FZD2 regulates cell proliferation and invasion in tongue squamous cell carcinoma

Abstract

Many studies have shown that FZD2 is significantly associated with tumor development and tumor metastasis. The purpose of the present study was to gain insight into the role of FZD2 in the cell proliferation and invasion of tongue squamous cell carcinoma. According to TCGA-HNSC dataset, among the 10 Frizzled receptors, FZD2 exhibited the highest degree of differential expression between cancer tissues and normal tissues, and the overall survival of patients with higher FZD2 levels was shown to be significantly shorter compared with those with lower FZD2 levels. The upregulation of FZD2 in clinical tongue cancer tissues was validated by real-time PCR. Knockdown of FZD2 inhibited the proliferation, migration and invasion of CAL-27 and TCA-8113 cells, whereas overexpression of FZD2 led to the opposite results. Further analysis revealed that FZD2 is positively correlated with WNT3A, WNT5B, WNT7A and WNT2 and is negatively correlated with WNT4. These results indicated that FZD2 may act as an oncogene in tongue squamous cell carcinoma. Therefore, FZD2 may be a target for the diagnosis, prognosis and gene therapy of tongue cancer.

Keywords: FZD2; Proliferation; Tongue cancer; WNT signaling pathway.

Figure 1

FZD2 is overexpressed in head and neck squamous cell carcinoma. The differential expression of Frizzled receptors in head and neck squamous cell carcinoma 501 cases of cancer and 41 cases of normal) and tongue cancer (149 cases of cancer and 15 cases of normal) from the TCGA database and the -Log10(P value) by t test was showed (A). The expression of FZD2 in head and neck squamous cell carcinoma and tongue cancer compared with normal tissues (B, N: normal tissues, C: cancer tissues). The overall survival of patients with HNSCC according to the different expression levels of FZD2 based on the TCGA database (C, 194 cases with high and 307 cases with low FZD2 expression, P=0.02 by Mantel-Cox test). Representative images of the upregulation of FZD2 in HNSCC from the Oncomine database (D). The expression of FZD2 in 44 pairs of tongue cancer tissues and adjacent tissues was detected by real-time PCR (E, P<0.05 by t test, n=44).

Figure 2

The siRNA-mediated knockdown of FZD2 inhibited the growth of CAL-27 cells in vitro. The expression of FZD2 in tongue cancer cell lines CAL-27 and TCA-8113 and normal human oral epithelial cell line HOEC was detected by Western blot (A). After siRNA transfection in CAL-27 cells, the expression of FZD2 was measured by real-time PCR (B) and Western blotting (C), the cell proliferation was detected by CCK8 assay (C, P < 0.01 by One-Way ANOVA followed by Tukey's multiple Comparison test from 96 h to 144 h) and colony-formation assay (D, P<0.005 by One-Way ANOVA followed by Tukey's multiple Comparison test, n=3).

Figure 3

Upregulation of FZD2 in CAL-27 cells promoted cells proliferation in vitro. After transfected with plasmid, the expression of FZD2 was measured by real-time PCR (A) and Western blotting (B), the cell proliferation was detected by CCK8 assay (C, P < 0.01 by One-Way ANOVA followed by Tukey's multiple Comparison test from 48 h to 120 h) and colony-formation assay (D, P<0.01 by One-Way ANOVA followed by Tukey's multiple Comparison test, n=3).

Figure 4

Knockdown of FZD2 represses tumorigenicity in vivo. After siRNA transfection in CAL-27 cells, the cells were injected into the flanks of nude mice, the tumor-bearing animals was were sacrificed in experimental observation for 28 days and the tumors were removed for weighing (A, P<0.001 by One-Way ANOVA followed by Tukey's multiple Comparison test) and photographing (B). The expression of FZD2 (upper panel), cleaved Caspase-9 (middle panel) and Ki-67 (lower panel) in the xenograft tumors were detected by immunohistochemistry (C) (DAB, 200 x) and the different groups of positive cells were counted and compared(D, P<0.001 by One-Way ANOVA followed by Tukey's multiple Comparison test).

Figure 5

Downregulation of FZD2 suppressed the migration and invasion of CAL-27 cells in vitro. After inhibition the expression of FZD2, the cells migration and invasion were detected by wound healing assay (A) and transwell assay coated with or without matrigel (B, P<0.005 by One-Way ANOVA followed by Tukey's multiple Comparison test), the representative images of transwell inserts coated without (upper panel) or with (lower panel) matrigel.

Figure 6

Upregulation of FZD2 in CAL-27 cells promoted cells migration and invasion in vitro. After transfected with plasmid, the cell motility and invasiveness were boosted, as detected by wound healing (A) and transwell assays (B, P<0.005 by One-Way ANOVA followed by Tukey's multiple Comparison test), the representative images of transwell inserts coated without (upper panel) or with (lower panel) matrigel.

Figure 7

Coexpression of FZD2 receptor and Wnt ligands in head and neck squamous carcinoma samples. The systematically map of FZD2-Wnt interactions in head and neck squamous carcinoma samples (A, n=501). The expression of WNT3A, WNT5B and WNT2 were significantly increased, while WNT4 was decreased in FZD2-high group (n=194) compared to FZD2-low (n=307) group (B, P<0.01 by t test).