Activation of the proteasome machinery by NRF2/mutant p53 axis and its therapeutic implications for triple negative breast cancer

TP53 is the most frequently mutated gene in human cancers. Great majority of these nucleotide changes are point mutations that occur within the DNA binding domain of p53. Point mutations of TP53 have been established to contribute to carcinogenesis by losing the tumour suppressor activities of the wild type, exerting dominant negative effects over the wild type allele and arming the mutant p53 with novel oncogenic gain-of-function (GOF) properties. Oncogenic mechanisms promoted by mutant p53 include: EMT, migration/invasion, tumour growth rate, apoptosis and chemoresistance. The study focuses on Triple-Negative Breast Cancers: model of an aggressive group of tumors in which the mutation rate in TP53 loci is over 50%. These tumours are prone to recurrence, metastasis, become resistant to chemotherapy and currently lack targeted therapies - therefore finding novel targets for possible treatment is crucial. In this thesis I describe the finding of a common pathway regulated in the mutant p53 dependent manner among different TNBC subtypes, represented by 5 TNBC cell lines with distinct TP53 point mutations. An overlap of mutant p53-depnedent transcriptome in the mentioned cell lines as well as a comparison of proteome, transcriptome and DNA-interactome (ChIP-seq) in the leading cell line of the study (MDA-MB-231), revealed both the 26S proteasome-ubiquitin pathway as the outstanding, conserved mutant p53-upregulated process. I show how the pathway is regulated at the transcriptional level by the cooperation of mutant p53 with transcription factors (TFs) among which NRF2 (NFE2L2), the master regulator of oxidative stress response was discovered as the common interactor for both structural and contact p53 mutant variants. Further validation, followed by functional and association studies revealed that p53 mutant variants together with NRF2 control the 26S proteasome level and activity. Moreover I found that the binding p53 mutants to the proteasome encoding genes promoters is facilitated by NRF2 \u2013 while wild type p53 counterpart did not possess this ability. Mutant p53 impact on proteasome genes transcription and proteasome activity was not dependent on other TFs that were known to regulate the expression of proteasome genes and activity (NRF1, STAT3, NF-YA, NF-kB). Upregulation of proteasome activity by p53 GOF mutants allows the TNBC cells to foster the chemoresistance to proteasome inhibitors. Aiming the p53 mutant proteins, either with siRNA or mutant p53 targeting drugs (APR-246) gives the opportunity to blunt the activity of an NRF2-mediated mechanism of an induced transcription of proteasome encoding genes in response to the treatment with proteasome inhibitors - known as the \u201cbounce back response\u201d. In summary, results of this thesis suggest that the combination of mutant p53 targeting drugs together with proteasome inhibitors might be a promising therapeutic solution tailored for treating TNBC and other tumours where p53 is mutated