The BRAF-inhibitor PLX4720 inhibits CXCL8 secretion in BRAFV600E mutated and normal thyroid cells: a further anti-cancer effect of BRAF-inhibitors

Abstract

CXCL8 is a chemokine secreted by normal and thyroid cancer cells with proven tumor-promoting effects. The presence of BRAFV600E mutation is associated with a more aggressive clinical behavior and increased ability to secrete CXCL8 by papillary-thyroid-cancer cells. Aim of this study was to test the effect of the BRAF-inhibitor (PLX4720) on the basal and TNF-α-induced CXCL8 secretions in BRAFV600E mutated (BCPAP, 8305C, 8505C), in RET/PTC rearranged (TPC-1) thyroid-cancer-cell-lines and in normal-human-thyrocytes (NHT). Cells were incubated with increasing concentrations of PLX4720 alone or in combination with TNF-α for 24-hours. CXCL8 concentrations were measured in the cell supernatants. PLX4720 dose-dependently inhibited the basal and the TNF-α-induced CXCL8 secretions in BCPAP (F: 14.3, p < 0.0001 for basal and F: 12.29 p < 0.0001 for TNF-α), 8305C (F: 407.9 p < 0.0001 for basal and F: 5.76 p < 0.0001 for TNF-α) and 8505C (F:55.24 p < 0.0001 for basal and F: 42.85 p < 0.0001 for TNF-α). No effect was found in TPC-1 (F: 1.8, p = 0.134 for basal; F: 1.6, p = 0.178 for TNF-α). In NHT an inhibitory effect was found only at the highest concentration of PLX4720 (F: 13.13 p < 0.001 for basal and F: 2.5 p < 0.01 for TNF-α). Cell migration assays showed that PLX4720 reduced both basal and CXCL8-induced cell migration in BCPAP, 8305C, 8505C and NHT but not in TPC-1 cells. These results constitutes the first demonstration that PLX4720 is able to inhibit the secretion of CXCL8 in BRAFV600E mutated thyroid cancer cells indicating that, at least some, of the anti-tumor activities of PLX4720 could be exerted through a lowering of CXCL8 in the thyroid-cancer-microenvironment.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Panel A PLX4720 inhibit the basal CXCL8 secretion in BCPAP (ANOVA F: 14.3; p p < 0.001) being significant only at the higher concentration of 10 µM (Post Hoc analysis by Bonferroni *p < 0.01 vs. basal). Panel F PLX4720 inhibit the TNF-𝛼-stimulated CXCL8 secretion in BCPAP (ANOVA F: 12.29 p < 0.0001), the inhibitory effect was significant starting by 1 10 µM (3.13; t anPost Hoc analysis by Bonferroni *p < 0.001 vs. TNF-𝛼). Panel G PLX4720 inhibit the TNF-α-stimulated CXCL8 secretion in 8305C (ANOVA F: 5.76 p < 0.0001), the inhibitory effect started from 1 rom Post Hoc analysis by Bonferroni *p < 0.001 vs. TNF-𝛼). Panel H PLX4720 inhibit the TNF-𝛼-stimulated CXCL8 secretion in 8505C (ANOVA F: 42.85 p < 0.0001), the inhibitory effect started from 0.1 rom Post Hoc analysis by Bonferroni *p < 0.001 vs. TNF-𝛼, **p < 0.001 vs. 0.1 ni *p < Panel I TNF-𝛼-stimulated CXCL8 secretion was not inhibited by PLX4720 in TPC-1 cell lines at any concentrations (ANOVA F: 1.6, p = 1.78). Panel E TNF-𝛼-stimulated CXCL8 secretion was inhibited by PLX4720 in NHT (ANOVA F: 2.5; p < 0.01) being significant only at the higher concentration of10 µM (Post Hoc analysis by Bonferroni *p < 0.001 vs. TNF-α alone).

