Abstract
The tumour suppressor gene p53 is the master sensor and regulator of the response to cell stress. Upon DNA damage, p53 can induce various outcomes including apoptosis, cell cycle arrest, senescence, and DNA repair in order to prevent tumorigenesis. Given these roles, p53 is frequently mutated in cancer, with missense mutations resulting in gain-of-function activity and tumour progression, including tumour cell migration, invasion and metastasis.
p53 exists as part of a complex network, including family members p63 and p73, all of which have multiple isoforms. The human p53 isoform ∆133p53 has been reported to be aberrantly expressed in a number of tumour types, and has been shown to be associated with poor patient outcome in some cancer types. ∆133p53 is involved in angiogenesis and tumour progression, and the ∆122p53 mouse is used as a model of ∆133p53, because it exhibits a pro-inflammatory profile, including elevated levels of IL-6, IFN-γ, and TNF-α.
The research outlined in this thesis explores the nature of the human ∆133p53 by utilising the ∆122p53 mouse, and validates ∆122p53 as a model for ∆133p53. The specific aims of this work were to determine the ability of ∆133p53 and ∆122p53 to promote proliferation and migration, to determine and validate the drivers of migration in cells expressing ∆122p53, and examine the ability of ∆122p53 to promote invasion and metastasis.
Chapter 3 details the characterisation of ∆133p53 and ∆122p53 overexpression cell lines, showing that both ∆133p53 and ∆122p53 promote proliferation and migration, and that these phenotypes are independent of each other. This is evidence that ∆133p53 and ∆122p53 behave in a similar manner and validates ∆122p53 as a model for ∆133p53.
Chapter 4 details further exploration of the migratory phenotype of ∆122p53, and shows that the migration phenotype of ∆122p53 is dependent on a secreted factor. Screening of conditioned media from ∆122p53 MEFs identified several candidate factors for the promotion of migration, including IL-6, IL-12 and CCL2, all of which were also elevated at the transcript level. Blocking antibodies to IL-6 and CCL2 reduced the migration of ∆122p53 cells, and acted co-operatively to further reduce migration. Using MEFs genetically null for IL-6, the migration of ∆122p53 MEFs was reduced in migratory assays, with a reduction in actin polarisation during migration, showing that IL-6 forms a key component of the migratory phenotype seen with ∆122p53 expression. Furthermore, differing results seen with ∆122/∆122 and ∆122/+ MEFs suggests that ∆122p53 may act co-operatively with wild type p53 to drive migratory outcomes.
Chapter 5 details the role of ∆122p53 in metastasis, and shows that ∆122p53 promotes invasion in an organotypic assay, and leads to extensive and early metastatic seeding. ∆122p53 promotes early invasion of tumour cells into associated tissue, as well as extensive lymphocytic infiltration. ∆122p53 mice on different IL-6 backgrounds have similar survival spectrums, though IL-6-/- mice developed fewer tumours than mice on an IL-6+/+ background. ∆122/∆122 IL-6-/- mice also developed fewer metastases than ∆122/∆122 IL-6+/+ mice, with fewer organs involved. This chapter showed that ∆122p53 alters the kinetics of metastasis, again a process involving IL-6 signalling. In addition, the ability of ∆122p53 to promote invasion and metastasis is reminiscent of classical p53 mutant gain-of-function activity.
Taken together, this thesis has demonstrated the parallels between the human ∆133p53 isoform and mouse ∆122p53 mutant; that ∆122p53 has the capacity to promote migration, invasion and metastasis in a similar manner to mutant p53, indicating ∆122p53 and by extension ∆133p53 have classical gain-of-function activity. These actions were found to be dependent, at least in part, on the pro-inflammatory cytokine IL-6. In sum, ∆122p53 was found to behave in a similar manner to classical gain-of-function p53 mutants and promote IL-6 dependent migration, invasion and metastasis, suggesting that targeting IL-6 may be a therapeutic strategy for patients with elevated ∆133p53 expression in cancer.