Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation

• Published in: Marine drugs 2015-06, Vol.13 (6), p.3625-3639
• Main Authors: Jossé, Rozenn, Zhang, Yong-Wei, Giroux, Valentin, Ghosh, Arun K, Luo, Ji, Pommier, Yves
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic Agents - isolation & purification; Antineoplastic Agents - pharmacology; Benzimidazoles - pharmacology; c-raf; Cell Line, Tumor; Chromatin - drug effects; Chromatin - metabolism; chromosome condensation; Chromosomes - drug effects; Chromosomes - metabolism; Embryo, Mammalian; Fibroblasts - drug effects; Fibroblasts - metabolism; Forcepia; Humans; lasonolide A; Macrolides - isolation & purification; Macrolides - pharmacology; Mice; myosin phosphatase; Niacinamide - analogs & derivatives; Niacinamide - pharmacology; Phenylurea Compounds - pharmacology; Phosphorylation - drug effects; Porifera - chemistry; Proto-Oncogene Proteins c-raf - metabolism; shRNA; Sorafenib
• ISSN: 1660-3397; 1660-3397
• DOI: 10.3390/md13063625

Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt's lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1-/- MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA.