Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma

H Wang, Q Wu, Z Liu, X Luo, Y Fan, Y Liu, Y Zhang, S Hua, Q Fu, M Zhao, Y Chen, W Fang, X Lv, H Wang, Q Wu, Z Liu, X Luo, Y Fan, Y Liu, Y Zhang, S Hua, Q Fu, M Zhao, Y Chen, W Fang, X Lv

Abstract

It is largely recognized that fibroblast activation protein (FAP) is expressed in cancer-associated fibroblasts (CAFs) of many human carcinomas. Furthermore, FAP was recently also reported to be expressed in carcinoma cells of the breast, stomach, pancreatic ductal adenocarcinoma, colorectum, and uterine cervix. The carcinoma cell expression pattern of FAP has been described in several types of cancers, but the role of FAP in oral squamous cell carcinoma (OSCC) is unknown. The role of endogenous FAP in epithelium-derived tumors and molecular mechanisms has also not been reported. In this study, FAP was found to be expressed in carcinoma cells of OSCC and was upregulated in OSCC tissue samples compared with benign tissue samples using immunohistochemistry. In addition, its expression level was closely correlated with overall survival of patients with OSCC. Silencing FAP inhibited the growth and metastasis of OSCC cells in vitro and in vivo. Mechanistically, knockdown of FAP inactivated PTEN/PI3K/AKT and Ras-ERK and its downstream signaling regulating proliferation, migration, and invasion in OSCC cells, as the inhibitory effects of FAP on the proliferation and metastasis could be rescued by PTEN silencing. Our study suggests that FAP acts as an oncogene and may be a potential therapeutic target for patients with OSCC.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
FAP expression in oral squamous cell carcinoma (OSCC) is associated with poor prognosis. (I) FAP expression examination in OSCC and control tissues (a) Negative FAP expression in oral cavity epithelium; (b, c) Negative expression of FAP in OSCC tissues; (d, e) Weakly positive expression of FAP in OSCC tissues; (f, g, and h) Positive and strong positive expression of FAP in OSCC tissues; (II) Immunofluorescent images of KB and Tca-8113 cells stained for FAP (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) (magnification, × 400); (III) Kaplan–Meier survival analysis of overall survival in all patients. The log-rank test was used to calculate P-values
Figure 2
Figure 2
Stable suppression of FAP attenuates the growth of OSCC cells in vitro and vivo. (a) Stably knocking down decreased the expression of FAP in shRNA-FAP-1 and -2 cells compared with shRNA-con cells by real-time PCR and western blotting. β-Actin was used as a loading control. (b) MTT cell viability assays were performed on days 1–6 of KB cells stably expressing the shRNA-FAP-1 and 2 or shRNA-con vectors. (c) Colony formation assay was performed on KB cells stably expressing the shRNA-FAP-1 and -2 or shRNA-con vectors for 2 weeks. (d) Cell-cycle analysis of KB cells stably expressing the shRNA-FAP-1 and 2 or shRNA-con vectors. (e) External whole-body fluorescence images of KB/shRNA-FAP-1 and -2 and KB/shRNA-con mice obtained 30 days after injection. Representative photographs of H&E staining of primary cancer tissues are shown. Data are presented as mean±S.D. for three independent experiments **P<0.01; ***P<0.001, compared with control
Figure 3
Figure 3
Transient suppression of FAP inhibits growth of OSCC cells in vitro. (a) Transiently knocking down FAP by siRNA reduced the expression of FAP by real-time PCR and western blotting. (b) MTT cell viability assays was performed on days 1–5 after the transfection of KB and Tca-8113 cells with either siRNA FAP-1 or the negative control. (c) Cell-cycle profiles were analyzed after KB and Tca-8113 cells were transfected with siRNA FAP-1 or the negative control for 48 h. Data are presented as mean±S.D for three independent experiments *P<0.05; **P<0.01; ***P<0.001, compared with control
Figure 4
Figure 4
Stable depletion of FAP suppresses cell migration and invasion in vitro and vivo. (a) Wound-healing assay showed KB cells stably expressing the shRNA-FAP-1 and -2 or shRNA-con vectors at 0 and 48 h after wounding. Bar chart showing the relative migration ability at 48 h. (b) Cell invasion assay showed decreased invasion from cells transfected with shRNA-FAP-1 and -2, with representative fields shown. (c) The ability of the cells to adhere to a solid surface was significantly increased in cells treated with shRNA-FAP-1 and -2, and a representative experiment is shown. Bar chart showing the adhesive rate quantified by MTT assay. (d). Phalloidin staining of F-actin in KB/shRNA-FAP-1 and KB/shRNA-con cells (magnification, × 1000). (e) External whole-body fluorescence images of liver were obtained 2 months after spleen injection, respectively. Metastatic cancer tissue (H&E staining, magnification, × 200). Data shown are means±S.D. of at least three independent experiments *P<0.05; **P<0.01, compared with control
Figure 5
Figure 5
Transient depletion of FAP reduces cell migration and invasion in vitro. (a) Transiently downregulated FAP dramatically inhibited KB and Tca-8113 cell migration in vitro. (b) Transiently suppressed FAP decreased in vitro invasiveness of KB and Tca-8113 cells. (c) Transient knockdown of FAP elevated the adhesion rate of KB and Tca-8113 cells. Data are presented as means±S.D. of at least three independent experiments *P<0.05; **P<0.01, compared with control
Figure 6
Figure 6
FAP regulates the expression of cell cycle, MMPs, and EMT-associated genes in OSCC through PTEN/PI3K/Akt and ERK signaling pathways. (a) Knocking down endogenous FAP expression reduced the activation of pRb(ser780), an oncogenic cell-cycle regulator including CCNE1, E2F1, and c-Myc, and elevated the expression of tumor suppressors including p27 and p21. However, the expression of CDK4, CCND1, and total Rb was not affected. (b) Suppressing FAP expression decreased the expression of MMP2, MMP9, and EMT-marker genes including Snail, Slug, N-cadherin, and Vimentin but increased E-cadherin expression. (c) Reduced FAP significantly decreased the expression of phosphorylated PI3K, AKT, MEK1/2, ERK1/2, and GSK3β, whereas total levels remained unchanged. β-Actin was used as a loading control
Figure 7
Figure 7
PTEN depletion or gain respectively elevates or decreases proliferation, migration, and invasion of cells with downregulated FAP. (a) Decreased PTEN mRNA and protein levels in KB cells with stably silenced FAP and transfected with siRNA were found by real-time PCR and western blotting. (b) MTT cell viability assays were performed on days 1–5 after the transfection of FAP knockdown KB cells with either siRNA PTEN-1 and -2 or the control. (c) Transiently downregulated PTEN dramatically enhanced the ability of KB cells treated with shRNA-FAP-1 invasion in vitro. (d) Transiently suppressed PTEN elevated in vitro migration of KB cells that had stable downregulated FAP. (e) MTT cell viability assays were performed on days 1–4 after the transfection of FAP knockdown KB cells with either the wild type (pEGFP-wt-PTEN) and the mutant type (pEGFP-G129R-PTEN) or empty vector (pEGFP-C1). (f) Introduction of PTEN decreased invasion of KB cells treated with shRNA-FAP-1. Data are presented as mean±S.D. for three independent experiments *P<0.05; **P<0.01; ***P<0.001, compared with control

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