Claudia Vitória de Moura Gallo

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils IMPLICATIONS FOR CANCER

Background: p53 function is lost in more than 50% of tumors. Results: p53 aggregates into amyloid oligomers and fibrils in vitro and in breast cancer tissues; mutant p53 seeds amyloid aggregation of WT p53, a behavior typical of a prion. Conclusion: Prion-like aggregation is crucial for the negative dominance of mutant p53. Significance: The inhibition of aggregation could be a target for cancer therapy. 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 β-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.

Cross‐talks between cyclooxygenase‐2 and tumor suppressor protein p53: Balancing life and death during inflammatory stress and carcinogenesis

Overexpression of Cyclooxygenase‐2 (COX‐2) is observed in most tumor types. Increased COX‐2 activity and synthesis of prostaglandins stimulates proliferation, angiogenesis, invasiveness and inhibits apoptosis. Many stress and proinflammatory signals induce COX‐2 expression, including oxyradicals or DNA‐damaging agents. The latter also induces p53, a transcription factor often inactivated by mutation in cancer. Several studies have identified complex cross‐talks between p53 and COX‐2, whereby p53 can either up‐ or down‐regulate COX‐2, which in turn controls p53 transcriptional activity. However, the molecular basis of these effects are open to debate, in particular since no p53 binding sequences have been identified in COX‐2 regulatory regions. In this review, we summarize the molecular mechanisms by which COX‐2 contributes to carcinogenesis and discuss the experimental set‐up, results and conclusions of studies analyzing cross‐talks between p53 and COX‐2. We propose 2 scenarios accounting for overexpression of COX‐2 in precursor and cancer lesions. In the “inflammatory” scenario, p53, activated by DNA damage induced by oxygen and nitrogen species, recruits NF‐kappaB to activate COX‐2, resulting in antiapoptotic effects that contribute to cell expansion in inflammatory precursor lesions. In the “constitutive proliferation” scenario, oncogenic stress due to activation of growth signaling cascades may upregulate COX‐2 promoter independently of NF‐kappaB and p53, synergizing with TP53 mutation to promote cancer progression. These 2 scenarios, although not mutually exclusive, may account for the diversity of the correlations between COX‐2 expression and TP53 mutation, which vary according to cancer types and biological contexts, and have implications for the use of COX‐2 inhibitors in cancer prevention and therapy.

CYP1A1, GSTM1, and GSTT1 polymorphisms and breast cancer risk in Brazilian women

The frequency of CYP1A1 (CYP1A1*2A), GSTM1, and GSTT1 polymorphisms, as well as the main risk factors associated with breast cancer were studied in Brazilian women, with malignant breast cancer (n=128), or age-matched controls (n=256). Only a family history of breast cancer presented a significant risk (OR=3.00, CI=1.27-7.06). Among non-whites, the CYP1A1*2A allele was underrepresented among patients. Statistical analysis indicated that this polymorphism may decrease the risk of breast cancer among these individuals, particularly after adjusting for the risk presented by selected risk factors (OR=0.30, 95% CI=0.12-0.76).

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.

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.

Interferon-inducible guanylate binding protein (GBP)-2: A novel p53-regulated tumor marker in esophageal squamous cell carcinomas

