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

Sharma A, Madhunapantula SV, Gowda R, Berg A, Neves RI, Robertson GP.
Source: Am J Pathol
Publication Date: (2013)
Issue: 182(4): 1151-62
Research Area:
Cancer Research/Cell Biology
Dermatology/Tissue Engineering
Gene Expression
Basic Research
Cells used in publication:
Species: human
Tissue Origin: dermal
1205 Lu
Species: human
Tissue Origin:
melanoma cell line, human (RIPA)
Species: human
Tissue Origin:
Nucleofector® I/II/2b
4D-Nucleofector® 96-well Systems
A first siRNA screen of 636 kinases was realized to detrmine kinases involved in melanoma signaling. BRAF kinase is involved in50% of melanoma ceancers. The first sreen identified 33 kinases that regulate cell proliferation in UACC903 cell line. To understand the role of BRAF and its downsttream targets, another siRNA screen was realized mutated BRAF (V600E), MEK1/2, ERK1/2. This screen resulted in reduced activity of the AURKB and WEE1. Inhitbiting the activvities of these two kinases by siRNA resulted in decreased tumor progression in mice injected with melanoma cells treated or not. The authors further identified that AURKB andWEE1 are involved in the cell cycle and identified that drugs like vemurafenib. The transefction were performed with the Nucleofector 96 well Shuttle device, using program CM-130, and solution SF and UACC 903 cells. This was used for the 636 kinases screen. For the further experiments, siRNA was introduced into melanoma cells via nucleofection using an Amaxa Nucleofector with Solution R/program K-17 for UACC 903 and 1205 Lu or Solution R/program A-23 for A375M. Transfection efficiency after nucleofectionwas>90%, with 80%to 90%cell viability.
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.