Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

Abstract

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF

“…It has been reported that activation of ERK leads to negative feedback regulation of MEK [ 30 ]. Our previous results also showed that long-term treatment with BRAF inhibitors significantly downregulated Spry2 in BRAF V600E–positive cell lines, and that this change is accompanied by rebound activation of the MAPK pathway [ 6 ]. As expected, the phospho-ERK levels were relatively unresponsive to PLX4720 in mimic-controltransfected A375P/Mdr cells.…”

“…Because it is known that A375P/Mdr cells are resistant to BRAF inhibitor-induced ERK inhibition [ 6 ], we used immunoblotting to examine phospho-MEK and phospho-ERK levels in mimic-control-RNA–transfected and miR-1246–transfected A375P/Mdr cells after PLX4720 treatment ( Fig. 5 ).…”

“…In general, reactivation of the mitogen-activated protein kinase (MAPK) pathway is considered a primary mechanism underlying the acquired resistance to BRAF inhibitors [ 5 ]. Our previous study indicated that induction of resistance to a BRAF inhibitor is associated with the inability of Spry2 to inhibit BRAF V600E activity in cells with mutant BRAF [ 6 ]. In fact, the relief of feedback after targeted therapy may be viewed as a key contributor to therapeutic resistance [ 7 ].…”

The Sprouty/Spred family as tumor suppressors: Coming of age

“…However, it does not bind to or inhibit BRAF V600E and is instead upregulated in BRAF‐mutant melanoma cells . Sprouty2 is also related to the induction of resistance to BRAF inhibitor in BRAF‐mutant melanoma cells . Salmonella typhimurium strain VNP20009 harboring Sprouty2‐expressing plasmid suppresses the s.c. growth of B16F10, a murine melanoma cell line, in vivo…”

“…2 Sprouty2 is also related to the induction of resistance to BRAF inhibitor in BRAF-mutant melanoma cells. 69 Salmonella typhimurium strain VNP20009 harboring Sprouty2-expressing plasmid suppresses the s.c. growth of B16F10, a murine melanoma cell line, in vivo. 70 The knockdown of Spred1 and Spred2 shows an effect similar to that of an MEK inhibitor and protects against apoptosis of BRAF V600E-positive melanoma cell lines.…”