Role of B-Raf(V600E) in differentiated thyroid cancer and preclinical validation of compounds against B-Raf(V600E)

Abstract

B-Raf(V600E), an oncogenic protein kinase, is the most frequent genetic alteration in papillary thyroid carcinomas (PTC). PTC represents 80-90% of all thyroid cancers and over the past five years, more than 200 manuscripts have been published about the relationship between "B-Raf(V600E) and thyroid cancer". B-Raf(V600E) genetically arises from a transversion point mutation (valine-to-glutamate substitution at amino acid residue-600, V600E) and leads to over activation of the mitogen-activated protein kinases (MAPK) signaling pathway. The MAPK pathway is essential for transmitting proliferation signals generated by cell surface receptors and cytoplasmic signaling elements to the nucleus. In many cancers, including thyroid cancer, B-Raf(V600E) appears to play a crucial role in cell proliferation, survival and de-differentiation. In thyroid cancer, the V600E mutation occurs with greater frequently in aggressive subtypes of PTC, and in individuals that present at advanced stages of disease with extra-thyroidal extension and/or lymph node metastases. B-Raf(V600E) is considered a marker of aggressive disease in both PTC (>1 cm) and micro-PTC (</=1 cm), and interestingly, is associated with both loss of I-131 avidity and PTC recurrence. Though treatment of patients with thyroid cancer is usually successful and most patients are rendered disease-free, to date there are no effective therapies for patients with invasive, non-radioiodine sensitive tumors or metastatic disease. In this article we will review the relation between B-Raf(V600E) and PTC, as well as both non-selective and selective pharmacological agents currently under investigation for treatment of B-Raf(V600E) positive PTC.

Fig.(1). Physiological mechanism and effects of wild-type B-Raf/MAP kinases signaling pathway

(A) 14-3-3 dimers bind and retain inactive B-Raf protein in the cytosol. (B) In response to exogenous stimuli, tyrosine kinase (TK) receptors activate the G protein RAS, which in turn binds the serine-threonine kinase B-Raf after inducing a conformational change by dephosphorylating the N-terminal 14-3-3-binding sites, thus allowing its recruitment to plasma membrane. Activated B-Raf then phosphorylates (PO₄⁻) and activates MAPKs/MAPKKs (phospho-ERK1/2), which translocate to the nucleus where they regulate chromatin remodelling and gene expression. Phospho-ERK1/2 then phosphorylates the transactivating domain of various transcription factors within the promoter region of early and delayed response genes. This process coordinates a highly interconnected and integrated cellular signaling networks that orchestrates the nuclear response to the extra-cellular microenvironment.

Fig.(2). Mechanism of activation and effects of activated phospho-ERK1/2 signaling pathway by B-Raf^V600E oncogene

When the B-Raf^V600E oncogene is generated through a point mutation, activation of the MAP kinase pathway becomes constitutive, inducing cells to proliferate indefinitely and initiate tumor formation. B-Raf^V600E shows a dramatically increased basal kinase activity (increased phosphorylation, PO₄⁻), ~480-folds higher than wild-type B-Raf activity. Response to B-Raf^V600E is independent of extra-cellular mitogens and Ras signaling, creating a deregulation of transcription mechanisms. The B-Raf^V600E-activated phospho-ERK1/2 pathway transforms human normal cells both in vitro and in vivo. In addition, B-Raf^V600E induces senescence in normal human foreskin fibroblasts by stimulation of the cell cycle inhibitor p16^INK4a.