(V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway

Abstract

Tumors with mutant BRAF and those with receptor tyrosine kinase (RTK) activation have similar levels of phosphorylated ERK, but only the former depend on ERK signaling for proliferation. The mitogen-activated protein kinase, extracellular signal-regulated kinase kinase (MEK)/ERK-dependent transcriptional output was defined as the genes whose expression changes significantly 8 h after MEK inhibition. In (V600E)BRAF cells, this output is comprised of 52 genes, including transcription factors that regulate transformation and members of the dual specificity phosphatase and Sprouty gene families, feedback inhibitors of ERK signaling. No such genes were identified in RTK tumor cells, suggesting that ERK pathway signaling output is selectively activated in BRAF mutant tumors. We find that RAF signaling is feedback down-regulated in RTK cells, but is insensitive to this feedback in BRAF mutant tumors. Physiologic feedback inhibition of RAF/MEK signaling down-regulates ERK output in RTK cells; evasion of this feedback in mutant BRAF cells is associated with increased transcriptional output and MEK/ERK-dependent transformation.

Fig. 1.

Identification of the transcriptional output of the MEK/ERK pathway in ^V600EBRAF tumor cells. (A) The plot identifies the 52 genes (probe sets in red) significantly changed in expression upon MEK inhibition, above background (probe sets for all other genes in gray) in ^V600EBRAF cells (n = 7), determined by SAM analysis. (B) Details of expression levels for the 52 genes (60 probe sets, rows), in each of the 7 cell lines (columns) with and without MEK inhibition (heatmap values: red = increased expression, blue = decreased expression). Parameters in SAM: final largest median FDR is 1.03% and δ cutoff is 1.505.

Fig. 2.

A molecular interaction map displaying the functional interaction of genes in the MEK/ERK output profile with established roles in ERK signaling. A molecular interaction map was constructed for major constituents of the RAS/RAF/MEK/ERK signaling pathway and downstream effectors. The genes in the output profile that have established functional relationships to ERK signaling are indicated by gray shading, with the nature of their relationship (i.e., direct phosphorylation, transcriptional activation) indicated by using the symbols described by Pommier and colleagues (43) and shown in the legend.

Fig. 3.

Expression of genes in the MEK/ERK output profile is down-regulated after MEK inhibition. The MEK dependence of genes identified as components of the MEK/ERK transcriptional output profile is confirmed by immunoblot and RT-PCR. (A) SkMel-5 cells in tissue culture were treated for the times indicated with 50 nM PD0325901 and lysates were subjected to immunoblotting with the indicated antibodies. (B) mRNA was isolated from SkMel-5 treated with drug as in A and analyzed by RT-PCR for levels of the indicated mRNAs. Values are relative mRNA level compared with those before treatment.

Fig. 4.

Quantitation of pERK and pMEK levels in tumor cell lines with mutant and WT BRAF. pERK and pMEK were quantitated by immunoblotting cell lysates from 13 tumor cell lines with ^WTBRAF or ^V600EBRAF. Cells grown in 10% FBS were harvested at 70–80% confluence, and lysates resolved by SDS/PAGE were probed for pERK (phosphorylated p44/ p42 MAPK), pMEK, total ERK, and total MEK 1/2. Band intensity was quantitatively measured by densitometry reading of the immunoblot [P value (one-sided Wilcoxon) is cited in text for difference in pMEK expression between groups].

Fig. 5.

MEK phosphorylation is feedback inhibited in tumor cells with activated receptor tyrosine kinases, but not in those with ^V600EBRAF. Shown are immunoblots of pMEK and pERK 1/2 in ^WTBRAF cell lines (A), ^V600EBRAF cell lines (B), and SkMel-28 and BT474 xenograft tumors (C) treated with MEK inhibitor as a function of time. Cell lines were treated with 50 nM PD0325901, and mice were treated with a single oral dose of 25 mg/kg PD0325901. Total ERK and total MEK were unchanged.

Fig. 6.

Models of RAF/MEK/ERK signaling in tumor cells with RTK activation and with ^V600EBRAF. (A) RTK-activated tumor cells: feedback inhibition occurs at multiple levels of the pathway, down-regulating both RAF/MEK activation and ERK phosphorylation levels. Steady-state levels of pERK are caused by low levels of both MEK activity and ERK dephosphorylation by DUSPs. Output of the pathway is low. (B) ^V600EBRAF cells: RAF kinase is active and insusceptible to negative feedback. Increased pathway output leads to increased DUSP6 expression. Steady-state levels of pERK are caused by high levels of MEK and DUSP6 dephosphorylation of ERK.