TFAP2B overexpression contributes to tumor growth and progression of thyroid cancer through the COX-2 signaling pathway

Abstract

Thyroid cancer is commonly seen in the clinic with a rapidly increasing incidence globally. COX-2 overexpression correlates with the pathologic type of thyroid carcinoma, and it has been suggested that COX-2 overexpression is associated with a poor prognosis. However, little is known about its upstream regulatory mechanism. Bioinformatics suggested that transcription factor AP-2 beta (TFAP2B) might specifically bind to the COX-2 promoter, which was confirmed by biotin-labeled COX-2 promoter pulldown and luciferase reporter assays. We performed western blot and immunohistochemical staining to detect the expression of TFAP2B/COX-2 in thyroid cancer tissues (T) and the matched adjacent noncarcinoma tissues (ANT), and investigated the relationship between TFAP2B/COX-2 expression and clinical pathological factors in thyroid cancer patients. Afterward, MTS, colony formation, cell-apoptosis assay, transwell-invasion and scratch assays were performed to examine the proliferation, apoptosis, invasion, and migration of thyroid cancer cells with TFAP2B knocked down or overexpressed. The mouse xenograft experiment was performed to study in vivo the proliferation of thyroid cancer cells with TFAP2B knocked down or overexpressed. We found that TFAP2B bound to the promoter of COX-2 to activate its expression. Western blot and immunohistochemistry showed that TFAP2B/COX-2 was highly expressed in thyroid cancer, and high TFAP2B and COX-2 expression was associated with aggressive clinicopathological features in thyroid cancer. TFAP2B mediated thyroid cancer cell proliferation, apoptosis, invasion, and migration via the COX-2 signaling pathway in vitro and in vivo. TFAP2B bound to the promoter of COX-2 to activate its expression, indicating that TFAP2B is a critical regulatory molecule in the COX-2 signaling pathway that promoted tumor progression in thyroid cancer.

Fig. 1. TFAP2B was identified as a COX-2 promoter-binding protein in thyroid cancer cells.

a Binding of TFAP2B on the 5′-biotin-labeled COX-2 promoter probe or a nonspecific probe (NSP) was detected by western blot using anti-TFAP2B antibody in thyroid cancer cell lines (BCPAP, TPC-1, KTC-1, K2). b Relative COX-2 promoter activity in TFAP2B knockdown BCPAP cells and TFAP2B overexpress K2 cells (n = 3). c COX-2 protein expression was upregulated in TFAP2B overexpressed TPC-1 cells, and relative COX-2 mRNA level was upregulated in TFAP2B overexpressed TPC-1 cells (n = 3). c COX-2 protein expression was downregulated in TFAP2B knockdown TPC-1 cells, and relative COX-2 mRNA level was downregulated in TFAP2B knockdown TPC-1 cells (n = 3)

Fig. 2. TFAP2B/COX-2 is highly expressed in thyroid cancer.

a The expression of the total TFAP2B/COX-2 proteins in normal thyroid gland cell (Nthy-ori 3–1) and thyroid cancer cells (BCPAP, TPC-1, KTC-1, and K2) were analyzed by western blot, and the relative protein level in different thyroid cancer cells is shown in a (right) (n = 3). b Protein subcellular localization of TFAP2B/COX-2 was examined by confocal microscopy analysis in BCPAP and Nthy-ori 3–1 cell line. c Both nucleic and cytoplasmic expression of TFAP2B can be detected in Nthy-ori-3-1 and BCPAP cell line. d The immunohistochemical results demonstrated the representative images of TFAP2B and COX-2 expression in thyroid cancer tissues and adjacent noncarcinoma tissues (ANTs) (x200 upper, x400 down). e The western blot results demonstrated TFAP2B and COX-2 expression in thyroid cancer tissues and ANTs. f The correlation between the expression of TFAP2B and COX-2 in human thyroid tissues from 252 patients (P < 0.05)

Fig. 3. TFAP2B promoted thyroid cancer cell proliferation and clonogenicity via the COX-2 signaling pathway in vitro.

a Cell proliferation and clonogenicity were suppressed by downregulating TFAP2B in TPC-1 cell line. b Cell proliferation and clonogenicity were promoted by upregulating TFAP2B in KTC-1 cell line. c, d Cell proliferation was suppressed by COX-2 inhibitor celecoxib; overexpression of TFAP2B promoted the expression of COX-2 and increased thyroid cancer cell proliferation, which was reversed by COX-2 knockdown or COX-2 inhibitor celecoxib in TPC-1 and BCPAP cell line. *P < 0.05 (n = 3). e, f Overexpression of TFAP2B promoted the thyroid cancer cell clonogenicity, which was reversed by COX-2 knockdown or COX-2 inhibitor celecoxib in TPC-1 and BCPAP cell line. *P < 0.05 (n = 3)

Fig. 4. TFAP2B promoted thyroid cancer cell growth via the COX-2 signaling pathway in vivo.

a TFAP2B knockdown suppressed thyroid cancer cell growth in a mouse xenograft. Images of the thyroid cancer cell tumor xenograft from each mouse (n = 7 mice/group). e TFAP2B overexpression promoted thyroid cancer cell growth in a mouse xenograft, but suppressed by COX-2 inhibitor celecoxib when feeded to mice. Images of the thyroid cancer cell tumor xenograft from each mouse (n = 5 mice/group). b, f Tumor volumes were analyzed. *P < 0.05. c, g Tumor weights were recorded and analyzed. *P < 0.05. d, h The expression of TFAP2B and COX-2 in tumor tissues was analyzed by immunohistochemistry (IHC) staining

Fig. 5. TFAP2B regulates thyroid cancer cell migration, invasion and cell apoptosis via the COX-2 signaling pathway in vitro.

a Knockdown of TFAP2B inhibited the cell migration and invasion in TPC-1 cells. b Knockdown of COX-2 reversed the promotion of cell migration and invasion by TFAP2B overexpression in BCPAP cells. c Cell apoptosis was measured by Annexin-V/PI assays in TFAP2B overexpression BCPAP cells (with or without COX-2 knockdown, 60 µM celecoxib, n = 3), histograms indicated subpopulation of cells positive for Annexin V/PI

Fig. 6. TFAP2B regulates COX-2 effects via the VEGF/PEDF signaling pathway.

a Knockdown of TFAP2B dramatically inhibited the expression of VEGF proteins and increased the expression of PEDF protein, and b the overexpression of TFAP2B resulted in the opposite results. c Quantification for the relevant expression level of proteins was also analyzed