Research IndicatorsGraph generated 16 March 2015 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 16 March, 2015 using data from PubMed, MeSH and CancerIndex
Specific Cancers (5)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
TICdb, Universidad de Navarra
Search the database of Translocation breakpoints In Cancer for "ZNF331"
Search the Epigenomics database and view relevant gene tracks of samples.
Latest Publications: ZNF331 (cancer-related)
Fiorini C, Cordani M, Padroni C, et al.Mutant p53 stimulates chemoresistance of pancreatic adenocarcinoma cells to gemcitabine.
Biochim Biophys Acta. 2015; 1853(1):89-100 [PubMed
] Related Publications
Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths worldwide; PDAC is characterized by poor prognosis, resistance to conventional chemotherapy and high mortality rate. TP53 tumor suppressor gene is frequently mutated in PDAC, resulting in the accumulation of mutated protein with potential gain-of-function (GOF) activities, such as genomic instability, hyperproliferation and chemoresistance. The purpose of this study was to assess the relevance of the p53 status on the PDAC cells response to the standard drug gemcitabine. We also examined the potential therapeutic effect of p53-reactivating molecules to restore the mutant p53 function in GEM treated PDAC cells. We showed that gemcitabine stabilized mutant p53 protein in the nuclei and induced chemoresistance, concurrent with the mutant p53-dependent expression of Cdk1 and CCNB1 genes, resulting in a hyperproliferation effect. Despite the adverse activation of mutant p53 by gemcitabine, simultaneous treatment of PDAC cells with gemcitabine and p53-reactivating molecules (CP-31398 and RITA) reduced growth rate and induced apoptosis. This synergistic effect was observed in both wild-type and mutant p53 cell lines and was absent in p53-null cells. The combination drug treatment induced p53 phosphorylation on Ser15, apoptosis and autophagosome formation. Furthermore, pharmacological inhibition of autophagy further increased apoptosis stimulated by gemcitabine/CP-31398 treatment. Together, our results show that gemcitabine aberrantly stimulates mutant p53 activity in PDAC cells identifying key processes with potential for therapeutic targeting. Our data also support an anti-tumoral strategy based on inhibition of autophagy combined with p53 activation and standard chemotherapy for both wild-type and mutant p53 expressing PDACs.
We have previously shown that gastrointestinal cancers display similar epigenetic aberrations. In a recent study, we identified frequently methylated genes for cholangiocarcinoma (CDO1, DCLK1, SFRP1 and ZSCAN18), where one of these genes, DCLK1, was also confirmed to be highly methylated in colorectal cancer. The aim of the present study was to determine whether these four genes, in addition to one gene found to be methylated in colon cancer cell lines (ZNF331), are commonly methylated across gastrointestinal malignancies, as well as explore their role as potential biomarkers. Quantitative methylation specific PCR (qMSP) of colorectal cancer (n=164) and normal colorectal mucosa (n=106) samples showed that all genes were frequently methylated in colorectal cancer (71-92%) with little or no methylation in normal mucosa (0-3%). Methylation of minimum two of these five genes identified 95% of the tumors with a specificity of 98%, and an area under the receiver operating characteristics curve (AUC) of 0.98. For gastric (n=25) and pancreatic (n=20) cancer, the same panel detected 92% and 90% of the tumors, respectively. Seventy-four cancer cell lines were further analyzed by qMSP and real time RT-PCR. In addition to the previously reported DCLK1, a high negative correlation between promoter DNA methylation and gene expression was observed for CDO1, ZNF331 and ZSCAN18. In conclusion, the high methylation frequency of these genes in colorectal- as well as in gastric-, pancreatic- and bile duct cancer confirmed an epigenetic similarity between gastrointestinal cancer types, and simultaneously demonstrated their potential as biomarkers, particularly for colorectal cancer detection.
BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells.
METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines.
RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%).
CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.
