Activating proto-oncogene mutations in human cutaneous melanoma

Abstract: Cutaneous melanoma arises from melanocytes located in the basal layer of the epidermis. The disease is known to progress through well-defined steps: 1) common nevus; 2) dysplastic nevus; 3) radial growth phase (RGP) primary melanoma; 4) vertical growth phase (VGP) primary melanoma; and 5) metastatic melanoma. Whereas RGP melanoma is confined to the epidermis, VGP melanoma expands into the dermis and has acquired the capacity to metastasize. If detected at the early RGP stage, melanoma is easily cured by surgical excision. However, metastatic melanoma is highly resistant to existing therapies and has a poor prognosis. Therefore a better understanding of the mechanisms underlying melanoma initiation and progression is required to allow the development of more effective therapies. The aim of this thesis was to determine the frequency and timing of CTNNB1, NRAS, BRAF and PIK3CA mutations in sporadic cutaneous melanoma. Beta-catenin (encoded by CTNNB1) is a molecule that plays central roles both in the Wnt/beta-catenin signaling pathway and in cell-cell adhesion. Using PCR-single strand conformation polymorphism analysis, we identified CTNNB1 mutations in 3% of melanoma tumors. Interestingly, cytoplasmic and nuclear beta-catenin was observed not only in tumors that harbored CTNNB1 mutations but also in about one third of primary tumors that were wild-type for CTNNB1. This suggests that there must exist other mechanisms of beta-catenin stabilization in cutaneous melanoma. The Ras GTPases transduce extracellular growth signals to a variety of different effector pathways, including the Raf-MEK-ERK and PI3K-Akt pathways. Signaling through these pathways is often deregulated in human cancers. Cutaneous melanomas are known to harbor NRAS and BRAF mutations at a high frequency. To determine the timing of these mutations in melanoma tumorigenesis we screened a large series of paired primary and metastatic melanomas from a total of 71 patients. NRAS mutations were found in 30% (21 of 71) and BRAF mutations in 59% (42 of 71) of melanoma patients. Interestingly, mutations in NRAS and BRAF were observed to be mutually exclusive and thus 89% (63 of 71) of the patients analyzed had mutations in either of these genes. We found that NRAS and BRAF mutations are present in early RGP lesions and preserved in later stage VGP and metastatic lesions. Only in rare cases did mutations arise at the metastatic stage. Together, these results indicate that NRAS and BRAF mutations correlate with melanoma initiation rather than progression, and that they are not responsible for the critical RGP to VGP transition. The finding that NRAS and BRAF mutations are preserved throughout melanoma progression suggests that they may be of importance for melanoma maintenance. Furthermore, we found that the PIK3CA gene, which encodes the p110alpha catalytic subunit of class IA PI3Ks, is mutated in approximately 3% of melanoma metastases. Interestingly, PIK3CA missense mutations were observed only in metastases that did not carry NRAS mutations, suggesting that PIK3CA and NRAS mutations may be mutually exclusive in melanoma. Finally, we analyzed a large series of melanoma metastases with known NRAS and BRAF mutation status for expression of phosphorylated (active) ERK and phosphorylated (active) Akt. We found that ERK and Akt are activated in the majority of melanoma metastases, irrespective of NRAS and BRAF mutation status. Taken together, our results confirm the involvement of both the Raf-MEK-ERK and PI3K-Akt signaling pathways in melanoma tumorigenesis and suggest that these pathways can be activated through multiple mechanisms. The frequent activation of both the Raf-MEK-ERK and PI3K-Akt pathways in melanoma suggest that therapy targeting only one of these pathways may not be effective, but rather that both pathways should be targeted.