The transcription cofactor c-JUN mediates phenotype switching and BRAF inhibitor resistance in melanoma

Abstract

Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAF(V600)-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.

Taking out the JNK: A window of opportunity to improve cancer therapy

“…In the case of BRAF V600 -mutant metastatic melanoma, we predict that phosphorylated c-JUN would be an ideal biomarker for monitoring the tumors following initial treatment with BRAF/MEK inhibitors to reveal the window of opportunity for combination treatment with JNK and BRAF/MEK inhibitors. 2 Matched samples from patients at progression of disease showed a reversion to reduced c-JUN levels and activation, 2 suggesting that this window is early during drug treatment. Our data also demonstrated that JNK inhibitor doses that did not result in cancer cell death were able to reduce drug-induced cell migration, 2 suggesting that therapeutic benefit may be obtained even in protected niches or compartments with reduced drug concentrations.…”

“…Our findings using cell line models revealed that a greater extent of cell killing could be attained with the same concentrations of the BRAF and JNK inhibitors if the cells were initially treated with the BRAF inhibitor that induced activation of the JNK pathway. 2 In essence this offers a way to 'prime' the surviving cancer cells and select for characteristics that confer enhanced responsiveness to the combination treatment. Our findings also revealed that the highest doses are not necessarily the best, as the greatest drug synergy was obtained at combinations within the mid-range of doses assessed.…”

“…2 The ability to target c-JUN through upstream inhibition of its activator enzyme c-JUN-N-terminal kinase (JNK) offers a significant opportunity to target both therapy resistance and phenotype-switching associated with enhanced metastatic potential. 2 The JNK-JUN pathway has been verified as a key pathway in mediating survival following BRAF and MEK inhibitor treatment in parallel studies by other research groups.…”

“…2 The ability to target c-JUN through upstream inhibition of its activator enzyme c-JUN-N-terminal kinase (JNK) offers a significant opportunity to target both therapy resistance and phenotype-switching associated with enhanced metastatic potential. 2 The JNK-JUN pathway has been verified as a key pathway in mediating survival following BRAF and MEK inhibitor treatment in parallel studies by other research groups. 3,4 The JNK signaling pathway has also been reported to confer therapy resistance in multiple tumor types, 5 including chemoresistance in ovarian, 6 colorectal, 7 and pancreatic 8 cancers, and has been linked to the stem cell-like properties of cancer cells.…”

“…Our findings also revealed that the highest doses are not necessarily the best, as the greatest drug synergy was obtained at combinations within the mid-range of doses assessed. 2 Animal models could be utilized to predict the best dosing and scheduling of single agents with combination therapies to guide strategies for achieving the best responses in patients.…”