Kupčinskaitė-Noreikienė R, Skiecevičienė J, Jonaitis L, et al.CpG island methylation of the MLH1, MGMT, DAPK, and CASP8 genes in cancerous and adjacent noncancerous stomach tissues.
Medicina (Kaunas). 2013; 49(8):361-6 [PubMed
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BACKGROUND AND OBJECTIVE. Many factors are involved in the development of gastric adenocarcinoma. The CpG island methylation of apoptosis and mismatch repair genes by the loss of their function is important in gastric adenocarcinoma. The aim of this study was to determine the methylation frequency of MLH1, MGMT, CASP8, and DAPK in cancerous and adjacent noncancerous stomach tissues, to determine possible associations with the selected clinicopathological characteristics, and to identify possible correlation between the methylation of individual genes. MATERIAL AND METHODS. The methylation status of MLH1, MGMT, DAPK, and CASP8 was investigated in 69 patients with gastric adenocarcinoma by using methylation-specific polymerase chain reaction. The associations between patients' clinical characteristics and methylation status were assessed. RESULTS. The methylation frequency of the MLH1, DAPK, MGMT, and CASP8 gene promoters in cancerous and adjacent noncancerous tissues was 31.9% and 27.5%; 47.8% and 46.4%; 36.2% and 44.9%; and 5.8% and 5.8%, respectively, but the differences were not significant. There was no significant association between the methylation status of the mentioned genes and clinicopathological characteristics, such as age, sex, tumor type by the Lauren classification, degree of differentiation G, and TNM staging. An inverse correlation between the methylation of the DAPK and MLH1 gene promoters in cancerous and surrounding noncancerous tissues was found. CONCLUSIONS. The methylation of the MLH1, MGMT, DAPK, and CASP8 genes was found to occur both in cancerous and noncancerous stomach tissues. These findings provide additional insights into gene methylation patterns in gastric adenocarcinoma.
Failli A, Legitimo A, Migheli F, et al.Efficacy and feasibility of the epithelial cell adhesion molecule (EpCAM) immunomagnetic cell sorter for studies of DNA methylation in colorectal cancer.
Int J Mol Sci. 2014; 15(1):44-57 [PubMed
] Free Access to Full Article Related Publications
The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.
Wang H, Chen G, Wang H, Liu CRITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis.
Oncol Res. 2013; 20(10):437-45 [PubMed
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RBP-J-interacting and tubulin-associated (RITA) is a novel RBP-J-interacting protein that downregulates Notch-mediated transcription. The current study focuses on the antitumor effect of RITA in human hepatocellular carcinoma (HCC) and aims to explore its molecular mechanism. Thirty paired HCC and adjacent non-tumoral liver samples were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RITA overexpression was induced by transfection of a pcDNA3.1-Flag-RITA plasmid into HepG2 cells. RITA knockdown was achieved by siRNA transfection. mRNA and protein expression of target genes were quantified by qRT-PCR and Western blotting, respectively. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry. Our results demonstrate that adjacent nontumoral liver samples exhibited increased RITA expression compared to HCC tissues (p < 0.05); RITA levels were associated with tumor differentiation status. Overexpression of RITA suppressed cell proliferation and promoted early apoptosis, while its silencing promoted cell growth dramatically (p < 0.05). RITA overexpression upregulated p53 and reduced cyclin E levels, whereas silencing of RITA had the opposite effect on p53 and cyclin E expression. Our in vitro results represent the first evidence that RITA might suppress tumor growth and induce apoptosis in HCCs, and may be a potent antitumoral agent for HCC treatment that deserves further exploration.
PURPOSE: We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL.
PATIENTS AND METHODS: By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival.
RESULTS: PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non-germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025).
CONCLUSION: STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype.
Burmakin M, Shi Y, Hedström E, et al.Dual targeting of wild-type and mutant p53 by small molecule RITA results in the inhibition of N-Myc and key survival oncogenes and kills neuroblastoma cells in vivo and in vitro.
