Identification of molecular targets of oncogenic NRAs and BRAF involved in regulation of melanoma cell proliferation

Petti, Carlotta (2007). Identification of molecular targets of oncogenic NRAs and BRAF involved in regulation of melanoma cell proliferation. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ea3b

Abstract

Mutated BRAF or NRAS genes have been found in ~90% of sporadic melanomas, with a frequency of ~65% of tumors harbouring a mutated BRAF and ~25% of tumors bearing an activated NRAS. However, NRAS^Q61R and BRAF^V600E are generally mutually exclusive, suggesting that occurrence of both mutations in the same tumor is selected against during tumorigenesis. Analysis of clones isolated from a rare melanoma bearing both mutations showed that the two activating mutations are indeed mutually exclusive at the single cell level. Forced co-expression of the two activating mutations in the same melanoma cell activated senescence and increased susceptibility to cell-mediated citotoxicity, consistent with a relationship of synthetic lethality between oncogenic NRAS and BRAF. A search for differentially expressed genes in double mutant (i.e. bearing NRAS^Q61R and BRAF^V600E) vs. single mutated cells (bearing only BRAF^V600E) allowed to identify LCN2 and LOXL3, by Microarray analysis, and activated AMPK, by Western blot, as potential targets involved in growth arrest of double mutant cells. AMPK was chosen for further analysis in panels of melanoma cells. Treatment of melanoma cells with either AICAR or Phenphormin, two specific activators of AMPK, led to inhibition of growth and of cell cycle progression in different human melanomas that was correlated to the up-regulation of p21 protein. Moreover, cells with activated AMPK showed evidence of induction of senescence. These results suggest that targeting genes associated with oncogenic NRAS and BRAF, and involved in regulation of cell growth and senescence, may allow the development of new therapeutic strategies for advanced melanoma.