Réparation par excision de nucléotides des dommages induits par rayons ultraviolets dans les mélanomes humains

Malignant melanoma (MM) is the second most frequent neoplasia among young Canadian adults (aged 20-44); moreover the incidence of this disease continues to rise annually at an alarming rate. Unless primary melanoma is diagnosed early and promptly resected the patient prognosis is dismal since this deadly tumour type metastasizes extremely aggressively and is highly refractory to conventional treatment protocols. It is well established that exposure to UV light, and subsequent induction of genotoxic DNA photoproducts, is a primary determinant in the initiation of MM. Furthermore nucleotide excision repair (NER) clearly represents a critical frontline defence against MM because it is the only human pathway designed to remove the aforementioned DNA photoproducts. Despite this, the potential contribution of NER defects to sporadic MM development in the general population has remained unclear. Our laboratory previously developed a novel flow cytometry-based assay to evaluate the efficiency of NER as a function of cell cycle. This method was employed to demonstrate that functional ATR kinase is strictly required for NER during S phase in primary human fibroblasts. Intriguingly we also reported that many model tumour cell lines are deficient in NER uniquely in S phase populations, raising the possibility that such a defect might be characteristic of certain types of cancers. We therefore hypothesized that a significant proportion of human MM cell lines may exhibit reduced NER capacity specifically during S phase, and that this in turn might be attributeable to reduced ATR signaling. To test this hypothesis, three major specific aims were proposed: (i) To measure the efficiency of NER as a function of cell cycle among a panel of  human MM cell lines and in primary melanocytes; (ii) To investigate whether any correlation exists between NER status and ATR activity during S phase in human MM cell lines; (iii) To investigate whether frequently mutated genes in melanoma (eg., PTEN, BRAF) might cooperate with ATR to regulate S phase-specific NER in MM cell lines. We were able to demonstrate that, in fact, 13/16 MM cell lines display remarkably diminished capacity to remove UV-induced DNA photoproducts specifically during S phase. Furthermore this defect correlates strongly with reduced activation of ATR kinase and, for a majority of MM, higher Akt phosphorylation levels. RNAi-mediated knockdown of the PTEN tumour suppressor, while stimulating Akt phosphorylation as expected, also engenders reductions in both photoproducts repair and ATR activation in S phase cells. In addition, (i) ectopic expression in PTEN-null strains of wild type PTEN but not of PTEN variants deficient in phosphatase activity, or (ii) pharmacological inhibition of Akt, significantly rescue S phase-specific repair as well as ATR activation. Our data indicate that reduced PTEN/Akt-dependent ATR signaling leading to defective repair of UV DNA photoproducts uniquely during S phase may represent an heretofore unrecognized major determinant in sunlight-induced melanoma development.

Keyphrases: UV, Cancer, ATR, PTEN, PI3K, Akt, Malignant melanoma, S phase, Nucleotide excision repair