Clin Cancer Res. 2013; 19(18):5092-103 [PubMed
] Related Publications
PURPOSE: Restoration of the p53 function in tumors is a promising therapeutic strategy due to the high potential of p53 as tumor suppressor and the fact that established tumors depend on p53 inactivation for their survival. Here, we addressed the question whether small molecule RITA can reactivate p53 in neuroblastoma and suppress the growth of neuroblastoma cells in vitro and in vivo.
EXPERIMENTAL DESIGN: The ability of RITA to inhibit growth and to induce apoptosis was shown in seven neuroblastoma cell lines. Mechanistic studies were carried out to determine the p53 dependence and the molecular mechanism of RITA-induced apoptosis in neuroblastoma, using cell viability assays, RNAi silencing, co-immunoprecipitation, qPCR, and Western blotting analysis. In vivo experiments were conducted to study the effect of RITA on human neuroblastoma xenografts in mice.
RESULTS: RITA induced p53-dependent apoptosis in a set of seven neuroblastoma cell lines, carrying wild-type or mutant p53; it activated p53 and triggered the expression of proapoptotic p53 target genes. Importantly, p53 activated by RITA inhibited several key oncogenes that are high-priority targets for pharmacologic anticancer strategies in neuroblastoma, including N-Myc, Aurora kinase, Mcl-1, Bcl-2, Wip-1, MDM2, and MDMX. Moreover, RITA had a strong antitumor effect in vivo.
CONCLUSIONS: Reactivation of wild-type and mutant p53 resulting in the induction of proapoptotic factors along with ablation of key oncogenes by compounds such as RITA may be a highly effective strategy to treat neuroblastoma.
The incidence of endometrial cancer, a common gynecological malignancy, is increasing in Japan. We have previously shown that the ER/MDM2/p53/p21 pathway plays an important role in endometrial carcinogenesis. In the present study, we investigated the effects of germline single nucleotide polymorphisms in murine double minute 2 (MDM2) SNP309, TP53 Arg72Pro, ESR1 PvuII and XbaI, and p21 codon 31 on endometrial cancer risk. We evaluated these polymorphisms in DNA samples from 125 endometrial cancer cases and 200 controls using polymerase chain reaction-based restriction fragment length polymorphism. The association of each genetic polymorphism with endometrial cancer was examined by the odds ratio and 95% confidence interval, which were obtained using logistic regression analysis. The SNP309 GG genotype non-significantly increased the risk of endometrial cancer. The 95% confidence interval for the GG genotype vs. the TT genotype of MDM2 SNP309 was 1.76 (0.93-3.30). Endometrial cancer was not associated with tested SNP genotypes for TP53, ESR1 and p21. The combination of SNP309 GG + TG and TP53 codon 72 Arg/Arg significantly increased endometrial cancer risk. The adjusted OR was 2.53 (95% confidence interval, 1.03-6.21) and P for the interaction was 0.04. This result was supported by in vitro data showing that endometrial cancer cell lines with the SNP309 G allele failed to show growth inhibition by treatment with RITA, which reduces p53-MDM2 binding. The presence of the SNP309 G allele and TP53 codon 72 Arg/Arg genotype is associated with an increased risk of endometrial cancer in Japanese women.
Giuseppe P, Daniele R, Rita BMCutaneous complications of Anderson-Fabry disease.
Curr Pharm Des. 2013; 19(33):6031-6 [PubMed
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Anderson-Fabry disease is an X-linked lysosomal storage disorder caused by a defect in the α-galactosidase A gene, which leads to the deficiency of the hydrolytic enzyme α-galactosidase A. The consequent inability to catabolize glycosphingolipids causes progressive accumulation of globotriaosylceramide in the vascular endothelium throughout the body. Fatalities in the classical phenotype may usually occur as a consequence of cerebral, cardiac or renal disease. Dermatological manifestations are a relevant feature of Fabry disease and include angiokeratomas, telangiectasiae, lymphedema, anhidrosis or hypohidrosis and pseudo-acromegalic facial appearance. The actual causal treatment for Fabry disease is the enzyme replacement therapy. Dermatologists have a key role, since cutaneous manifestations may lead to the diagnosis. This may help an early therapeutic intervention, reducing both morbidity and mortality.
