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Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival.

ABSTRACT: Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro-apoptotic member of the BCL-2 family, is inhibited by BRAF-->MEK-->ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to elevated BIM expression. However, re-addition of trophic factors or activation of a conditional form of BRAF(V600E) leads to rapid inhibition of BIM expression. In both cases, inhibition of BIM expression was dependent on the activity of MEK1/2 and the proteasome. Consistent with these observations, pharmacological inhibition of BRAF(V600E) or MEK1/2 in human melanoma cells (using PLX4720 and CI-1040 respectively) led to a striking elevation of BIM expression. Re-activation of BRAF-->MEK-->ERK signaling led to phosphorylation of BIM-EL on serine 69 and its subsequent degradation. Interestingly, endogenous expression of BIM in melanoma cells was insufficient to induce apoptosis unless combined with serum deprivation. Under these circumstances, inhibition of BIM expression by RNA interference provided partial protection from apoptosis. These data suggest that regulation of BIM expression by BRAF-->MEK-->ERK signaling is one mechanism by which oncogenic BRAF(V600E) can influence the aberrant physiology of melanoma cells.

SUBMITTER: Cartlidge RA

PROVIDER: S-EPMC4916913 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival.

Cartlidge Robert A RA Thomas G R GR Cagnol Sebastien S Jong Kimberly A KA Molton Sarah A SA Finch Andrew J AJ McMahon Martin M

Pigment cell & melanoma research 20070728 5

Somatic activating mutations of BRAF are the earliest and most common genetic abnormality detected in the genesis of human melanoma. However, the mechanism(s) by which activated BRAF promotes melanoma cell cycle progression and/or survival remain unclear. Here we demonstrate that expression of BIM, a pro-apoptotic member of the BCL-2 family, is inhibited by BRAF-->MEK-->ERK signaling in mouse and human melanocytes and in human melanoma cells. Trophic factor deprivation of melanocytes leads to el ...[more]

PMID: 18715233

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Project description:BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

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Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival.

Publications

Oncogenic BRAF(V600E) inhibits BIM expression to promote melanoma cell survival.

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