TP53 mutations are common in esophageal squamous cell carcinomas (SCC). To identify biological markers of possible relevance in esophageal SCC, we (i) searched for genes expressed in a p53‐dependent manner in TE‐1, an esophageal SCC cell line expressing the temperature‐sensitive TP53 mutant V272M, and (ii) investigated the expression of one of those genes, the interferon‐inducible Guanylate Binding Protein 2 (GBP‐2), in esophageal SCC tissues. Clontech Human Cancer 1.2 arrays containing 1,176 human cancer gene‐related sequences were used to identify differentially expressed genes in TE‐1 cells at permissive (32°C) and nonpermissive (37°C) temperatures. The expression of GBP‐2 and IRF‐1, its main transcriptional regulator, was analyzed by immunohistochemistry in a retrospective series of 41 esophageal SCC cases with a clear transition zone from noncancer, apparently normal epithelium to invasive cancer. The expression of the GBP‐2 gene is consistently increased in TE‐1 at 32°C in a p53‐dependent manner, as confirmed by inhibition of p53 expression by RNA interference. Increase in GBP‐2 is accompanied by an increase in protein levels of IRF‐1, the main transcriptional regulator of GBP‐2, and in the formation of complexes between p53 and IRF‐1. GBP‐2 expression is significantly higher in esophageal SCC than in adjacent normal epithelium (p < 0.01), in which GBP‐2 staining is limited to the basal layer. Our results suggest that p53 up‐regulates GBP‐2 by cooperating with IRF‐1. The association of GBP‐2 expression with proliferative squamous cells suggests that GBP‐2 may represent a marker of interest in esophageal SCC.

Part III. Molecular changes induced by high nitric oxide adaptation in human breast cancer cell line BT-20 (BT-20-HNO): a switch from aerobic to anaerobic metabolism

Nutrient deprivation and reactive oxygen species (ROS) play an important role in breast cancer mitochondrial adaptation. Adaptations to these conditions allow cells to survive in the stressful microenvironment of the tumor bed. This study is directed at defining the consequences of High Nitric Oxide (HNO) exposure to mitochondria in human breast cancer cells. The breast cancer cell line BT-20 (parent) was adapted to HNO as previously reported, resulting in the BT-20-HNO cell line. Both cell lines were analyzed by a variety of methods including MTT, LDH leakage assay, DNA sequencing, and Western blot analysis. The LDH assay and the gene chip data showed that BT-20-HNO was more prone to use the glycolytic pathway than the parent cell line. The BT-20-HNO cells were also more resistant to the apoptotic inducing agent salinomycin, which suggests that p53 may be mutated in these cells. Polymerase chain reaction (PCR) followed by DNA sequencing of the p53 gene showed that it was, in fact, mutated at the DNA-binding site (L194F). Western blot analysis showed that p53 was significantly upregulated in these cells. These results suggest that free radicals, such as nitric oxide (NO), pressure human breast tumor cells to acquire an aggressive phenotype and resistance to apoptosis. These data collectively provide a mechanism by which the dysregulation of ROS in the mitochondria of breast cancer cells can result in DNA damage.

XRCC1 gene polymorphisms in a population sample and in women with a family history of breast cancer from Rio de Janeiro (Brazil)

The X-ray repair cross-complementing Group1 (XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes. This gene is highly polymorphic, and the most extensively studied genetic changes are in exon 6 (Arg194Trp) and in exon 10 (Arg399Gln). These changes, in conserved protein sites, may alter the base excision repair capacity, increasing the susceptibility to adverse health conditions, including cancer. In the present study, we estimated the frequencies of the XRCC1 gene polymorphisms Arg194Trp and Arg399Gln in healthy individuals and also in women at risk of breast cancer due to family history from Rio de Janeiro. The common genotypes in both positions (194 and 399) were the most frequent in this Brazilian sample. Although the 194Trp variant was overrepresented in women reporting familial cases of breast cancer, no statistically significant differences concerning genotype distribution or intragenic interactions were found between this group and the controls. Thus, in the population analyzed by us, variants Arg194Trp and Arg399Gln did not appear to have any impact on breast cancer susceptibility.

Potential Correlation between Tumor Aggressiveness and Protein Expression Patterns of Nipple Aspirate Fluid (NAF) Revealed by Gel-Based Proteomic Analysis

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

TP53 and XRCC1 polymorphisms and breast cancer prognosis: a case-case study

XRCC1 is important in the base excision repair process(BER). The gene has two common polymorphisms in codons194 (Arg194Trp) and 399 (Arg399Gln) that affect the aminoacid sequence. Codon 194 is located in the linker region thatconnects the domains that interact with poly(ADP-ribose)polymerase (PARP) and DNA polymerase b.