BACKGROUND: The significant mortality associated with breast cancer (BCa) suggests a need to improve current research strategies to identify new genes that predispose women to breast cancer. Differential allele-specific expression (DASE) has been shown to contribute to phenotypic variables in humans and recently to the pathogenesis of cancer. We previously reported that nonsense-mediated mRNA decay (NMD) could lead to DASE of BRCA1/2, which is associated with elevated susceptibility to breast cancer. In addition to truncation mutations, multiple genetic and epigenetic factors can contribute to DASE, and we propose that DASE is a functional index for cis-acting regulatory variants and pathogenic mutations, and that global analysis of DASE in breast cancer precursor tissues can be used to identify novel causative alleles for breast cancer susceptibility.
RESULTS: To test our hypothesis, we employed the Illumina(®) Omni1-Quad BeadChip in paired genomic DNA (gDNA) and double-stranded cDNA (ds-cDNA) samples prepared from eight BCa patient-derived normal mammary epithelial lines (HMEC). We filtered original array data according to heterozygous genotype calls and calculated DASE values using the Log ratio of cDNA allele intensity, which was normalized to the corresponding gDNA. We developed two statistical methods, SNP- and gene-based approaches, which allowed us to identify a list of 60 candidate DASE loci (DASE ≥ 2.00, P ≤ 0.01, FDR ≤ 0.05) by both methods. Ingenuity Pathway Analysis of DASE loci revealed one major breast cancer-relevant interaction network, which includes two known cancer causative genes, ZNF331 (DASE = 2.31, P = 0.0018, FDR = 0.040) and USP6 (DASE = 4.80, P = 0.0013, FDR = 0.013), and a breast cancer causative gene, DMBT1 (DASE=2.03, P = 0.0017, FDR = 0.014). Sequence analysis of a 5' RACE product of DMBT1 demonstrated that rs2981745, a putative breast cancer risk locus, appears to be one of the causal variants leading to DASE in DMBT1.
CONCLUSIONS: Our study demonstrated for the first time that global DASE analysis is a powerful new approach to identify breast cancer risk allele(s).
Michaelis M, Rothweiler F, Agha B, et al.Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.
Cell Death Dis. 2012; 3:e294 [PubMed
] Free Access to Full Article Related Publications
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.
Yu J, Liang QY, Wang J, et al.Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer.
Oncogene. 2013; 32(3):307-17 [PubMed
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Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene.
A rapid and easy method to screen for aberrant cDNA would be a very useful diagnostic tool in genetics since a fraction of the DNA variants found affect RNA splicing. The currently used RT-PCR methods require new primer combinations to study each variant that might affect splicing. Since MLPA is routinely used to detect large genomic deletions and successfully detected exon skipping events in Duchenne muscular dystrophy in cDNA, we performed a pilot study to evaluate its value for BRCA1 cDNA. The effect of puromycin, DNase I and two different DNA cleaning protocols were tested in the RNA analysis of lymphocyte cultures. We used two samples from unrelated families with two different BRCA1 exon deletion events, two healthy unrelated controls and six samples from hereditary breast/ovarian cancer syndrome (HBOC) patients without BRCA1/2 mutations. Using RNA treated with DNase I and cleaned in a column system from puromycin-treated fractions, we were able to identify the two BRCA1 deletions. Additional HBOC patients did not show additional splice events. However, we were not able to get reproducible results. Therefore, the cDNA-MLPA technique using kit BRCA1 P002 is in our hands currently not reliable enough for routine RNA analysis and needs further optimization.
