Role of ubiquitination of ERCC1 in DNA repair in melanoma
Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.