The Role of B-RAF Mutations in Melanoma and the Induction of EMT via Dysregulation of the NF-κB/Snail/RKIP/PTEN Circuit

Abstract

Melanoma is a highly metastatic cancer, and there are no current therapeutic modalities to treat this deadly malignant disease once it has metastasized. Melanoma cancers exhibit B-RAF mutations in up to 70% of cases. B-RAF mutations are responsible, in large part, for the constitutive hyperactivation of survival/antiapoptotic pathways such as the MAPK, NF-κB, and PI3K/AKT. These hyperactivated pathways regulate the expression of genes targeting the initiation of the metastatic cascade, namely, the epithelial to mesenchymal transition (EMT). EMT is the result of the expression of mesenchymal gene products such as fibronectin, vimentin, and metalloproteinases and the invasion and inhibition of E-cadherin. The above pathways cross-talk and regulate each other's activities and functions. For instance, the NF-κB pathway directly regulates EMT through the transcription of gene products involved in EMT and indirectly through the transcriptional up-regulation of the metastasis inducer Snail. Snail, in turn, suppresses the expression of the metastasis suppressor gene product Raf kinase inhibitor protein RKIP (inhibits the MAPK and the NF-κB pathways) as well as PTEN (inhibits the PI3K/AKT pathway). The role of B-RAF mutations in melanoma and their direct role in the induction of EMT are not clear. This review discusses the hypothesis that B-RAF mutations are involved in the dysregulation of the NF-κB/Snail/RKIP/PTEN circuit and in both the induction of EMT and metastasis. The therapeutic implications of the dysregulation of the above circuit by B-RAF mutations are such that they offer novel targets for therapeutic interventions in the treatment of EMT and metastasis.

Figure 1.

The role of the B-RAF/NF-κB/Snail/RKIP/PTEN circuitry in the regulation of EMT in melanoma. B-RAF induces EMT in melanoma through the constitutive activation of NF-κB and resulting in the up-regulation of the metastasis inducer, Snail. Inhibition of B-RAF by either up-regulation of RKIP or PTEN inhibits Snail expression. Expression of Snail down-regulates both RKIP and PTEN, thus providing a positive feedback loop for self-amplification and EMT.

Figure 2.

Conformations and mutations in RAF proteins. (A)Conformations of A-RAF, B-RAF, and C-RAF. The presence of an aspartic acid in B-RAF just upstream of the conserved region CR3 is not present in either A-RAF or C-RAF; this difference accounts for B-RAF’s natural propensity towards activation. Oncogenic mutations often occur in the activation segment. (B) Common mutations in the activation segment of B-RAF. The activation segment is the region in which oncogenic mutations commonly occur. Common mutations occur in the first few codons of the activation segment (boxed region). The most common mutation occurs at codon 600 in exon 15; valine (V) is substituted for glutamic acid (E).

Figure 3.

Regulation of survival pathways by mutant B-RAF. (A) Mutant B-RAF activation of the mitogen-activated protein kinase (MAPK) pathway. B-RAF mutants, with elevated kinase activity, interact with the microenvironment to hyperactively phosphorylate MAP, which results in the phosphorylation downstream of the kinase MEK and the translocation of ERK into the nucleus. B-RAF mutants with lowered kinase activity regulate the MAPK pathway indirectly through C-RAF. Translocation of ERK to the nucleus via B-RAF/C-RAF signaling results in the expression of metalloproteinases and EMT. (B) Mutant B-RAF activation of the NF-κB pathway. The constitutively active mutant B-RAF activates the NF-κB pathway via IKKβ activation. Activation of NF-κB results in EMT via up-regulation of the metastasis inducer, Snail, as well as other metastatic gene products. NF-κB is inhibited directly through the metastasis suppressor, RKIP. RKIP also indirectly inhibits NF-κB through inhibition of B-RAF activity. (C) Up-regulation of mutant B-RAF via PTEN deletion, PI3K signaling, and AKT overexpression. RAS signaling results in PI3K activation and AKT expression. Normally, PI3K-induced expression of AKT is antagonized by PTEN. However, deletion of PTEN as well as signals from the microenvironment can result in uncontrolled AKT production. Higher levels of AKT expression inhibit mutant B-RAF signaling levels. Inhibition of B-RAF signaling by AKT counterintuitively promotes EMT. When mutant B-RAF signaling is extremely high, cellular defense mechanisms will induce the cells into senescence. AKT inhibition of B-RAF lowers B-RAF signaling to levels high enough to promote EMT and low enough to evade senescence.

Figure 4.

Activation of NF-κB by the PI3K/AKT and MAPK pathways. (A) Activation of NF-κB via the PI3K/AKT pathway. Activation of the PI3K pathway by RAS results in the production of AKT. AKT activates NF-κB through the activation of IKK. Activation of NF-κB results in Snail-induced EMT. PI3K signaling is antagonized by PTEN; however, up-regulation in Snail inhibits PTEN, resulting in a self-amplification cycle. (B) Activation of NF-κB via the MAPK pathway NF-κB is regulated through MEK-induced activation of IKK and ERK-induced up-regulation of Snail. (C) Regulation of Snail via RKIP and PTEN up-regulation of Snail through the PI3K/AKT and MAPK pathways inhibits both PTEN and RKIP, which then, in turn, amplifies the expression of Snail.