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

Jossé R, Zhang YW, Giroux V, Ghosh AK, Luo J, Pommier Y - Mar Drugs (2015)

Bottom Line: 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.Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA. rozenn.josse@gmail.com.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

shRNA screen for genetic determinants of lasonolide A (LSA) sensitivity. (A) Chemical structure of LSA; (B) Scheme of the shRNA screen aiming to identify genes that modulate LSA sensitivity. A pooled retroviral shRNA library directed against kinases and phosphatases was transduced into HCT-116 colorectal cancer cells. Cells were cultured in the absence (control) or presence of LSA at two concentrations, 10 nM for 24 days or 100 nM for 14 days, respectively. Library compositions in starting and end samples were deconvoluted by deep sequencing to identify shRNAs that selectively dropped out or enriched in the LSA-treated samples; (C) Top candidates are enriched in known protein-protein interactions as determined by STRING; (D) shRNAs that enriched and dropped out selectively in the LSA treated samples. Results are expressed as the difference between the log2 ratio of (LSA/start) and the log2 ratio of (control/start). LSA low and high indicates the 10 nM and 100 nM treatment conditions, respectively. Genes whose shRNAs enriched with LSA treatment are potentially associated with LSA sensitivity, while genes whose shRNAs depleted with LSA treatment are potentially associated with resistance.
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marinedrugs-13-03625-f001: shRNA screen for genetic determinants of lasonolide A (LSA) sensitivity. (A) Chemical structure of LSA; (B) Scheme of the shRNA screen aiming to identify genes that modulate LSA sensitivity. A pooled retroviral shRNA library directed against kinases and phosphatases was transduced into HCT-116 colorectal cancer cells. Cells were cultured in the absence (control) or presence of LSA at two concentrations, 10 nM for 24 days or 100 nM for 14 days, respectively. Library compositions in starting and end samples were deconvoluted by deep sequencing to identify shRNAs that selectively dropped out or enriched in the LSA-treated samples; (C) Top candidates are enriched in known protein-protein interactions as determined by STRING; (D) shRNAs that enriched and dropped out selectively in the LSA treated samples. Results are expressed as the difference between the log2 ratio of (LSA/start) and the log2 ratio of (control/start). LSA low and high indicates the 10 nM and 100 nM treatment conditions, respectively. Genes whose shRNAs enriched with LSA treatment are potentially associated with LSA sensitivity, while genes whose shRNAs depleted with LSA treatment are potentially associated with resistance.

Mentions: Lasonolide A (LSA) (Figure 1A), which was first extracted from the Caribbean marine sponge Forcepia sp., was noted for its high potency and unique profile of cytotoxic activity in the National Cancer Institute cell screen panel (the NCI60) [8]. The anticancer potential of LSA has remained relatively unexplored at least in part because of its low chemical abundance and challenging chemical synthesis due to its complex chemical structure comprising multiple chiral centers (Figure 1A) [9,10,11]. However, its biological effects are remarkable. LSA has rapid and profound effects on critical aspects of cell biology including inhibition of cell adhesion, activation of PKC, and activation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38 [8,12,13].


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

Jossé R, Zhang YW, Giroux V, Ghosh AK, Luo J, Pommier Y - Mar Drugs (2015)

shRNA screen for genetic determinants of lasonolide A (LSA) sensitivity. (A) Chemical structure of LSA; (B) Scheme of the shRNA screen aiming to identify genes that modulate LSA sensitivity. A pooled retroviral shRNA library directed against kinases and phosphatases was transduced into HCT-116 colorectal cancer cells. Cells were cultured in the absence (control) or presence of LSA at two concentrations, 10 nM for 24 days or 100 nM for 14 days, respectively. Library compositions in starting and end samples were deconvoluted by deep sequencing to identify shRNAs that selectively dropped out or enriched in the LSA-treated samples; (C) Top candidates are enriched in known protein-protein interactions as determined by STRING; (D) shRNAs that enriched and dropped out selectively in the LSA treated samples. Results are expressed as the difference between the log2 ratio of (LSA/start) and the log2 ratio of (control/start). LSA low and high indicates the 10 nM and 100 nM treatment conditions, respectively. Genes whose shRNAs enriched with LSA treatment are potentially associated with LSA sensitivity, while genes whose shRNAs depleted with LSA treatment are potentially associated with resistance.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4483648&req=5

marinedrugs-13-03625-f001: shRNA screen for genetic determinants of lasonolide A (LSA) sensitivity. (A) Chemical structure of LSA; (B) Scheme of the shRNA screen aiming to identify genes that modulate LSA sensitivity. A pooled retroviral shRNA library directed against kinases and phosphatases was transduced into HCT-116 colorectal cancer cells. Cells were cultured in the absence (control) or presence of LSA at two concentrations, 10 nM for 24 days or 100 nM for 14 days, respectively. Library compositions in starting and end samples were deconvoluted by deep sequencing to identify shRNAs that selectively dropped out or enriched in the LSA-treated samples; (C) Top candidates are enriched in known protein-protein interactions as determined by STRING; (D) shRNAs that enriched and dropped out selectively in the LSA treated samples. Results are expressed as the difference between the log2 ratio of (LSA/start) and the log2 ratio of (control/start). LSA low and high indicates the 10 nM and 100 nM treatment conditions, respectively. Genes whose shRNAs enriched with LSA treatment are potentially associated with LSA sensitivity, while genes whose shRNAs depleted with LSA treatment are potentially associated with resistance.
Mentions: Lasonolide A (LSA) (Figure 1A), which was first extracted from the Caribbean marine sponge Forcepia sp., was noted for its high potency and unique profile of cytotoxic activity in the National Cancer Institute cell screen panel (the NCI60) [8]. The anticancer potential of LSA has remained relatively unexplored at least in part because of its low chemical abundance and challenging chemical synthesis due to its complex chemical structure comprising multiple chiral centers (Figure 1A) [9,10,11]. However, its biological effects are remarkable. LSA has rapid and profound effects on critical aspects of cell biology including inhibition of cell adhesion, activation of PKC, and activation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38 [8,12,13].

Bottom Line: 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.Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute (CCR-NCI), NIH, Bethesda, MD 20892-9760, USA. rozenn.josse@gmail.com.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus