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A novel ERCC1 mRNA has been identified in mouse skin. Subsequent characterisation of the transcript demonstrated that the difference between the normal and skin-specific ERCC1 mRNA was at the 5' end and is due to differential initiation of transcription. As with the normal ERCC1 transcript the novel skin-specific transcript did not appear to be induced by UV irradiation. A functional ERCC1 minigene was constructed to facilitate subsequent analysis of the upstream promoter region and identification of the sequences involved in the regulation of the observed skin-specific pattern of expression. The minigene corrected the UV sensitivity of ERCC1-deficient cultured cells but did not exhibit the characteristic skin-specific expression pattern at the level of mRNA analysis. A pilot study for a potential gene therapy approach to increasing the lifespan of the ERCC1 knockout mice was completed. Recombinant ecotropic retroviruses containing ERCC1 coding sequences were produced using a murine leukaemia virus derived viral vector and viral packaging cell line. The resultant ERCC1 retrovirus was shown to partially correct the UV sensitivity associated with pools of ERCC1-deficient mouse embryonic fibroblasts. Clones, subsequently isolated from the transduced pools, were shown to be phenotypically corrected to wild type levels. The observed phenotypic correction correlated with expression of a retroviral ERCC1 mRNA. A transthyretin regulated ERCC1 transgene was used to bring about the targeted expression of ERCC1 in the liver. Pronuclear injection of the- transgene was used to produce a transgenic mouse line containing ~5 copies of the transgene integrated at a single site. Expression of this transgene on an ERCC1 -deficient background resulted in the correction of the lethal liver phenotype. Transgene positive nulls were not as severely runted and survived for between 9 and 12 weeks compared to the three-week survival of the transgene negative ERCC1-deficient mice. The consequences of ERCC1 deficiency in other tissues were studied. Abnormalities were identified in the skin and kidneys of adult transgene positive nulls. An investigation into the consequences of DNA repair deficiency on UV-B induced immunosuppression revealed that antigen presenting epidermal Langerhans cells, in the transgene positive nulls, did not show the normal pattern of accumulation in the lymph nodes following UV irradiation.

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.

Cutaneous melanoma is associated with significant morbidity and mortality representing the most significant cutaneous malignancy. As it is known that early diagnosis and treatment are the most efficient approaches to cure cutaneous melanoma, an improved understanding of the molecular pathogenesis of melanoma and exploration of more reliable molecular biomarkers are particularly essential. Two different types of molecular biomarker for melanoma have been investigated in this thesis. microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotides in length that are found in both animal and plant cells. miRNAs are involved in the RNA interference (RNAi) machinery to regulate gene expression posttranscriptionally. miRNAs have important roles in cancer: by controlling the expression level of their target genes they can affect cell signalling pathways and have been shown to have both prognostic and therapeutic potential. Importantly for melanoma research, reproducible miRNA expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues can be obtained that are comparable to those from fresh-frozen samples. The aims of the miRNA project were: first, to identify a melanoma-specific miRNA expression profile; secondly, to investigate roles of some of the melanoma-specific miRNAs identified in melanomagenesis. Using miRNA microarray on FFPE samples, I obtained a melanoma-specific miRNA expression profile. 9 of these differentially expressed miRNAs between benign naevi and melanomas (7 downregulated, 2 upregulated in malignancies) were verified by qRT-PCR and the functions of four of these miRNAs were studied. Ectopic overexpression of miR- 200c and miR-205 in A375 melanoma cells inhibited colony forming ability in methylcellulose, an in vitro surrogate assay for tumourigenicity. Moreover, elevation of miR-200c resulted in increased expression levels of E-cadherin through negative regulation of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Ectopic overexpression of miR-211 in A375 melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-200c, miR-205 and miR-211 act as tumour suppressors in melanomagenesis. The second biomarker investigated, mutated BRAF, has been seen in 50-70% of spontaneous cutaneous melanoma. The commonest mutation in melanoma is a glutamic acid for valine substitution at position 600 (V600E). Oncogenic BRAF controls many aspects of melanoma cell biology. The aim of this part of the work was: firstly, to study BRAF V600E mutation status in our melanoma tissue microarray (TMA) panel; secondly, to correlate this mutation to various clinicopathological features and evaluate its prognostic value through statistical analyses. BRAF V600E mutations were seen in 20% of the primary and 69% of the metastatic melanomas, respectively. More BRAF V600E mutations were seen in males relative to females. The mutation was also related to cell pigmentation, but not to age, ulceration or solar elastosis. Melanoma patients with the BRAF V600E mutation relapse earlier than patients without this mutation. However, no significant association between the BRAF V600E mutation and overall survival and melanoma specific survival was found.

Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously generated oxidative lesions. Defects in NER result in the human inherited disorder xeroderma pigmentosum (XP), which is characterised by UV hypersensitivity and a 1000-fold increased risk of skin cancer. ERCC1 is essential for the NER pathway where it acts in a complex with the XPF protein to make the incision 5' to the DNA lesion. The normal 1.1kb Ercc1 transcript is expressed in all tissues. Our group has discovered a second larger 1.5 kb transcript, which initiates from an alternative promoter, and is the most abundant Ercc1 transcript in mouse skin. The aims of this project were: 1, To investigate the role of ERCC1 and of the 1.5kb skin specific Ercc1 transcript in protecting the skin against UV-induced DNA damage. 2, To study the importance of ERCC1 in melanoma skin cancer and investigate ERCC1as a possible target for therapy against melanoma. Using a panel of Ercc1 wild-type and deficient cells, we established a quantitative western blotting system to study the expression of ERCC1 in a range of mouse tissues and mouse and human cell types. Although the skin-specific Ercc1 transcript was found to be present at much higher levels in the skin of albino compared to pigmented mouse strains, this did not result in an elevated level of ERCC1 protein. We were also unable to demonstrate that UV-irradiation, or other stress-inducing treatments resulted in increased levels of ERCC1 protein in cultured mouse keratinocytes. We investigated the DNA methylation status of the normal Ercc1 promoter and that of two potential upstream promoter regions that were candidates for the source of the 1.5kb skin-specific Ercc1 transcript. We found no evidence that they were the source and, instead, used 5' RACE analysis to locate the skin-specific promoter to a polymorphic region 500bp upstream of the normal initiation site. In albino strains this region contains a SINE element, which we hypothesize could be involved in the production of the skin-specific Ercc1 transcript. We also investigated the protein level of ERCC1 and other DNA repair proteins, including XPF, MSH2, MSH6 and MLH1 in human melanoma cells and ovarian tumour cells. Significantly elevated protein levels of ERCC1 and XPF, as well as the mismatch repair protein MLH1 were found in melanoma cells. This could possibly contribute to the higher resistance to chemotherapy in melanoma, although the melanoma cell lines we tested did not show increased resistance to UV and cisplatin compared to the ovarian cancer cells tested. When Ercc1 proficient mouse melanoma cells were xenografted into nude mice the xenografts grew rapidly. Cisplatin treatment caused an initial shrinkage of the tumours, but re-growth rapidly followed. Cells re-isolated into culture from cisplatin treated xenografts had significantly higher levels of ERCC1 protein than either input cells, or cells re-isolated from untreated xenografts. An isogenic Ercc1 deficient derivative of the Ercc1 proficient mouse melanoma cell line grew as rapidly as the parent line in vitro, but grew much more slowly as xenografts. In addition, the xenografts shrank completely following cisplatin treatment and did not recover. This suggests that ERCC1 could be a drug target for melanoma therapy.