Introduction
Tongue cancer is a form of oral cancer. The majority of cases of tongue cancer are squamous cell carcinomas which originate from the surface layer of the tongue. At present, tongue squamous cell carcinoma is the most common form of oral cancer and is associated with early lymph node metastasis and poor survival. Early tongue cancer is often treatable with surgery or radiation therapy, depending upon the location and size. Therefore, a growing emphasis has recently been placed on inhibiting the invasion and metastasis of tongue cancer. However, the effect of inhibition remains unclear.
Focal adhesion kinase (FAK) (1) functions as a phosphorylation-regulated signaling scaffold and is important for adhesion turnover, Rho-family GTPase activation, cell migration and cross-talk between growth-factor signalling and integrins (Mitra et al., 2005) (2). This ubiquitously expressed, essential protein contains an N-terminal FERM domain, a central kinase domain, proline-rich regions and a C-terminal focal-adhesion-targeting domain that interacts with paxillin (3). p130Cas-Crk signaling is regulated by interacting proteins. Cells utilize FAK-Src activity to recruit a p130Cas-Crk complex that promotes Rac GTPase activity, leading to lamellipodia formation in migrating cells (4–6). Rates of focal adhesion formation/turnover are affected by FAK-Src complex phosphorylation of paxillin, paxillin kinase linker and p130Cas. Elevated extracellular signal regulated kinase 2 and c-Jun N-terminal kinase (JNK) promote increased gene transcription of target proteins leading to enhanced secretion of matrix metalloproteinase (MMP)-2 and -9. Protease localization and activation at the leading edge of migrating tumor cells is regulated by integrins and facilitates matrix degradation. However, the mechanism of FAK in invasion and metastasis of tongue cancer remains unclear.
In the present study, Tca-8113 cells were selected to analyze the effect of FAK knockdown on tongue cancer cells, demonstrating that FAK promotes Tca-8113 invasion and metastasis by JNK signaling.
Materials and methods
Cell culture
Human tongue cancer TCA-8113 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in complete DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C, in a 5% CO2-95% O2 atmosphere and passaged every 3–5 days. The study was approved by the ethics committee of Liaoning medical college, Jinzhou city, China.
Knockdown of FAK
shRNAs against FAK were designed and synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China). The sequence of the sense strand was as follows: 5′-GCTAGTGACGTATGGATGT-3′. TCA-8113 cells in exponential growth phase were plated in six-well plates and allowed to adhere for 24 h prior to transfection. Transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). Briefly, cells were cultured to 80% confluence in a six-well culture plate and transfected at a 1:4 ratio (plasmid DNA:transfection reagent = 5 μg:20 μl) (7). Control cells were transfected with control RNA under the same conditions. Transfection efficiency was analyzed by western blot analysis at 72 h.
Transwell assay
The transwell assay was performed using 24-well transwells (Costar, Cambridge, MA, USA). Following 48 h transfection, cells were seeded into fibronectin gel pre-coated, porous upper chamber inserts (1×104 cells/well) and allowed to invade for 24 h. Following 24 h, inserts were inverted and stained with Hoechst 33258. The number of invaded cells were observed and quantified using a Zeiss Axio Scope A1 fluorescent microscope (Carl Zeiss AG, Oberkochen, Germany; magnification, ×100). Five fields were randomly selected and the number of penetrated cells was quantified.
Wound healing assay
Following 48 h transfection, cells were harvested and seeded in six-well plates (1×105 cells/well) and cultured until cells reached. The cells were scratched using a yellow pipette tip, washed with PBS three times and cultured in serum-free medium for 24 h. The status of cell migration was represented by the rate of wound closure.
Western blot analysis
Following 48 h siRNA transfection, cells were harvested, washed twice with ice-cold PBS, then lysed in RIPA buffer [150 mM NaCl, 1% NP-40, 1% SDS, 1 mM PMSF, 10 μg/ml leupeptin, 1 mM aprotinin and 50 mM Tris-Cl (pH 7.4)]. The cell lysate was cleared by centrifugation at 12,000 × g for 15 min. Cell lysate containing 50 μg protein was separated by 10% SDS-PAGE and the protein was transferred onto polyvinylidene fluoride membranes. Following blocking with 5% non-fat dry milk in Tris-buffered saline, the membrane was incubated overnight with primary antibodies against FAK (FAK sampler kit, 9330; Cell Signaling, Danvers, MA, USA)., MMP-2 (4022; Cell Signaling), MMP-9 (3852; Cell Signaling), MMP-14 (Ab51074; Abcam, Cambridge, MA, USA), N-cadherin (sc-59987; Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (sc-52328; Santa Cruz), Paxillin (2542; Cell Signaling), p-Tyr118 (2541; Cell Signaling) and p-S178 (ab51617; Abcam). Following this, the membrane was incubated with appropriate secondary antibodies for 1 h and stained with ECL. Protein expression levels of the targeted proteins were analyzed by UVP gel analysis system and images of the membranes were captured (7).
