title: Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer.
abstract: BACKGROUND
  Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors.
  
  
  METHODS
  EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used.
  
  
  RESULTS
  In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005).
  
  
  CONCLUSIONS
  Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: van der Mijn
authors.affiliation: Neuro-Oncology Research Group, Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Nik
authors.initials: N
authors.lastname: Sol
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Wouter
authors.initials: W
authors.lastname: Mellema
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jimenez
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: Dekker
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Lisette M
authors.initials: LM
authors.lastname: Schutte
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Egbert F
authors.initials: EF
authors.lastname: Smit
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Broxterman
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
authors.forename: Johan
authors.initials: J
authors.lastname: Skog
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
authors.forename: Bakhos A
authors.initials: BA
authors.lastname: Tannous
authors.affiliation: Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands; Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wurdinger
authors.affiliation: Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands; h.verheul@vumc.nl.
authors.forename: Henk M W
authors.initials: HM
authors.lastname: Verheul
keywords: biomarker; cancer; kinase inhibitor; plasma; signalling