CXCR4/CXCR7/CXCL12-Axis in Follicular Thyroid Carcinoma

Abstract

Background: Follicular thyroid carcinoma's (FTC) often benign course is partially due to adjuvant radioactive iodine (RAI) treatment. However, once the tumour has spread and fails to retain RAI, the therapeutic options are limited and the outcome is poor. In this subset of patients, the identification of novel druggable biomarkers appears invaluable. Here, we investigated the stage dependent expression and functional role of the C-X-C chemokine receptors type 4 and 7 (CXCR4/7) in FTC. Methods: CXCR4/7 expression was examined in 44 FTC and corresponding non-neoplastic thyroid specimens as well as 10 FTC distant metastases and 18 follicular adenomas using tissue microarray technology. Expression levels were correlated with clinicopathological variables as well as overall and recurrence free survival. Changes regarding cell cycle activation, tumour cell invasiveness and mRNA expression of genes related to epithelial-mesenchymal transition (EMT) were investigated after treatment with recombinant human SDF1α/CXCL12 (rh-SDF1α) and CXCR4 antagonists AMD3100 and WZ811. Results: CXCR4/7 expression was associated with large tumour size, advanced UICC stage as well as shorter overall and recurrence free survival. CXCR4 was significantly higher expressed in distant metastases than in primary tumour cores. In addition, rh-SDF1α induced invasive growth, cell cycle activation and EMT, while CXCR4 antagonists significantly reduced FTC invasiveness in vitro. Conclusion: Here we provide first evidence of the biological importance of the CXCR4/CXCR7/CXCL12 axis in FTC. Our findings underscore the therapeutic potential of this chemokine receptor family in advanced FTC and offer new valuable insight into the oncogenesis of metastatic FTC.

Keywords: CXCL12; CXCR4; CXCR7; FTC; metastasis.

Figure 1

Expression of CXCR4 and CXCR7 in FTC. (A) Representative tissue samples of FTC, distant metastasis, follicular adenoma and non-neoplastic thyroid tissue, immunohistochemically stained for CXCR4 and CXCR7. All samples were classified as strong expression of the given marker. The bar in the top left corner indicates 50 μm. (B/C) Boxplots display the expression levels of CXCR4 and CXCR7 in non-neoplastic thyroid tissue specimens, follicular adenomas as well as FTC and distant metastases according to the IRS. Boxplots illustrate the median IRS with the upper and lower quartile, as well as maximum and minimum of the given marker. Expression levels of CXCR4 and CXCR7 in the different tissue specimens were compared between groups using the Wilcoxon matched-pairs signed rank test and the non-parametric Mann-Whitney U test as indicated. Bars mark the respective pairs. IRS: immunoreactivity score; CXCR4/7: C-X-C chemokine receptor type 4/7; NT: non-neoplastic thyroid gland; FTC: follicular thyroid carcinoma; DM: distant metastases; *p<0.05; **p<0.01; ***p<0.001.

Figure 2

Association between CXCR4/7 expression and clinicopathological variables as well as overall and recurrence free survival. (A, B) Boxplots display the median IRS with the upper and lower quartile, as well as maximum and minimum for CXCR4 and CXCR7 in the primary tumour core grouped according to tumour size and UICC stage. CXCR4/7 expression levels were compared across groups employing the non-parametric Mann-Whitney U test. (C-F) For the overall and recurrence free survival analysis, patients were grouped according to their CXCR4/7 expression in their primary tumour cores into high ≥median and low <median and groups were compared using the log-rank (Mantel Cox) test. (G) The survival regression tree analysis displays subgroups of patients with very high risk of death. Each junction represents a decision point with an optimal cut-off value for the respective variable and the numbers under the vertical lines represent hazard ratios. IRS: immunoreactivity score; CXCR4/7: C-X-C chemokine receptor type 4/7; UICC: Union internationale contre le cancer; *p<0.05; **p<0.01).

Figure 3

Expression levels of CXCR4 and CXCR7 in FTC cell line TT2609-C02. (A) FTC cell line TT2609-C02 exhibits both chemokine receptors CXCR4 and CXCR7. Immunocytochemical staining of CXCR4 and CXCR7 was conducted by using Alexa Fluor® 488 as secondary antibody and DAPI for visualization of the nucleus. Compositions of both images are illustrated as overlays. Isotype controls were used to confirm antibody specificity (Control). Images were captured using a fluorescence microscope at 400x magnification. Bar at the top left corner indicates 50 µm. (B) FTC cell line TT2609-C02 demonstrates a strong protein expression of both chemokine receptors as displayed in the western blot analysis. GAPDH served as loading control. CXCR4/7: C-X-C chemokine receptor type 4/7; DAPI: 4′,6-diamidin-2-phenylindol; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Rh-SDF1α induces tumour cell invasion, cell cycle activation and EMT. (A) Pictures display representative results of the change of invading cells after incubation of human FTC cell line TT2609-C02 with different concentrations of CXCR4 antagonists AMD3100 and WZ811 as well as rh-SDF1α as receptor agonist. Cells were visualized by DAPI staining. (B) Invading cells were counted in five visual fields of at least three different membranes. Differences after treatment are illustrated as fold change to control. (C) FACS after PI staining revealed cell cycle changes in the respective cell populations after rh-SDF1α treatment. Cells are grouped according to their specific cell cycle phase. (D) After incubation with rh-SDF1α, mRNA expression changes of genes associated with EMT were investigated using qRT-PCR. GAPDH functioned as housekeeping gene. The 2^-∆∆CT method was employed to estimate fold changes to control treated cells. Plots display the mean +SEM. Numerical data were analysed using the non-parametric Mann-Whitney U test. Ctrl: control; *p<0.05; **p<0.01; ***p<0.001.