Aggregation as a mechanism for p53 oncogenic transformation: clinical relevance and new tools for its study

The p53 tumor suppressor is one of the most frequently mutated genes in human cancers, up to 50% of all cancers. This event has three different consequences: (i) Loss-of- function, (ii) Dominant-negative activity, i.e. reduced amount of active p53 due to the incorporation of wild-type and mutant monomers into the mixed tetramer, and (iii) Gain-of-function, that is, the ability of mutant p53 to interact with other proteins and inactivate them. It was recently discovered that protein aggregation explains the gain- of-function activity of mutant p53, through the exposure of an aggregation-prone region (APR) within the protein. Nonetheless, the clinical impact of p53 aggregation remains largely unknown. In this thesis, I investigated p53 aggregation phenotypes in tumor biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. P53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system. Colon cancer patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyzes proteostatic dysregulation. Since the presence of p53 nIBs seems to be a relatively frequent phenotype in the clinic, I next investigated Patient-Derived tumor Xenografts (PDXs) as an in vivo model to study p53 aggregation in cancer. A first exploratory screening of 99 PDXs revealed p53 nIBs in 5.05% of the samples analyzed, encompassing three different tumor types (melanoma, breast and uterine leiomyosarcoma). Similar to the observations in patients, the presence of nIBs occurs in both wild-type and mutant p53 samples. This preliminary screening serves as a starting point for the future characterization and usefulness of PDXs to study the causality of p53 aggregation and to interrogate the response to therapies targeting this phenotype. In addition, I developed a new tool, so called p53 gammabody (P53Gb), by grafting the p53 APR into a single VH antibody domain. P53Gb captures p53 aggregates in a transient overexpression system, revealed by the binding to structurally destabilized mutants and not to well-folded contact mutants or to p53 wild-type, suggestive of conformational sequence specificity. Even though interaction was not evident in p53 endogenous cell lines, toxicity was apparent in those tumor cells depending on the presence of aggregated p53 for their survival. This suggests a new potential therapeutic approach based on APR-APR specificity by grafting APRs into single VH domains that will be further investigated. Overall, this thesis highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration, as well as, the relevance of p53 aggregation in cancer and new tools to further study this phenotype.

Jaar van publicatie:2017

Toegankelijkheid:Closed