p53 is a well-known tumor suppressor that is mutated in over 50% of human cancers. These mutations were shown to exhibit gain of oncogenic function compared with the deletion of the gene. Additionally, p53 has fundamental roles in differentiation and development; nevertheless, mutant p53 mice are viable and develop malignant tumors only on adulthood. We set out to reveal the mechanisms by which embryos are protected from mutant p53-induced transformation using ES cells (ESCs) that express a conformational mutant of p53. We found that, despite harboring mutant p53, the ESCs remain pluripotent and benign and have relatively normal karyotype compared with ESCs knocked out for p53. Additionally, using high-content RNA sequencing, we show that p53 is transcriptionally active in response to DNA damage in mutant ESCs and elevates p53 target genes, such as p21 and btg2. We also show that the conformation of mutant p53 protein in ESCs is stabilized to a WT conformation. Through MS-based interactome analyses, we identified a network of proteins, including the CCT complex, USP7, Aurora kinase, Nedd4, and Trim24, that bind mutant p53 and may shift its conformation to a WT form. We propose this conformational shift as a novel mechanism of maintenance of genomic integrity, despite p53 mutation. Harnessing the ability of these protein interactors to transform the oncogenic mutant p53 to the tumor suppressor WT form can be the basis for future development of p53-targeted cancer therapy.T he tumor protein 53 (p53) transcription factor (encoded by the human gene TP53/TRP53) is a key tumor suppressor and a master regulator of genomic stability, cell cycle, DNA repair, senescence, and apoptosis (1). p53 function is frequently compromised during tumorigenesis, usually as a result of somatic mutations, which occur in more than 50% of human cancers (2). These mutants were shown to exhibit gain of oncogenic functions in addition to the loss of WT activity, leading to an aggressive malignant phenotype (2). Most TP53 mutations can be classified into two main categories: DNA contact and conformational mutations. The first group is composed of mutations in residues that directly bind the DNA, the second group of mutations causes distortion of the core domain folding and inhibits p53 from binding the DNA and transactivating its target genes. These mutations affect p53 conformation in a dynamic fashion, which at least partially depends on its binding partners in a cell context-dependent manner (3).Over the years, researchers have developed several mouse models as tools for investigating p53, including p53 KO mice (4) and mice knocked in for mutant p53 (Mut) (5, 6). These models showed the role of p53 as a regulator of developmental and differentiation processes. For instance, p53 KO mice were found to display developmental abnormalities, such as upper incisor fusion, ocular abnormalities, polydactyly of the hind limbs, and exencephaly (7). On the cellular level, ES cells (ESCs) were found to express high levels of p53 mRNA and protein, which...
Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.
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