Identification of aurora kinase B and Wee1-like protein kinase as downstream targets of (V600E)B-RAF in melanoma

Am J Pathol. 2013 Apr;182(4):1151-62. doi: 10.1016/j.ajpath.2012.12.019. Epub 2013 Feb 12.

Abstract

BRAF is the most mutated gene in melanoma, with approximately 50% of patients containing V600E mutant protein. (V600E)B-RAF can be targeted using pharmacological agents, but resistance develops in patients by activating other proteins in the signaling pathway. Identifying downstream members in this signaling cascade is important to design strategies to avoid the development of resistance. Unfortunately, downstream proteins remain to be identified and therapeutic potential requires validation. A kinase screen was undertaken to identify downstream targets in the (V600E)B-RAF signaling cascade. Involvement of aurora kinase B (AURKB) and Wee1-like protein kinase (WEE1) as downstream proteins in the (V600E)B-RAF pathway was validated in xenografted tumors, and mechanisms of action were characterized in size- and time-matched tumors. Levels of only AURKB and WEE1 decreased in melanoma cells, when (V600E)B-RAF, mitogen-activated protein kinase 1/2, or extracellular signal-regulated kinase 1/2 protein levels were reduced using siRNA compared with other identified kinases. AURKB and WEE1 were expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. Targeting these proteins reduced tumor development by approximately 70%, similar to that observed when inhibiting (V600E)B-RAF. Furthermore, protein or activity levels of AURKB and WEE1 decreased in melanoma cells when pharmacological agents targeting upstream (V600E)B-RAF or mitogen-activated protein kinase were used to inhibit the (V600E)B-RAF pathway. Thus, AURKB and WEE1 are targets and biomarkers of therapeutic efficacy, lying downstream of (V600E)B-RAF in melanomas.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics*
  • Animals
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects
  • Aurora Kinase B
  • Aurora Kinases
  • Biomarkers, Tumor / metabolism
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Cell Transformation, Neoplastic / metabolism
  • Cell Transformation, Neoplastic / pathology
  • Flow Cytometry
  • G2 Phase / drug effects
  • Glycogen Synthase Kinase 3 / metabolism
  • Humans
  • MAP Kinase Signaling System / drug effects
  • Melanoma / drug therapy
  • Melanoma / enzymology*
  • Melanoma / pathology*
  • Mice
  • Mice, Nude
  • Mitosis / drug effects
  • Molecular Targeted Therapy
  • Neoplasm Staging
  • Nuclear Proteins / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins B-raf / genetics*
  • RNA, Small Interfering / metabolism
  • Signal Transduction / drug effects

Substances

  • Antineoplastic Agents
  • Biomarkers, Tumor
  • Cell Cycle Proteins
  • Nuclear Proteins
  • RNA, Small Interfering
  • Protein-Tyrosine Kinases
  • WEE1 protein, human
  • AURKB protein, human
  • Aurkb protein, mouse
  • Aurora Kinase B
  • Aurora Kinases
  • BRAF protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins B-raf
  • Glycogen Synthase Kinase 3
  • glycogen synthase kinase 3 alpha