Epigenetic modifications, chromatin distribution and TP53 transcription in a model of breast cancer progression.

In the present paper we aimed to characterize epigenetic aspects and analyze TP53 transcription in the 21 T series, composed of breast cell lines: non-cancerous H16N2; Atypical Ductal Hyperplasia 21PT; Ductal Carcinoma in situ 21NT and Invasive Metastatic Carcinoma 21MT1. We detected a global genomic hypomethylation in 21NT and 21MT1. The histone modification markers analysis showed an important global decrease of the active chromatin mark H4Ac in 21MT1 relative to the other cell lines while the repressive mark H3K9Me3 were not significantly altered. The mRNA levels of DNA methylation and histone modification key enzymes are consistent with the observed genomic hypomethylation and histone hypoacetylation. The expression of DNMT3A/B increased at the initial stages of oncogenesis and the expression of DNMT1 and HAT1 decreased at the advanced stages of breast cancer. Using a confocal immunofluorescent assay, we observed that H4Ac was mostly located at the periphery and the repressive mark H3K9Me3, at the center of 21NT and 21MT1 cells nuclei. TP53 P1 promoter was found to be in an open chromatin state, with a relatively high enrichment of H4Ac and similar TP53 transcription levels in all 21 T cell lines. In conclusion, we observed epigenetic alterations (global genome hypomethylation, global hypoacetylation and accumulation of pericentric heterochromatin) in metastatic breast cancer cells of the 21 T series. These alterations may act at later stages of breast cancer progression and may not affect TP53 transcription at the P1 promoter.

Prion-like aggregation of mutant p53 in cancer.

p53 is a master regulatory protein that participates in cellular processes such as apoptosis, DNA repair, and cell cycle control. p53 functions as a homotetrameric tumor suppressor, and is lost in more than 50% of human cancers. Recent studies have suggested that the formation of mutant p53 aggregates is associated with loss-of-function (LoF), dominant-negative (DN), and gain-of-function (GoF) effects. We propose that these phenomena can be explained by a prion-like behavior of mutant p53. We discuss the shared properties of cancer and neurodegenerative diseases and how the prion-like properties of p53 aggregates offer potential targets for drug development.

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor.

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.

Mutant p53 aggregates into prion-like amyloid oligomers and fibrils: implications for cancer.

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross ß-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.

Loop domain organization of the p53 locus in normal and breast cancer cells correlates with the transcriptional status of the TP53 and the neighboring genes.

P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines. We have found that in the control breast epithelial cell line, HB2, the TP53 gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231, and BT474, in which the domain size was increased and TP53 transcription was decreased. We propose a model in which the organization of the TP53 gene domain correlates with the transcriptional status of TP53 and neighboring genes.

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.

Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation.

The tumor suppressor protein p53 is a nuclear protein that serves as an important transcription factor. The region responsible for sequence-specific DNA interaction is located in its core domain (p53C). Although full-length p53 binds to DNA as a tetramer, p53C binds as a monomer since it lacks the oligomerization domain. It has been previously demonstrated that two core domains have a dimerization interface and undergo conformational change when bound to DNA. Here we demonstrate that the interaction with a consensus DNA sequence provides the core domain of p53 with enhanced conformational stability at physiological salt concentrations (0.15 M). This stability could be either increased or abolished at low (0.01 M) or high (0.3 M) salt concentrations, respectively. In addition, interaction with the cognate sequence prevents aggregation of p53C into an amyloid-like structure, whereas binding to a nonconsensus DNA sequence has no effect on p53C stability, even at low ionic strength. Strikingly, sequence-specific DNA binding also resulted in a large stabilization of full-length p53, whereas nonspecific sequence binding led to no stabilization. The effects of cognate DNA could be mimicked by high concentrations of osmolytes such as glycerol, which implies that the stabilization is caused by the exclusion of water. Taken together, our results show an enhancement in protein stability driven by specific DNA recognition. When cognate DNA was added to misfolded protein obtained after a pressurization cycle, the original conformation was mostly recovered. Our results may aid the development of therapeutic approaches to prevent misfolded species of p53.

TP53 mutations in breast cancer tumors of patients from Rio de Janeiro, Brazil: association with risk factors and tumor characteristics.

Somatic mutations in the TP53 gene are the most frequently observed genetic alterations in human malignancies, including breast cancer, which is one of the leading causes of death among women in Brazil. In our study, we determined the frequency and the pattern of TP53 mutations in malignant breast tumors from 120 patients living in Rio de Janeiro, Brazil. TP53 mutations were found in 20% of the tumors, which contained a diversity of mutation types: missense (62.5%), nonsense (8.3%), silent (4.2%), intronic (12.5%), insertion (4.2%) and deletion (8.3%). Of a total of 15 missense mutations, 4 were observed at Arg248 and 2 at Cys242, which are directly involved in DNA binding and in zinc binding, respectively. A subgroup of 51 patients was analyzed with respect to the relation between the presence of TP53 mutations and classical risk factors and with tumor and patient characteristics. For this analysis, we used logistic regression and, in order to obtain more precise confidence intervals, they were recalculated using a bootstrap resampling technique. Our results demonstrate that these mutations are not statistically associated with the risk factors or the patients' characteristics. However, the presence of TP53 mutations is strongly associated with the aggressiveness of the tumors, measured by Elston classification (OR = 11.97 and 95% CI of 2.24-307.05). This finding is in agreement with previous studies, which report the presence of TP53 mutations in tumors with poor prognosis. This correlation between tumor aggressiveness and TP53 mutations could be a crucial variable for the treatment and prognosis of breast cancer.