The NF1 protein, neurofibromin 1, negatively regulates RAS proteins through GTPase activity. Additionally, mutations in NF1 genes can lead to melanoma. Aside from BRAF mutations, NRAS mutations have also been described in about 15% of melanoma patients, and result in the reduction of the intrinsic GTPase activity and in the constitutive activation of NRAS. More than 90% of BRAF mutations result in the valine to glutamic acid substitution (V600E), associated with a 400-fold increased activity of the protein. BRAF is a serine-threonine kinase, which transfers growth signals to the nucleus of the cells. Perhaps, the most remarkable progress has been made after the identification of BRAF mutations in melanoma. The recognition of driving mutations in multiple melanoma oncogenes allowed the successful implementation of targeted therapies. In the recent years, several molecular alterations have been identified as occurring during melanoma initiation and progression. ConclusionĬell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.Ĭutaneous melanoma is an aggressive disease representing one of the leading causes of mortality related to human cancers worldwide. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. 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. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. 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 initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. BRAF V600E RNA expression was assessed by qPCR. Cell proliferation was assessed by MTT colorimetric assay. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). MethodsĭNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. Patient management is often based on the use of specific inhibitors targeting this mutation. Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution.
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