The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK activators in the treatment of MM.
Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.
Textor S, Fiegler N, Arnold A, et al.Human NK cells are alerted to induction of p53 in cancer cells by upregulation of the NKG2D ligands ULBP1 and ULBP2.
Cancer Res. 2011; 71(18):5998-6009 [PubMed
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Natural killer (NK) cells are immune cells sensing and eliminating foreign, stressed, transformed, and senescent cells through specialized surface receptors, such as NKG2D, that interacts with several virus- or stress-inducible ligands, including ULBP1 and -2, which are expressed on target cell surfaces. For example, induction of DNA damage or cellular senescence pathways in tumor cells led to upregulation of NKG2D ligands that activate NK cells. Although, both pathways activate p53, the relationship of p53 activation to upregulation of NKG2D ligands has not been addressed. In this study, we report that induction of wild-type p53, but not mutant p53, strongly upregulated mRNA and cell surface expression of ULBP1 and -2, whereas expression of other NK cell ligands was not affected. We defined intronic p53-responsive elements in these two novel p53 target genes. Coculture of wild-type p53-induced human tumor cells with primary human NK cells enhanced NKG2D-dependent degranulation and IFN-γ production by NK cells. Accordingly, treatment of certain wild-type p53-expressing tumor cell lines with the p53-reactivating small molecular compound RITA resulted in upregulation of ULBP2 mRNA and cell surface protein expression. Taken together, our findings define the involvement of p53 in the regulation of specific NKG2D ligands that enhance NK cell-mediated target recognition. One implication of our work is that activating p53 after adoptive transfer of NK cells might constitute an effective combinatorial strategy of NK cell-based immunochemotherapy in cancers in which wild-type p53 function is preserved.
Kazemi A, Safa M, Shahbazi ARITA enhances chemosensivity of pre-B ALL cells to doxorubicin by inducing p53-dependent apoptosis.
Hematology. 2011; 16(4):225-31 [PubMed
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The use of low-molecular-weight, non-peptidic molecules that disrupt the interaction between the p53 tumor suppressor and its negative regulator MDM2 has provided a promising alternative for the treatment of different types of cancer. Here, we used small-molecule reactivation of p53 and induction of tumor cell apoptosis (RITA) to sensitize leukemic NALM-6 cells to doxorubicin by upregulating p53 protein. RITA alone effectively inhibited NALM-6 cells viability in dose-dependent manner as measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay and induced apoptosis as evaluated by flow cytometry, whereas RITA in combination with doxorubicin enhanced NALM-6 cells to doxorubicin-sensitivity and promoted doxorubicin induced apoptosis. Levels of p53 protein and its proapoptotic target genes, quantified by western blot and real-time PCR respectively, showed that expression of p53 was significantly increased after RITA treatment. Using p53 inhibitors PFT-alpha and PFT-mu it was shown that p53-mediated apoptosis induced by RITA can be regulated by both p53-transcription-dependent and -independent pathways. Moreover, RITA-induced apoptosis was accompanied by the activation of caspase-3 and PARP cleavage. Therefore, exploiting synergistic effects between RITA and chemotherapeutics might be an effective clinical strategy for leukemia chemotherapy.
Brandão RD, van Roozendaal K, Tserpelis D, et al.Characterisation of unclassified variants in the BRCA1/2 genes with a putative effect on splicing.
