Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis

Abstract

Background: Genes of the Raf family encode kinases that are regulated by Ras and mediate cellular responses to growth signals. Recently, it was shown that activating mutations of BRaf are found with high frequency in human melanomas. The Ras family member most often mutated in melanoma is NRas.

Methods: The constitutive activation of the Ras/Raf signaling pathway suggests an impact on the clinical course of the tumor. To address this notion, we analyzed tumor DNA from 114 primary cutaneous melanomas and of 86 metastatic lesions obtained from 174 patients for mutations in BRaf (exons 15 and 11) and NRas (exons 1 and 2) by direct sequencing of PCR products and correlated these results with the clinical course.

Results: In 57.5% of the tumors either BRaf or NRas were mutated with a higher incidence in metastatic (66.3%) than in primary lesions (50.9%). Although the majority of BRaf mutations affected codon 599, almost 15% of mutations at this position were different from the well-described exchange from valine to glutamic acid. These mutations (V599R and V599K) also displayed increased kinase and transforming activity. Surprisingly, the additional BRaf variants D593V, G465R and G465E showed a complete loss of activity in the in vitro kinase assay; however, cells overexpressing these mutants displayed increased Erk phosphorylation. The correlation of mutational status and clinical course revealed that the presence of BRaf/NRas mutations in primary tumors did not negatively impact progression free or overall survival. In contrast, however, for metastatic lesions the presence of BRAF/NRAS mutations was associated with a significantly poorer prognosis, i.e. a shortened survival.

Conclusion: We demonstrate a high - albeit lower than initially anticipated - frequency of activating BRaf mutations in melanoma in the largest series of directly analyzed tumors reported to date. Notably, the clinical course of patients harboring activating BRaf mutations in metastatic melanoma was significantly affected by the presence of a constitutive BRaf activation in these.

Figure 1

Mutation of two adjacent nucleotides. Following cloning of the PCR product and sequencing of independent clones we always observed either wild type or double mutants.

Figure 2

Mutations V599R and V599K like V599E increase BRaf kinase and transforming activity. NIH3T3 cells were transiently transfected with expression constructs harbouring a wild type BRaf cDNA or the indicated mutant. A) Five days post transfection the cells per well were counted. Shown are the mean values (±SD) of 3 wells each. B) In vitro Raf kinase assay: Immunoprecipitated BRaf was subjected to a kinase reaction with purified MEK and ERK proteins as series connected substrates. B-Raf and Phospho-ERK were detected on a western blot. A representative experiment is shown. C) Focus assay. The number of transformed foci overgrowing the cell monolayer was counted 18 days post transfection. For this assay the cells were split 2 days post transfection from a well of a 6 well plate to a 10 cm dish. Shown are the mean values (±SD) of 6 plates each.

Figure 3

Analysis of the BRaf mutants D593V, G463R, G465R and G465E. NIH3T3 cells were transiently transfected with expression constructs harbouring a wild type BRaf cDNA or the indicated mutant. A) Cells were lysed two days post transfection and the lysates were analysed for BRaf and Phospho-Erk on a western blot. B) Quantified Phospho Erk signals from blots shown in A. Given are the mean values (±SD) from two experiments. C) In vitro Raf kinase assay: Immunoprecipitated BRaf was subjected to a kinase reaction with purified MEK and Erk proteins as series connected substrates. B-Raf and Phospho-Erk were detected on a western blot. A representative experiment is shown.

Figure 4

Influence of BRaf or NRas mutation on overall survival. Kaplan-Meier plots for overall survival for (A) primary tumors and (B) metastases stratified for absence (full line) or presence of either a BRAF or a NRAS mutation (dotted line). A), i.e., as well as B) Overall survival was measured from removal of primary tumor time or the respective metastasis to time of death.