37B across the filter membrane while C3 and Y-27632 blocked this activity (P<0.05;
Western blotting indicated that CCL19-pretreated PCI-37B cells had an increased expression of p-Pyk2 (
A critical problem in SCCHN therapy is metastasis, particularly to lymph nodes, lung, liver and bone. Metastasis is based on chemotaxis and the migratory ability of tumor cells. CCR7 induces chemotaxis and invasion of SCCHN cells via the activation of several signaling pathways (
RhoA is upregulated in various human tumor types. RhoA stimulates cell cycle progression and cytokinesis, as well as cell migration (
Rho GTPases participate in various signal transduction pathways via the activation of multiple downstream effector proteins. An important downstream effector is ROCK (
Tyrosine kinase Pyk2 is involved in signaling from chemokine receptors in different cells (
In conclusion, CCR7 promotes tumor migration and invasiveness via the RhoA/ROCK pathway in metastatic SCCHN. Direct inhibition of ROCK, a downstream molecule of Rho GTPase, using a pharmacological inhibitor (Y-27632) or a molecular approach (dominant-negative expression vector) can produce substantial therapeutic effects suggesting that direct targeting of the RhoA/ROCK pathway, alone or in combination with other targets, may be promising as a chemotherapeutic target (
This study was supported by grants from the National Natural Science Foundation of China (no. 81372877), the National Young Scholars Science Foundation of China (no. 81102058), the Foundation of Education Bureau of Liaoning Province (nos. 2009A755 and L2014317), the Public Welfare Fund Project for Science of Liaoning Province (no. 2011002001), the Natural Science Foundation of Liaoning Province (no. 2014021096), and the Excellent Talent Fund Project of Higher Education of Liaoning Province (LJQ2014087).
Immunohistochemical staining of CCR7 and RhoA in normal tissues, primary SCCHN and metastatic lymph nodes, normal lymph nodes and normal human oral mucosal tissues (original magnification, ×200) indicate immunoreactivity for (A) CCR7 and (B) RhoA. CCR7 immunoreactivity was observed mainly in the cell membrane and cytoplasm of tumor cells, conforming to RhoA immunoreactivity. CCR7 and RhoA expressed in normal lymph nodes and normal human oral mucosal tissues were weak or absent. CCR7, chemokine receptor 7; SCCHN, squamous cell carcinoma of the head and neck.
CCR7 stimulated the activation of RhoA. CCR7, chemokine receptor 7.
Role of RhoA and ROCK inhibitors in CCL19-induced cell invasion. ROCK, Rho-associated kinase.
Role of RhoA and ROCK inhibitors after CCL19-induced cell migration. ROCK, Rho-associated kinase.
Roles of RhoA and ROCK inhibitors after CCL19-induced scrape wound-healing. ROCK, Rho-associated kinase.
Western blotting shows the role of RhoA and ROCK inhibitors after CCL19-induced p-Pyk2 expression. PCI-37B cells pretreated with/without C3 (50 ng/ml) or Y-27632 (10 M, at 37°C for 1 h) were stimulated with CCL19 (200 ng/ml for 5 min). ROCK, Rho-associated kinase.
Western blotting shows the role of RhoA, ROCK and Pyk2 inhibitors after CCL19-induced p-cofilin expression. ROCK, Rho-associated kinase. PCI-37B cells pretreated with/without C3 (50 ng/ml) or Y-27632 (10 M) CCR7mAb (10 ng/ml) or tyrphostin (10 M, at 37°C for 1 h) were stimulated with CCL19 (200 ng/ml for 5 min).
Western blotting shows the role of RhoA, ROCK and Pyk2 inhibitors after CCL19-induced p-MLC expression. PCI-37B cells pretreated with/without C3 (50 ng/ml) or Y-27632 (10 M) CCR7mAb (10 ng/ml) or tyrphostin (10 M, at 37°C for 1 h) were stimulated with CCL19 (200 ng/ml for 5 min). ROCK, Rho-associated kinase.
Correlation between CCR7 expression and SCCHN clinicopathological characteristics.
Characteristics | Cases | +-+++ | + (%) | χ2 |
---|---|---|---|---|
Age (years) |
39/36 | 29/25 | 74.4/69.4 | 0.224 |
Male/female | 42/33 | 29/25 | 69.0/75.8 | 0.413 |
Tumor size | ||||
T1, T2 | 24 | 16 | 66.7 | 0.498 |
T3, T4 | 51 | 38 | 74.5 | 0.595 |
High diff | 48 | 36 | 75 | |
Low diff | 27 | 18 | 66.7 | |
Clinical stage | ||||
I, II | 20 | 10 | 50 | 6.548 |
III, IV | 55 | 44 | 80 | |
Nodal metastases | ||||
Yes | 30 | 27 | 90 | 8.036 |
No | 45 | 27 | 60 |
P<0.05, difference of CCR7 expression within clinicopathological characteristics.
CCR7, chemokine receptor 7; SCCHN, squamous cell carcinoma of the head and neck; diff, difference.
Correlation between RhoA expression and SCCHN clinicopathological characteristics.
Characteristics | Cases | +-+++ | + (%) | χ2 |
---|---|---|---|---|
Age (years) |
39/36 | 22/24 | 56.4/66.7 | 0.830 |
Male/female | 42/33 | 29/17 | 69.0/51.5 | 2.395 |
Tumor size | ||||
T1, T2 | 24 | 13 | 54.2 | 0.764 |
T3, T4 | 51 | 33 | 64.7 | 0.077 |
High diff | 48 | 30 | 62.5 | |
Low diff | 27 | 16 | 59.3 | |
Clinical stage | ||||
I, II | 20 | 6 | 30 | 11.29 |
III, IV | 55 | 40 | 72.7 | |
Nodal metastases | ||||
Yes | 30 | 24 | 80 | 7.346 |
No | 45 | 22 | 48.9 |
P<0.05, difference of RhoA expression within clinicopathological characteristics.
SCCHN, squamous cell carcinoma of the head and neck; diff, difference.
Correlations between the CCR7 and RhoA expression in SCCHN primary tumor.
RhoA | |||
---|---|---|---|
|
|||
CCR7 | +-+++ | - | Total |
+-+++ | 36 | 18 | 54 |
- | 10 | 11 | 21 |
Total | 46 | 29 | 75 |
CCR7, chemokine receptor 7; SCCHN, squamous cell carcinoma of the head and neck.