Association between Circulating Fibroblast Growth Factor 21 and Aggressiveness in Thyroid Cancer

Abstract

Fibroblast growth factor 21 (FGF21) plays important roles in regulating glucose, lipid, and energy metabolism; however, its effects in tumors remain poorly understood. To understand the role of FGF21 in regulating tumor aggressiveness in thyroid cancer, serum levels of FGF21 were measured in healthy subjects and patients with papillary thyroid cancer (PTC), and expression levels of FGF21, FGF receptors (FGFRs), and β-klotho (KLB) were investigated in human thyroid tissues. The cell viability, migrating cells, and invading cells were measured in PTC cells after treatment with recombinant FGF21. Higher serum levels of FGF21 were found in patients with thyroid cancer than in control participants, and were significantly associated with body mass index (BMI), fasting glucose levels, triglyceride levels, tumor stage, lymphovascular invasion, and recurrence. Serum FGF21 levels were positively correlated with the BMI in patients with PTC, and significantly associated with recurrence. Recombinant FGF21 led to tumor aggressiveness via activation of the FGFR signaling axis and epithelial-to-mesenchymal transition (EMT) signaling in PTC cells, and AZD4547, an FGFR tyrosine kinase inhibitor, attenuated the effects of FGF21. Hence, FGF21 may be a new biomarker for predicting tumor progression, and targeting FGFR may be a novel therapy for the treatment of obese patients with PTC.

Keywords: FGF21; FGFR; cell proliferation; metastasis; obesity; thyroid cancer.

Figure 1

Thyroid cancer and serum levels of fibroblast growth factor 21 (FGF21). (A) The overall study design, including the numbers of participants included in this study. (B) Serum FGF21 levels in patients with papillary thyroid cancer (PTC) were significantly higher than in control subjects (* p < 0.05). (C) Serum FGF21 levels in PTC patients correlated positively with the body mass index.

Figure 2

Associations of serum levels of FGF21 with recurrence-free survival and overall survival in patients with PTC. (A) Comparison of the five-year recurrence-free survival rate between the high and low FGF21 groups. (B) Comparison of overall survival between the high and low FGF21 groups.

Figure 3

Expression of FGF receptors (FGFRs) in human thyroid tissue and the effects of FGF21 on the viability, migration, and invasion of thyroid carcinoma cells. (A) Western blot analyses of β-klotho (KLB) and FGFR protein levels in total cell lysates from paired clinical specimens of normal (N) and tumor (T) tissues from four patients with PTC (white bar, normal; black bar, tumor). (B) Gene expressions of FGFR1, FGFR2, FGFR3, FGFR4, KLB, and FGF21 between normal tissues and paired tumor tissues from a database of 59 patients in TCGA. (Data are given in matrix format, in which rows represent individual genes and columns represent each patient.) (C) Protein expression of KLB and all FGFRs in normal thyroid cells (Nthy-ori3-1) and thyroid carcinoma cells (BCPAP, TPC-1, and 8505C). (D) The effects of FGF21 on the cell viability of BCPAP cells treated with recombinant FGF21 (rFGF21) or 0.1% bovine serum albumin (BSA; negative control) for 12 h, as determined by WST-1 assay. (E) The effects of FGF21 on the cell viability of TPC-1 cells treated with rFGF21 or 0.1% BSA for 12 h, as determined by WST-1 assay. (F) The effects of FGF21 on the migration of BCPAP cells treated with rFGF21 or 0.1% BSA for 12 h, as determined by Transwell chamber assay. (G) The effects of FGF21 on the invasion of BCPAP cells in chambers coated with Matrigel after treatment with rFGF21 or 0.1% BSA for 12 h. (H) The effects of FGF21 on the migration of TPC-1 cells treated with rFGF21 or 0.1% BSA for 12 h, as determined by Transwell chamber assay. (I) The effects of FGF21 on the invasion of TPC-1 cells in chambers coated with Matrigel after treatment with rFGF21 or 0.1% BSA for 12 h. The experiments were performed in duplicate, and all experiments were performed at least three times. * p < 0.05; ** p < 0.01.

Figure 4

Effects of FGF21 in the FGFR signaling axis, including epithelial–mesenchymal transition (EMT)-associated proteins in thyroid cancer cell lines. (A) Representative images of Western blot analyses for the detection of p-FGFR, p-AKT, AKT, p-ERK, ERK, N-cadherin, E-cadherin, vimentin, SLUG, TWIST, and beta-actin in BCPAP cells treated with rFGF21 or 0.1% BSA. (B) Representative images of Western blot analyses for the detection of p-FGFR, p-AKT, AKT, p-ERK, ERK, N-cadherin, E-cadherin, vimentin, SLUG, TWIST, and beta-actin in TPC-1 cells treated with rFGF21 or 0.1% BSA. Experiments were performed in duplicate, and all experiments were performed at least three times. ** p < 0.01, * p < 0.05 compared to the 0.1% BSA control.

Figure 5

AZD4547 attenuates the effects of FGF21 in thyroid cancer via downregulation of FGFR signaling. (A) The effects of AZD45487 on the migration of BCPAP cells after treatment with only rFGF21 or rFGF21 and AZD4547 for 12 h, as determined by Transwell chamber assay. (B) The effects of AZD4547 on the invasion in BCPAP cells in chambers coated with Matrigel after treatment with only rFGF21 or rFGF21 and AZD4547 for 12 h. (C) The effects of AZD45487 on the migration in TPC-1 cells after treatment with only rFGF21 or rFGF21 and AZD4547 for 12 h, as determined by Transwell chamber assay. (D) The effects of AZD4547 on invasion in TPC-1 cells in chambers coated with Matrigel after treatment with only rFGF21 or rFGF21 and AZD4547 for 12 h. (E) Representative images of Western blot analyses for the detection of p-FGFR, N-cadherin, E-cadherin, vimentin, p-AKT, AKT, p-ERK, ERK, SLUG, TWIST, and beta-actin in BCPAP cells treated only with rFGF21 or rFGF21 and AZD4547. (F) Representative images of Western blot analyses for the detection of p-FGFR, N-cadherin, E-cadherin, vimentin, p-AKT, AKT, p-ERK, ERK, SLUG, TWIST, and beta-actin in TPC-1 cells treated only with rFGF21 or rFGF21 and AZD4547. (G) Proposed model of the interaction between serum levels of FGF21 and tumor aggressiveness in PTC based on this study. Experiments were performed in duplicate and all experiments were performed at least three times.