Mechanisms of V600EBRAF-induced tumour development

BRAF is a serine/threonine protein kinase that functions as a component of the RAS/RAF/MEK/ERK signalling pathway. Gain-of-function BRAF mutations have been found in ~50%-70% of malignant melanomas and in a variety of other cancer types, including thyroid, colon, and lung cancers. The activating V600EBRAF mutation is the most frequent mutation comprising a single amino acid substitution (V600E) that results in constitutive activation of BRAF kinase activity. Here, the CreERT system is used to induce endogenous V600EBRaf expression from a single knockin allele after 4-hydroxytamoxifen treatment in MEFs. V600EBRaf induced a faster G1/S phase progression by a significant upregulation of Cyclin D1 at the transcriptional level and was reversed by MEK inhibition. V600EBRaf also suppressed apoptosis at the premitochondrial level following serum withdrawal by a drastic downregulation in BimEL expression at the post-transcriptional level which was rescued by MEK inhibition and proteasomal inhibition. In vivo, expression of endogenous V600EBRaf in mice induced senescence and autophagy in lung adenomas and this prevented lung tumour progression. Senescence was primarily mediated by the p53/p21Cip1 and p16INK4a pathways while autophagy was regulated by inhibition of the mTOR pathway. Mass spectrometry was used to screen for components of the senescence-messaging secretome induced by V600EBRaf in the lung. The cholesterol binding protein Npc2 was identified as being significantly induced by V600EBRaf expression. The role of Npc2 in tumour suppression was investigated and it was found that this protein induced upregulation of the p53/p21Cip1 pathway and triggered autophagy in a paracrine/autocrine manner. Knockout of Npc2 abrogated senescence and promoted growth of lung tumours by downregulation of the p53/p21Cip1 pathway in vivo, but did not prevent the induction of autophagy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo