Near-genomewide RNAi screening for regulators of BRAF(V600E) -induced senescence identifies RASEF, a gene epigenetically silenced in melanoma.
The activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-β-galactosidase activity, interleukins, and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.
Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Joanna
J
Kaplon
Cornelia
C
Hömig-Hölzel
Linda
L
Gao
Katrin
K
Meissl
Els M E
EM
Verdegaal
Sjoerd H
SH
van der Burg
Remco
R
van Doorn
Daniel S
DS
Peeper
D019943:Amino Acid Substitution; D059447:Cell Cycle Checkpoints / Q000235:genetics; D045744:Cell Line, Tumor; D016922:Cellular Senescence; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020125:Mutation, Missense; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*
C529641:RASEF protein, human; D025521:Tumor Suppressor Proteins; D020663:ras Guanine Nucleotide Exchange Factors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
BRAF; RASEF; melanoma; methylation; screen; senescence