Figure 2

CXCL8 concentrations progressively increased throughout the 72-h time course in untreated thyroid cells. Panel A BCPAP cells (ANOVA: F = 13.39; p 

Figure 3

Time course of PLX4720 inhibitory effect on CXCL8 secretion. No significant change in the inhibitory power of PLX4720 at a given concentration was found throughout the 72-h time course. Panel A BCPAP (ANOVAs: PLX4720 0.1 µM F: 0.6; NS; 1 µM F: 1.7; NS; 2 µM F: 1.7; p = 0.2; 5 µM F: 2.7; p = 0.7; 10 µM F: 1.0, NS). Panel B NHT (ANOVAs: PLX4720 0.1 µM F:1.7, NS; 1 µM F:2.8; NS; 2 µM F: 3; NS; 5 µM F: 1; NS; 10 µM F: 2.3, NS). Panel C 8305C (ANOVAs: PLX4720 0.1 µM F:1.6, NS; 1 µM F: 2.6; NS; 2 µM F: 0.5; NS; 5 µM F: 0.4; NS; 10 µM F: 1.3, NS). Panel D 8505C (ANOVAs: PLX4720 0.1 µM F:0.02, NS; 1 µM F: 0.2; NS; 2 µM F: 0.1; NS; 5 µM F: 1.2; NS; 10 µM F: 2.9, NS).

Figure 4

Effect of PLX4720 on the basal and rh-CXCL8-induced migration in BCPAP, 8305C, 8505C, NHT and TPC-1 thyroid cancer cells. Representative images and the respective histograms after 16 hours of migration within the trans-well migration chamber system in BCPAP, 8305C, 8505C, NHT and TPC-1 (Panels A–E respectively). Panel (A) in BCPAP the treatment with PLX4720 10 µM reduced the basal migration (ANOVA F = 27.9, p Post Hoc °p < 0.005 vs. basal). The incubation of BCPAP with rh-CXCL8 produced a significant increase of BCPAP migration (Post Hoc *p < 0.0001 vs. basal). The co-incubation with rh-CXCL8 and PLX4720 10 µM significantly inhibited the BCPAP migration induced by rh-CXCL8 (Post Hoc#p < 0.001 vs. CXCL8). Panel (B) in 8305C, the treatment with PLX4720 10 µM reduced the basal migration of 8305C (ANOVA F = 161.7, p < 0.0001; Post Hoc °p < 0.005 vs. basal). The incubation of 8305C with rh-CXCL8 produced a significant increase of 8305C migration (Post Hoc *p < 0.0001 vs. basal). The co-incubation with rh-CXCL8 and PLX4720 10 µM significantly inhibited the 8305C migration induced by rh-CXCL8 (Post Hoc#p < 0.0001 vs. CXCL8). Panel (C) in 8505C the treatment with PLX4720 10 µM reduced the basal migration of 8505C (ANOVA F = 21.6, p < 0.0001; Post Hoc °p < 0.005 vs. basal). The incubation of 8505C with rh-CXCL8 produced a significant increase of 8505C migration (Post Hoc *p < 0.0001 vs. basal). The co-incubation with rh-CXCL8 and PLX4720 10 µM significantly inhibited the 8505C migration induced by rh-CXCL8 (Post Hoc#p < 0.0001 vs. CXCL8). Panel (D) in NHT, the treatment with PLX4720 10 µM reduced the basal migration of NHT (ANOVA F = 25.3, p < 0.0001; Post Hoc °p < 0.005 vs. basal). The incubation of NHT with rh-CXCL8 produced a significant increase of NHT migration (Post Hoc *p < 0.05 vs. basal). The co-incubation with rh-CXCL8 and PLX4720 10 µM significantly inhibited the NHT migration induced by rh-CXCL8 (Post Hoc#p < 0.0001 vs. CXCL8). Panel (E) in TPC-1 the treatment with PLX4720 10 µM did not reduce the basal migration of TPC-1 (ANOVA F = 4.4, p < 0.01), Post Hoc NS vs. basal). The incubation of TPC-1 with rh-CXCL8 produced a significant increase of TPC-1 migration (Post Hoc *p < 0.05 vs. basal). The co-incubation with rh-CXCL8 and PLX4720 10 µM did not reduce the TPC-1 migration induced by rh-CXCL8 (Post Hoc # NS vs. CXCL8). Bar graphs show the corresponding analysis of migrated cells on the lower side of the transwell filter. Basal migration was conventionally estimated as 100%.