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The aggregation of p53 in response to changes in the proteostasis network

Final Report Summary - P53-AGGR-CANCER (The aggregation of p53 in response to changes in the proteostasis network)

Project Objectives:
The objective of this project is to develop a model system that allows us to study the aggregation of mutant p53 proteins in cells of various differentiation stages, and thereby to test our hypothesis and gain information on the key factors keeping p53 aggregation, and tumorigenesis, at bay.
p53 is a potent tumor suppressor, and it is the most frequently mutated gene in human cancer (approximately half of the cases). The former study on the aggregation of p53 is the first report of protein aggregation leading directly to increased cell proliferation of the same cell in which the aggregation occurs by a gain of function mechanism. Protein misfolding and subsequent aggregation had already been thought to be the main cause of neuronal death in neurodegenerative diseases, but the mentioned study has added cancer, as well, to the list of protein aggregation diseases.
The ability of the cell’s proteostasis network (PN) to cope with mutant or damaged biological material varies with the differentiation stage of cells, as well as deteriorates with cellular senescence. According to our hypothesis, the decline of the PN, particularly its decreasing ability to control aggregation-prone mutant p53 proteins, is one of the contributing factors to the steadily increasing incidence of cancer with age.
A further objective of our project is to compare the PNs of cells that are capable of suppressing p53 aggregation to cell that contain aggregated in order to determine the key factors necessary for suppression of p53 aggregation.
Work performed since the beginning of the project:
We have generated a new targeting vector for performing Flippase-mediated cassette exchange reaction in a pluripotent hESC cell line established at the Stem Cell Institute Leuven (SCIL).
In collaboration with researchers at the SCIL then we used this targeting vector to establish three hESC lines, one expressing no p53, one expressing an aggregating p53 mutant, and one expressing a non-aggregating p53 mutant.
We subjected the hESCs lines generated to differentiation protocols developed at the SCIL to produce hepatocytes and neuronal cells.
We used blue native polyacrylamide gel electrophoresis (BN-PAGE) and immunofluorescent staining to monitor aggregation status of p53 during the course of differentiations. As a control, we also monitored p53 aggregation status by BN-PAGE in the hESC lines during their culture under conditions promoting pluripotency.
We engineered targeting vectors encoding fusion proteins of the aggregating- and the non-aggregating mutant of p53 with a fluorescent protein, and established hESC lines expressing these fusion proteins.
We have set up an assay to monitor function of the p53 transgene during differentiation by treating the cells with cisplatin (a DNA-damaging drug causing p53 activation) or nutlin (a compound causing p53 activation by inhibiting the major negative regulator of p53). We assessed the increase in the expression level of p53-target genes and checked the aggregation status of p53 using BN-PAGE in response to p53 activation in undifferentiated hESCs, in hESCs at the beginning and at the end of the hepatocytic differentiation, as well as hESC at the end of the neuronal differentiation.
Main results achieved so far:
The aggregating mutant and the non-aggregating mutant of p53 showed similar electrophoretic mobility on BN-PAGE in pluripotent hESCs, confirming that undifferentiated hESCs are indeed capable of suppressing the aggregation of the aggregating mutant, as we hypothesized. High molecular weight (HMW) complexes of the aggregating p53 arise in the cells during differentiation.
A moderate amount of HMW aggregating p53 arised in hESCs a few weeks following the establishment of the cell line, as well, despite being cultured in conditions supporting pluripotency.
In order to create a way to sort live intact cells containing aggregated p53, we engineered targeting vectors encoding fusion proteins of the aggregating- and the non-aggregating mutant of p53 with a fluorescent protein, and established hESC lines expressing these fusion proteins. Unfortunately, these cell lines did not perform according to our hopes, as they both produced highly fluorescent clumpy structures during differentiation, regardless of the nature of the mutant p53 over-expressed in them, perhaps due to the fluorescent fusion partner used.
The results of the analysis of final analysis of the data is still ongoing, but it is clear so far that the two treatment have different effect on the appearance of HMW aggregating mutant p53, and they show different profiles of the activation of p53 target genes following treatment.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far):
The preliminary results are compatible with the interpretation that aggregation of p53 has a detrimental effect on the ability of the cell to mount an adequate p53 response. It also appears that nutlin, a novel p53-activating chemotherapeutic agent in clinical trials now, has much less effect in cells carrying either of the two mutant forms of p53 tried than cisplatin, an older chemotherapeutic agent. If these conclusions are confirmed, it can have an immediate impact on the choice of chemotherapeutic agent used by physicians on the short term, as well as it opens up new therapeutic opportunities based on preventing or reversing p53 aggregation on the long term. Both can have a great impact on the outcome of oncologic care.