Jossé Rozenn, Zhang Yong-Wei, Giroux Valentin, Ghosh Arun K, Luo Ji, Pommier Yves
Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA.
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA.
Mar Drugs. 2015 Jun 5;13(6):3625-39. 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.
拉索内酯A（LSA）是一种从海洋海绵Forcepia sp.中提取的强效抗肿瘤聚酮化合物，在纳摩尔浓度下可诱导快速且可逆的蛋白质过度磷酸化和早熟染色体凝集（PCC），且不依赖细胞周期蛋白依赖性激酶。为了确定LSA的细胞靶点，我们筛选了针对一组人类激酶和磷酸酶（1140个RefSeq）的2951个短发夹RNA（shRNA），以识别响应LSA调节PCC的基因。这导致鉴定出RAF1（C-RAF）是LSA诱导PCC的介质，因为针对RAF1的shRNA赋予了对LSA的抗性。我们发现，LSA在数分钟内即可诱导人结肠直肠癌HCT-116、卵巢癌OVCAR-8和伯基特淋巴瘤CA46细胞系中丝氨酸338位点的RAF1磷酸化。通过小干扰RNA（siRNA）消耗RAF1可减弱LSA在HCT-116和OVCAR-8细胞中诱导的PCC。此外，Raf1纯合缺失的小鼠胚胎成纤维细胞（MEF）对LSA诱导的PCC具有抗性，而相关激酶Braf缺失的MEF则不然。用野生型人RAF1而非激酶失活的RAF1突变体补充Raf1-/- MEF可恢复LSA诱导的PCC。最后，Raf抑制剂索拉非尼可有效抑制LSA诱导的PCC，而MEK抑制剂AZD6244则不能。我们的研究结果表明，RAF1在染色质凝集中具有先前未知的、不依赖丝裂原活化蛋白激酶（MAPK）的作用，且LSA可有效激活该途径。