Immunofluorescence
Following 48 h siRNA transfection, cells were harvested and replated on fibronectin pre-coated coverslips in 6-well plates. After 24 h, cells were fixed in 3.7% formaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with PBS containing 1% BSA and incubated with specific primary antibodies for 60 min. Next, the slides were mounted by 95% glycerol and observed using the Zeiss A-1 fluorescent microscope (Carl Zeiss AG).
Cytoskeleton staining
Cells were harvested and replated on fibronectin-coated coverslips (10 μg/ml). Following 24 h, cells were fixed in 3.7% formaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with PBS containing 1% BSA and incubated with TRITC-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The slides were mounted by 95% glycerol and observed using the Zeiss Axio Scope A1 fluorescent microscope.
Gelatin zymography
Conditioned medium was collected and concentrated by 2-fold using a centrifugal concentrator. Equal amounts of protein were loaded and separated by 10% polyacrylamide gel containing 1 g/l gelatin. The gels were re-natured in 2.5% Triton-X-100 with gentle agitation for 30 min at room temperature. The gel was pretreated with developing buffer [5 mM CaCl2, 50 mM Tris, 0.2 mM NaCl and 0.02% Brij-35 (pH 7.5)] for 30 min at room temperature, then developed in developing buffer overnight at 37°C. Following this, the gel was stained with Coomassie Brilliant Blue R-250 for 30 min and destained with destaining solution. Protease activity was analyzed using a gel imaging and analysis system (9,10).
Statistical analysis
SPSS 13.0 statistical software was used for analysis of the data (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.
Results
Knockdown of FAK inhibits the invasion and metastasis of TCA-8113
To determine whether FAK knockdown affects the invasion and metastasis of TCA-8113, FAK knockdown was performed by transfection of FAK-specific shRNA and the effect of RNA interference was determined using western blot analysis (Fig. 1C). The invasion and metastasis potential of FAK knockdown cells was analyzed further by wound healing and transwell assays (11). Wound healing assay revealed that the metastasis of FAK knockdown cells was significantly decreased (~50%) compared with the control cells (Fig. 1B). Transwell assay demonstrated that knockdown of FAK in TCA-8113 cells reduced the invasion potential to 46% compared with control cells (Fig. 1A).
Downregulation of FAK in TCA-8113 inhibits epithelial-mesenchymal transition (EMT)
EMT is essential for the invasion and metastasis of TCA-8113 (12). To elucidate the underlying mechanisms, the effects of FAK knockdown on EMT were examined by analyzing the expression of E-cadherin and N-cadherin in FAK knockdown cells. Knockdown of FAK increased the levels of E-cadherin, whereas the expression of N-cadherin was reduced, indicating that FAK knockdown inhibited EMT.
Knockdown of FAK leads to cytoskeletal rearrangement and inhibition of cell adhesion and spreading
To determine the role of FAK in the regulation of cytoskeletal dynamics, cytoskeletal dynamics (13) were analyzed by TRITC-conjugated phalloidin staining (Fig. 2A). Actin filaments were distributed mainly on the periphery and no marked stress fiber formation was observed in the cell cortex in FAK knockdown cells, indicating that knockdown of FAK inhibits stress fiber formation. The status of cell adhesion and spreading was analyzed further in FAK knockdown cells using cell adhesion and spreading assays (Fig. 2B). The cell adhesion assay revealed that knockdown of FAK inhibited cell adhesion to fibronectin-coated substrate. In addition, the cell spreading assay demonstrated that knockdown of FAK reduced the spreading of tumor cells.
Knockdown of FAK induces paxillin redistribution and inhibits the activity of JNK
To identify whether FAK knockdown affects extracellular matrix degradation (14), the activity of MMP-2 and -9 was determined by gelatin zymography in FAK knockdown cells (Fig. 3A). Knockdown of FAK decreased the activity of MMP-2 and -9. In addition, the expression of several MMP family members, including MMP-2, -9 and -14, and TIMP-2, was determined in FAK knockdown cells. FAK knockdown decreased the levels of MMP-2, -9 and -14, and TIMP-2 (Fig. 3B).
Paxillin is an important signal transduction adaptor protein involved in the regulation of tumor invasion and metastasis (15). The distribution of paxillin in FAK knockdown cells was analyzed and paxillin was observed to be distributed homogeneously in the cytoplasm in FAK knockdown cells. However, paxillin was found to accumulate at the cell membrane in control cells. In addition, the phosphorylation levels of paxillin at residues Y118 and S178 were analyzed by western blot analysis (16). Western blot analysis revealed that the phosphorylation levels of Y118 and S178 were significantly decreased (Fig. 4).
JNK is a well-known Ser and Thr protein kinase. Therefore, JNK activity was analyzed in FAK knockdown cells by western blot analysis. The results revealed that the phosphorylation level of JNK was significantly decreased in FAK knockdown cells as compared with control cells, indicating that FAK knockdown inhibited the activity of JNK. However, the expression of JNK was comparable in FAK knockdown and control cells.
Discussion
Due to its characteristic feature of early lymph node metastasis, tongue cancer is difficult to treat. Of note, surgical resection has been found to correlate with tissue and organ dysfunction leading to a poorer quality of life for patients (17,18). Therefore, the identification of novel therapeutic target molecules to inhibit invasion and metastasis in tongue cancer is vital. At present, FAK is known to be overexpressed in various forms of cancer (19,20). A number of studies have demonstrated that FAK is useful as a molecular marker of nodal metastasis in cancer cells (21–23). In the present study, shFAK was used to treat Tca-8113 cells to determine the mechanisms associated with invasion and metastasis of tongue cancer.
Firstly, western blot analysis was used to analyze the knockdown efficiency of shFAK in Tca-8113. Next, the invasive abilities of shFAK-treated and control Tca-8113 cells was determined by wound healing assay. shFAK was found to inhibit the invasion of Tca-8113 cells. To further validate this result, the activity of a number of MMPs was analyzed by western blot analysis. MMPs are important for tumor invasion and metastasis, and among numerous MMPs, MMP-2 and -9 are well characterized in invasion and metastasis (24). Western blot analysis revealed that shFAK decreases the expression of MMP-2, -9 and -14 in cells. In addition, knockdown of FAK was found to decrease the expression of TIMP-2.
To elucidate the effect of shFAK on the cytoskeleton (25,26), shFAK-treated TCA-8113 cells were compared with control cells. The results indicate that shFAK inhibits stress fiber formation and results in rearrangement of the cytoskeleton. In addition, shFAK was found to initiate paxillin redistribution and promote changes in paxillin expression from the cell membrane to the cytoplasm. These observations indicate that paxillin functions as an anchor for the FAK-Src complex and activates downstream signaling (27–29). Downregulated FAK promotes paxillin redistribution. Western blot analysis also revealed that inhibition of FAK downregulates paxillin expression and phosphorylation.
To elucidate the mechanism by which shFAK inhibits the invasion and metastasis of tongue cancer cells, the expression of JNK was analyzed as JNK is known to regulate MMP-2 and -9. shFAK was found to inhibit the activity of JNK. As JNK cannot activate c-jun, which is downstream of JNK, the expression of MMP-2/9 and MMP-14 was downregulated.
In summary, the results of the current study indicate that the downregulation of FAK inhibits Tca-8113 invasion and metastasis by JNK and c-Jun signaling. Elevated JNK promotes increased gene transcription of target proteins leading to enhanced secretion of MMP-2, -9 and -14 (30). Following this, the leading edge of migrating tumor cells is activated, cytoskeletal rearrangement occurs and invadopodia formation is enhanced. The inhibition of FAK leads to the reduction of JNK phosphorylation, inhibiting c-jun activity, which is an important TF in the nucleus. The reduction of c-jun inhibits MMP-2 activity, as well as decreasing cell invasion
As shFAK inhibits the invasion and metastasis of tongue cancer cells, the molecular pathway may provide a potential therapeutic target for tongue tumor.