Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition.
Ontology highlight
ABSTRACT:
SUBMITTER: Wilcken R
PROVIDER: S-EPMC3427094 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a β-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.
| S-EPMC6255152 | biostudies-literature
Project description:Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.
| S-EPMC5423176 | biostudies-literature
Project description:Protein misfolding and aggregation contributes to the development of a wide range of diseases. In cancer, over 50% of diagnoses are attributed to p53 malfunction due to missense mutations, many of which result in protein misfolding and accelerated aggregation. p53 mutations also frequently result in alteration or loss of zinc at the DNA-binding site, which increases aggregation via nucleation with zinc-bound p53. Herein, we designed two novel bifunctional ligands, LI and LH , to modulate mutant p53 aggregation and restore zinc binding using a metallochaperone approach. Interestingly, only the incorporation of iodine function in LI resulted in modulation of mutant p53 aggregation, both in recombinant and cellular environments. Native mass spectrometry shows a protein-ligand interaction for LI , as opposed to LH , which is hypothesized to lead to the distinct difference in the p53 aggregation profile for the two ligands. Incorporation of a di-2-picolylamine binding unit into the ligand design provided efficient intracellular zinc uptake, resulting in metallochaperone capability for both LI and LH . The ability of LI to reduce mutant p53 aggregation results in increased restoration of p53 transcriptional function and mediates both caspase-dependent and -independent cell death pathways. We further demonstrate that LI exhibits minimal toxicity in non-cancerous organoids, and that it is well tolerated in mice. These results demonstrate that iodination of our ligand framework restores p53 function by interacting with and inhibiting mutant p53 aggregation and highlights LI as a suitable candidate for comprehensive in vivo anticancer preclinical evaluations.
| S-EPMC7006507 | biostudies-literature
Project description:The metabolic function of p53 is important for its oncosuppressive function. Mutant p53 (mutp53) with gain of oncogenic function can regulate cell metabolism. Our recent study revealed a novel transcription-independent mechanism for a gain-of-function mutp53 that directly inhibits activation of adenosine monophosphate-activated protein kinase (AMPK) to promote cancer cell metabolism.
| S-EPMC4904903 | biostudies-other
Project description:The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.
| S-EPMC9352912 | biostudies-literature
Project description:To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6?kcal?mol(-1) (15.1?kJ?mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the ?-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.
| S-EPMC3792646 | biostudies-literature
Project description:Somatic mutations in p53, which inactivate the tumour-suppressor function of p53 and often confer oncogenic gain-of-function properties, are very common in cancer1,2. Here we studied the effects of hotspot gain-of-function mutations in Trp53 (the gene that encodes p53 in mice) in mouse models of WNT-driven intestinal cancer caused by Csnk1a1 deletion3,4 or ApcMin mutation5. Cancer in these models is known to be facilitated by loss of p533,6. We found that mutant versions of p53 had contrasting effects in different segments of the gut: in the distal gut, mutant p53 had the expected oncogenic effect; however, in the proximal gut and in tumour organoids it had a pronounced tumour-suppressive effect. In the tumour-suppressive mode, mutant p53 eliminated dysplasia and tumorigenesis in Csnk1a1-deficient and ApcMin/+ mice, and promoted normal growth and differentiation of tumour organoids derived from these mice. In these settings, mutant p53 was more effective than wild-type p53 at inhibiting tumour formation. Mechanistically, the tumour-suppressive effects of mutant p53 were driven by disruption of the WNT pathway, through preventing the binding of TCF4 to chromatin. Notably, this tumour-suppressive effect was completely abolished by the gut microbiome. Moreover, a single metabolite derived from the gut microbiota-gallic acid-could reproduce the entire effect of the microbiome. Supplementing gut-sterilized p53-mutant mice and p53-mutant organoids with gallic acid reinstated the TCF4-chromatin interaction and the hyperactivation of WNT, thus conferring a malignant phenotype to the organoids and throughout the gut. Our study demonstrates the substantial plasticity of a cancer mutation and highlights the role of the microenvironment in determining its functional outcome.
| S-EPMC7116712 | biostudies-literature
Project description:The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.
| S-EPMC7030899 | biostudies-literature
Project description:The tumor suppressor protein p53 is inactivated in the majority of human cancers and remains a prime target for developing new drugs to reactivate its tumor suppressing activity for anticancer therapies. The oncogenic p53 mutant Y220C accounts for approximately 125,000 new cancer cases per annum and is one of the most prevalent p53 mutants overall. It harbors a narrow, mutationally induced pocket at the surface of the DNA-binding domain that destabilizes p53, leading to its rapid denaturation and aggregation. Here, we present the structure-guided development of high-affinity small molecules stabilizing p53-Y220C in vitro, along with the synthetic routes developed in the process, in vitro structure-activity relationship data, and confirmation of their binding mode by protein X-ray crystallography. We disclose two new chemical probes displaying sub-micromolar binding affinity in vitro, marking an important milestone since the discovery of the first small-molecule ligand of Y220C in 2008. New chemical probe JC744 displayed a K d = 320 nM, along with potent in vitro protein stabilization. This study, therefore, represents a significant advance toward high-affinity Y220C ligands for clinical evaluation.
| S-EPMC9667543 | biostudies-literature
Project description:Mutant p53 tends to form aggregates with amyloid properties, especially amyloid oligomers inside the nucleus, which are believed to cause oncogenic gain-of-function (GoF). The mechanism of the formation of the aggregates in the nucleus remains uncertain. The present study demonstrated that the DNA-binding domain of p53 (p53C) underwent phase separation (PS) on the pathway to aggregation under various conditions. p53C phase separated in the presence of the crowding agent polyethylene glycol (PEG). Similarly, mutant p53C (M237I and R249S) underwent PS; however, the process evolved to a solid-like phase transition faster than that in the case of wild-type p53C. The data obtained by microscopy of live cells indicated that transfection of mutant full-length p53 into the cells tended to result in PS and phase transition (PT) in the nuclear compartments, which are likely the cause of the GoF effects. Fluorescence recovery after photobleaching (FRAP) experiments revealed liquid characteristics of the condensates in the nucleus. Mutant p53 tended to undergo gel- and solid-like phase transitions in the nucleus and in nuclear bodies demonstrated by slow and incomplete recovery of fluorescence after photobleaching. Polyanions, such as heparin and RNA, were able to modulate PS and PT in vitro. Heparin apparently stabilized the condensates in a gel-like state, and RNA apparently induced a solid-like state of the protein even in the absence of PEG. Conditions that destabilize p53C into a molten globule conformation also produced liquid droplets in the absence of crowding. The disordered transactivation domain (TAD) modulated both phase separation and amyloid aggregation. In summary, our data provide mechanistic insight into the formation of p53 condensates and conditions that may result in the formation of aggregated structures, such as mutant amyloid oligomers, in cancer. The pathway of mutant p53 from liquid droplets to gel-like and solid-like (amyloid) species may be a suitable target for anticancer therapy.
| S-EPMC8171334 | biostudies-literature