Poster Presentation 25th Lorne Cancer Conference 2013

Phosphoproteomics of cellular response to Vemurafenib reveals reprogramming by oncogenic B-RAF protein in Thyroid Cancer (#304)

Rob Parker 1 , Roderick Clifton-Bligh 2 , Mark Molloy 1
  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW, Australia
  2. Kolling Institute of Medical Research, University of Sydney, Sydney, NSW, Australia

An activating mutation (V600E) in the B-raf serine/threonine MAP kinase protein is a prevalent driver of several cancers (melanoma, thyroid, colon). The chief consequence of the mutation is a hyperactive ERK1/2 MAP kinase able to promote cell proliferation, producing critical hallmarks of metastatic disease. In mammals ERK’s control fundamental cellular mechanisms, determining cell-fate decisions by mediating differentiation in response to extracellular stimuli. Biochemically, the ERK pathway is well characterised, however how the pathway achieves different outcomes in the face of different stimuli or genetic aberrations of cancer is not clear. In this work a thyroid cancer cell line harbouring V600EBRAF has been treated with the mutant selective V600EBRAF inhibitor Vemurafenib (PLX-4032) and a potent inhibitor of MEK MAP kinase inhibitor (AZD-6244) in an attempt to distinguish oncogenic signals from V600EBRAF. After treatment for 30 minutes phospho-peptides were affinity purified by TiO2 and the response to inhibition identified using LC-MS and a label free quantitative approach. In total 59252 spectra could be assigned as peptides with a FDR at 1.13%, within these ~90% had phosphorylated residues. For quantitation ~3700 nr peptides were identified of which ~1900 could be quantified across all samples after manual validation. Statistical analysis revealed 55 V600EBRAF and 94 MEK inhibitor effects. As expected many of BRAF regulated phosphorylation events are dependent of MEK-ERK activation, the role of these phospho-proteins in cancer is discussed.