Genetic changes in melanoma progression

Abstract

Melanoma is a highly aggressive tumour with a poor prognosis for patients with advanced disease because it is resistant to current therapies. Therefore, the development of novel strategies for melanoma treatment is important. The characterization of the molecular mechanisms underlying melanoma proliferation, progression, and survival could help the development of novel targeted melanoma treatments. The MAPK and PI3K pathways both play important roles in melanoma progression. In the MAPK pathway, DUSP6, which acts as a phosphatase to negatively control the activation of ERK1/2, is involved in the development of human cancers. The MAPK pathway also regulates expression of the DNA repair gene ERCC1 following EGF treatment. ERCC1 is essential for nucleotide excision repair, which is one of the major systems for removal of cisplatin induced DNA lesions. The aims of this project were: 1, to investigate the molecular changes in our immortal mouse melanocyte cell lines that were needed for them to form tumours in a xenograft model; 2, to investigate whether the MAPK pathway regulates ERCC1 following cisplatin treatment and protects melanoma cells from death. Through comparison of the RAS/RAF/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways between our immortal mouse melanocyte cell lines and their tumour derivatives in our xenograft model, we identified a molecularly distinct subtype of mouse melanoma characterized by reduced ERK and AKT activity and increased expression of DUSP6. Functional analyses employing ectopic overexpression indicated that increased expression of DUSP6 enhanced anchorage independent growth ability and invasive ability in our mouse melanocytes, suggesting that increased DUSP6 expression may contribute to melanoma formation in the xenograft assay. We also demonstrated that higher expression of p-ERK suppressed invasion, but not anchorage independent growth, in our subtype of mouse melanoma by enforced expression of constitutively active MEK1 and MEK2. In addition, the role of DUSP6 in classical human melanoma was investigated in this Genetic changes in melanoma progression study. Inhibition of anchorage independent growth and invasion were observed after exogenous expression of DUSP6 in human melanoma cells. This suggested that DUSP6 played different roles in classic human melanoma than in our distinct subtype of mouse melanoma. Our study also investigated the phosphorylation level of ERK1/2 and the mRNA and protein level of ERCC1 and its partner XPF in the human melanoma cell line following cisplatin treatment. Significant increases in expression of p-ERK, ERCC1 and XPF were found in cisplatin treated cells. Moreover, a MEK inhibitor inhibited ERCC1 induction by cisplatin, but did not significantly affect XPF induction. This suggested that the MAPK pathway was involved in regulation of ERCC1 but not XPF. Furthermore, the DUSP6 level decreased after cisplatin treatment and overexpression of DUSP6 inhibited ERCC1 and XPF induction and reduced resistance to cisplatin. DUSP6 seems to play a crucial role in resistance of melanoma to cisplatin. In addition, a novel larger ERCC1 transcript was identified in human cell lines and was found to be upregulated by cisplatin. The ratio of larger ERCC1 transcript relative to the normal ERCC1 transcript increased following cisplatin treatment. The functions of this larger ERCC1 transcript in cisplatin resistance deserve further study.