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

The cartographic methods of presentation and GIS tool in analysis of historical data on the example of Great War cemeteries in southern Poland

The paper deals with the application of Geographic Information System software in cartographic data presentation in the field of historical data mining. Lists of soldiers buried in the I world war cemeteries near Jasło were used. The prepared database helped to create series of maps, mainly diagram maps, that serve as a useful statistical and demographic characteristics of the phenomena. The authors stressed advantages of the solution used and the usefulness of historical data in spatial database preparation process. The shortages of the GIS software itself were also pointed out, especially as long as cartographic editing and methodology principles are concerned

Mutant p53–Nrf2 axis regulates the proteasome machinery in cancer

The proteasome machinery is a common target of gain-of-function p53 missense mutants. Upregulation of the proteasome fosters chemoresistance to proteasome inhibitors. In triple negative breast cancer cells this resistance mechanism, namely the Nrf2-regulated “bounce-back” response to proteasome inhibitors, can be overcome by targeting p53 mutant proteins with APR-246/PRIMA-1Met

Nuclear architecture dictates HIV-1 integration site selection

International audienceLong-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle

Mechanical cues control mutant p53 stability through a mevalonate-RhoA axis

Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, metastatic potential and drug resistance (gain-of-function)1. Mutant p53 protein stabilization is a prerequisite for gain-of-function manifestation; however, it does not represent an intrinsic property of p53 mutants, but rather requires secondary events2. Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours2,3. By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-mediated proteolysis, we found that HDAC6/Hsp90-dependent mutant p53 accumulation is sustained by RhoA geranylgeranylation downstream of the mevalonate pathway, as well as by RhoA- and actin-dependent transduction of mechanical inputs, such as the stiffness of the extracellular environment. Our results provide evidence for an unpredicted layer of mutant p53 regulation that relies on metabolic and mechanical cues

Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer

In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53-proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP-microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53