Characterization and functional analysis of mutant p53 secretome in human cancer

Among several genetic alterations in human cancer, mutations in the TP53 tumour suppressor gene represent the most common, occurring in approximately 50% of all human cancers. The majority of these mutations in p53 are missense mutations, resulting in cancer cells expressing stable, full-length mutated p53 proteins. Missense mutant p53’s exhibit loss of tumour suppressive property of wild-type p53, dominant negative effects that can inactivate any wild-type p53 protein, and gain-of-function (GOF) properties that promote tumour progression and metastasis. Evidence suggests that cancer cells depend on the sustained expression of mutant p53 GOF. Thus, identifying the common downstream factor that drive mutant p53 GOF can provide an attractive approach to therapeutically target mutant p53 expressing tumours. This thesis presents the study of the characterization and functional analysis of mutant p53 secreted factors called “the mutant p53 secretome”. In particular, the thesis aims at identifying the critical secreted effector of mutant p53 GOF that can serve as a potential therapeutic target for treatment of mutant p53 expressing tumours. Furthermore, the thesis investigates the association of the identified factor within the secretome with clinical parameters such as patient’s survival. This thesis makes several original contributions to the field of cancer research, which are briefed below. Firstly, the mutant p53 induced secretome was characterized using quantitative proteomics of conditioned medium from mutant p53 expressing inducible H1299 human lung cancer cells. The majority of the identified secreted proteins were the transcriptional targets influenced by mutant p53. Alpha-1 antitrypsin (A1AT) was selected for further investigation, as it was the protein showing the highest expression in the mutant p53 secretome. The role of A1AT in driving the oncogenic activity of mutant p53 in human lung cancer cells was explored. A1AT was shown to drive mutant p53 induced invasion in lung cancer cell lines. Ablation of A1AT using antibodies and gene knockdown approaches inhibited the mutant p53 driven invasion, providing a rational to investigate the development of antibody-based cancer therapies that target A1AT. The clinical association of A1AT was further investigated in tissue microarray (TMA) samples of lung adenocarcinoma (ADC) patients. Mutant p53 expression was shown to correlate with A1AT, which validates in vivo that A1AT is a bonafide target of mutant p53. Furthermore, elevated expression of A1AT was demonstrated to correlate with increased local invasion and poor prognosis of lung ADC patients. Mutant p53 is reported to function as an aberrant transcription factor that can interact with other transcription factors to reprogram the cellular transcriptome of cancer cells. The mechanism of regulation of A1AT by mutant p53 was confirmed to involve p63. The role of A1AT in driving the mutant p53 induced invasive behavior of breast cancer cells was also explored, and a relationship of A1AT with p53 status and with different subtypes of breast cancer was established. In p53 mutant basal-like subtypes, A1AT expression was shown to drive invasion and treatment with anti-A1AT antibodies inhibited invasion. This suggests that the A1AT-targeted are potential therapies in various cancer types and its regulation in breast cancer may also extend beyond p53. Collectively, these studies provide new insights into the invasive behavior of mutant p53 that are manifested through aberrant secretion of extracellular proteins. The identification of A1AT as a critical and indispensable effector of mutant p53 gain-of-function offers a new therapeutic options for treatment of p53 mutant tumours. The findings in this thesis involve significant elements of novelty describing how mutant p53 influences the cellular secretome.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2016

Impact of pre-therapy glioblastoma multiforme microenvironment on clinical response to autologous CMV-specific T-cell therapy

Objectives: Clinical response to antibody-based immunotherapies targeting checkpoint inhibitors is critically dependent on the tumor immune microenvironment (TIME).\ua0However, the precise impact of the TIME\ua0on adoptive cellular immunotherapy remains unexplored. Here we have conducted a long-term follow-up analysis of patients with recurrent glioblastoma multiforme (GBM) who were treated with autologous CMV-specific T-cell therapy to delineate the potential impact of the TIME on their clinical response. Methods: Multiplexed immunohistochemical analysis of CD3, PD-L1 and Sox-2 in GBM tissue biopsies obtained before autologous T-cell therapy was carried out and correlated with long-term survival of GBM patients adoptively treated with T-cell therapy. Results: Tumor microenvironment analyses revealed that the pre-treatment cellular composition of the tumor tissue may influence the subsequent response to adoptive T-cell therapy. GBM patients who showed prolonged overall survival following T-cell therapy had a significantly lower number of tumor-infiltrating CD3 T cells in recurrent tumors than that in patients with short-term survival. Furthermore, long-term surviving patients showed low or undetectable PD-L1 expression in tumor cells in recurrent GBM biopsies. Conclusion: We hypothesise that lack of PD-L1-mediated immunosuppression in the TIME may allow efficient immune control following adoptive T-cell therapy. Future studies combining anti-PD-L1 or genetically modified T cells with PD-1 receptor knockdown could be considered to improve clinical responses in patients who have high PD-L1 expression in their tumors