p53 family members and apoptosis in malignant melanoma: a role in chemoresistance?

We are investigating why melanoma, an aggressive form of skin cancer, is so resistant to chemotherapy, and how new treatments might be able to overcome this resistance. In the UK 8,100 new cases of melanoma are diagnosed annually with the incidence predicted to treble in the next 30 years. Melanoma arises from the pigment-producing cells (melanocytes) in skin and both its development and resistance to standard cancer treatments is thought in part due to the resistance of cells to apoptosis - a form of cell death. Essential to apoptosis is the p53 gene and loss of p53 activity is frequently implicated in other cancers. p53 also plays a crucial role in protecting against skin cancer induced by ultraviolet radiation (UVR). The aim of this project is to study the p53 family of proteins and their interacting roles in UVR-induced cell death in melanocytes and melanoma. Melanoma represents a major burden on public health and healthcare resources. This project will determine the mechanism of p53 inactivation in melanoma. Information obtained will further our understanding of changes in melanocytes as they undergo malignant progression and provide new ways to develop treatments that are effective.

Malignant melanoma is the most aggressive form of skin cancer. Its incidence has increased more rapidly than that of any other cancer in the last 30 years. Advanced disease has poor prognosis and is notoriously resistant to all current therapeutic modalities. Unlike many other malignancies, p53 mutations are infrequent in melanoma implicating aberrant upstream or downstream components of the p53 signalling pathway. In the proposed study the hypothesis that the p53 family members are determinants of chemosensitivity and chemoresistance in melanoma will be tested.

The specific aims of the study are to assess the integrity of the p53 signalling pathway including downstream target genes and co-activators of p53 family members in normal melanocytes and melanoma cell lines and to relate this to cellular response to genotoxins including ultraviolet radiation (UV) and cytotoxic drugs. Experiments will first establish the existence of post-translational modifications (specifically phosphorylation and acetylation) of p53 and p73 following genotoxic stress in melanocytes and melanoma cell lines. Using p53 target gene micro-arrays the p53 transcriptosome will be analysed following genotoxic stress in primary melanocytes and melanoma cell lines of varying sensitivity to DNA damage. Expression patterns of pro-apoptotic co-activators will be monitored (in collaboration) to seek correlations between patterns of target gene expression, co-activator expression and cellular response to DNA damage.

Expression patterns of each p53 family member and their splice variants will be established in control cultures and in tissue samples obtained prospectively. The role of individual p53 family members in UV-induced apoptosis will be examined by FACS analysis by exposing p53 wild-type and p53 null melanoma cell lines to different doses of UVB, UVA and DNA-damaging agents. Experiments to upregulate (by transfection or lentiviral infection) and/or deplete p63, p73 and their splice variants (by siRNAi and micro-siRNAi) will determine the chemosensitivity of melanoma cells to relevant genotoxic stimuli. Melanoma cell lines and a spectrum of melanocytic lesions from the patient cohort will also be screened for p53 polymorphic variants to investigate their synergistic roles with p53 family members in UV-induced apoptotic cell death and correlation with clinical outcomes respectively.

The proposed work combines both functional/mechanistic studies and the molecular pathological analysis of tumour samples to assess the clinical significance of in vitro results. Determining the molecular basis of the intrinsic resistance of melanocytes to apoptosis during malignant progression may provide insight into potential therapeutic targets for overcoming their acquired chemoresistance.