Previous data have unveiled a novel autoregulatory feedback loop between iASPP and p63 in the stratified epithelia; this involves two microRNAs, miR-574-3p and miR-720, and is critical for epidermal homeostasis. The iASPP oncoprotein, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is a key inhibitor of p53 and NF-κB and is highly expressed in many cancers. Non-melanoma skin cancer, comprising of cutaneous squamous carcinoma (cSCC) and basal cell carcinoma, is currently the most common malignancy in the UK. In view of this newly-identified iASPP-p63 axis, I hypothesised a potential role for dysregulation of this feedback loop in the pathogenesis of cSCC and aimed to assess the role of iASPP in human cSCC. Protein and mRNA expression patterns were assessed in a panel of 10 cSCC cell lines generated by our group. In addition, immunostaining of iASPP and p63 was performed in 107 cSCC clinical samples of variable differentiation status. The data reveal an overall increase in expression of iASPP and ΔNp63 in cSCC but also suggest a significant alteration of the cellular localisation of iASPP dependent on the differentiation status of the tumour. To further assess the effects mediated by the iASPP/p63 axis, iASPP and p63 have been silenced by RNAi technology in a subset of cSCC cell lines. Whilst data shows the direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop at the microRNA level may be occurring via a novel p63 regulator, miR-211-5p. Functionality of iASPP in cSCC (proliferation, apoptosis, cell motility/migration and invasiveness) provides evidence for a role of iASPP in preventing epithelial-mesenchymal transition in cSCC via a p63/miR-205-5. These findings provide potential future directions for development of clinical biomarkers and novel therapeutic targets for cSCC and may ultimately provide the tools for tackling the increasing morbidity and mortality associated with this malignancy.

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Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet need with a 5-year survival following diagnosis of 3% with limited surgical, radiotherapy and chemotherapy treatment options. Central to PDAC tumorigenesis is acquisition of an oncogenic Kras mutation to drive acinar-to-ductal metaplasia and progression to PDAC that is potentiated by NF-kB deregulation. However, PDAC requires the additional loss of tumour suppressors such as p53, SMAD4 or p16. ASPP family members ASPP2 and iASPP regulate both p53 and NF-kB, and are classified as a tumour suppressor and oncogene respectively. However, the precise roles of ASPP2 and iASPP in pancreatic cancer are unknown. In this thesis I demonstrate that ASPP2 suppresses metastasis and iASPP suppresses the pro-inflammatory tumour microenvironment. In a mouse model of PDAC development, ASPP2-deficiency does not alter metaplasia, PanIN progression or primary PDAC onset. However, median survival due to metastasis is significantly reduced in an ASPP2-deficient PDAC model. I demonstrate ASPP2-deficient PDAC can result in increased squamous differentiation defined histologically or via increased p63 expression. I propose ASPP2 is a key suppressor ΔNp63 and the squamous PDAC subtype in vivo. Conversely, iASPP is a putative oncogene and high expression in cancer associates with poor prognosis. However, in a mouse model of PDAC, loss of iASPP accelerates PDAC onset and metastasis. I demonstrate that iASPP is a functional tumour suppressor of a pro-inflammatory phenotype in response to oncogenic Kras and pancreatitis. I propose ASPP2- and iASPP-deficient mouse models of PDAC represent in vivo the squamous and immunogenic subtypes of PDAC respectively; and are relevant tools to study mechanisms of metastasis and inflammation-driven carcinogenesis.

Desmosomes are intercellular junctions that anchor intermediate filaments to the sites of intercellular contacts. They are critical for maintaining the integrity of tissues that experience constant mechanical and structural stresses, like the skin and heart. Perturbation of desmosomal adhesion can lead to devastating epidermal and myocardial diseases. However, little is known about the regulators of desmosomes and the role of desmosomes in cell signalling events. Recent work has suggested that iASPP, an inhibitor of the p53 family of proteins, localises at the intercalated discs where desmosomes reside. However, its role at the desmosomes has remained elusive. Thus, in this thesis, it was investigated whether iASPP is a dual function protein that links desmosome adhesion to gene expression and if desmosome-related diseases develop in the absence of iASPP. iASPP was found to be a novel regulator of desmosomes, co-localising with them by physically interacting with the desmosomal components desmoplakin and K5 intermediate filaments. Loss of iASPP resulted in increased phosphorylation and solubilisation of desmoplakin, leading to the formation of K5 aggregates. This culminated in disrupted intercellular adhesion and enhanced cellular migration. Consistent with the role of iASPP in the maintenance of desmosomal adhesion integrity, focal palmoplantar keratoderma was observed in iASPP-deficient mice — a disorder often associated with desmosome dysfunction. This was accompanied by disrupted intracellular signalling, as exemplified by the disrupted expression of differentiation markers; an increase in the thickness of cell layers expressing differentiation marker K1 was noted, and K5 and K6 cells were ectopically expressed throughout the diseased palmoplantar epidermis. Impaired intercellular adhesion and migration had consequences for wound healing, as iASPP-deficient mice exhibited delayed wound closure. Furthermore, defects in eyelid closure in iASPP-deficient mice were found to be due to increased apoptosis. The localisation of apoptotic cells at the leading edge of the eyelid epidermis implied that apoptosis might have occurred due to a loss of cell-matrix or cell-cell contact, i.e. anoikis. Taken together, these results suggest that iASPP is involved in pathological (palmoplantar keratoderma), physiological (wound healing) and developmental processes (embryonic eyelid closure) through its regulation of desmosomes and their dynamics. Therefore, iASPP represents a new candidate gene in cutaneous disorders and could be implicated in a variety of epidermal and myocardial diseases.