Breast Cancer Res Treat. 2011; 129(3):971-82 [PubMed
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A subset of the unclassified variants (UVs) identified during genetic screening of BRCA1/2 genes may affect splicing. We assessed at RNA level the effect of four BRCA1 and ten BRCA2 UVs with a putative splice effect, as predicted in silico. The variants selected for this study were beyond the positions -1, -2 or +1, +2 from the exon, and were not previously described (n = 8) or their effect on splicing was not assessed previously (n = 6). Lymphocytes from UV carriers and healthy controls were cultured and treated with puromycin to prevent nonsense-mediated mRNA decay. The relative contribution of each allele to the various transcripts was assessed using combinations of allele-specific and transcript-specific primers. BRCA2 c.425G>T, c.7976+3_7976+4del and c.8754+3G>C give rise to aberrant transcripts BRCA2Δ4, BRCA2Δ17 and retention of 46nt of intron 21, respectively, and were considered pathogenic. BRCA1 c.4987-3C>G gives rise to BRCA1Δ17 that is likely pathogenic; however, residual expression of the full-length transcript from the variant allele could not be excluded. BRCA1 c.692C>T, c.693G>A and BRCA2 c.6935A>T, besides expressing the full-length transcript, increased expression of BRCA1Δ11 and BRCA2Δ12, respectively. As these are naturally occurring isoforms, also observed in controls, the clinical relevance is unclear. The seven remaining UVs did not affect splicing and three intronic variants were therefore classified as neutral. In conclusion, the RNA analysis results clarified the clinical relevance of 6 of the 14 studied UVs and thereby greatly improve the genetic counselling of high-risk breast/ovarian cancer patients carrying these classified variants.
Guimarães JL, Ayrizono Mde L, Coy CS, Lima CSGene polymorphisms involved in folate and methionine metabolism and increased risk of sporadic colorectal adenocarcinoma.
Tumour Biol. 2011; 32(5):853-61 [PubMed
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This pilot study has compared the polymorphic genotype frequencies of methylenetetrahydrofolate reductase (MTHFR A1298C and C677T), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), and thymidylate synthase (TS 2R/3R) in 113 patients with sporadic colorectal adenocarcinoma (SCA) and 188 healthy blood donors, used as matched controls. The aim was to assess the role of these genotypes in the increased risk of SCA among the southeastern Brazilian population. Carriers of genotype MTRR 66GG, or the combined variants MTHFR 1298AC + CC plus 677CT + TT, or MTHFR 677CT + TT plus MTR 2756AG + GG, or MTHFR 1298AC + CC plus 677CT + TT plus MTR 2756AG + GG, or yet, MTHFR 1298AC + CC plus 677CT + TT plus MTRR 66AG + GG, respectively, showed an increased risk of the order of 1.99-, 3.26-, 2.22-, 10.92-, and 14.88-fold of developing SCA when compared with carriers of the other studied polymorphic genotypes, whether in isolation or in combination. In addition, individuals with the MTHFR 677CT + TT or the MTR 2756AG + GG genotypes had a 2.12- and a 1.42-fold increased risks of SCA onset before 50 years of age. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk of SCA while individuals with the MTR 2756AG + GG and the MTHFR 677CT + TT genotypes showed a 2.11- and a 1.62-fold increased risk of undifferentiated and advanced tumors at diagnosis, respectively. Carriers of genotype MTHFR 1298AC + CC or MTHFR 1298AC + CC plus MTRR 66AG + GG had a 1.42- and a 3.07-fold increased risk of rectal tumor, respectively. Additionally, carriers of MTHFR 677CT + TT or MTHFR 677CT + TT plus TS 2R/3R + 3R/3R had a 1.55- and a 5.39-fold increased risk for colon tumor, respectively, in comparison with carriers of the wild genotypes. These data suggest that all polymorphisms coding for folate and methionine-dependent enzymes, particularly when present in combination with other polymorphisms, have consistent roles in the increased risk of SCA among the southeastern population of Brazil.
We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.
Chiu HC, Chou DL, Huang CT, et al.Suppression of Stat3 activity sensitizes gefitinib-resistant non small cell lung cancer cells.
Biochem Pharmacol. 2011; 81(11):1263-70 [PubMed
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Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3.
Saha MN, Jiang H, Mukai A, Chang HRITA inhibits multiple myeloma cell growth through induction of p53-mediated caspase-dependent apoptosis and synergistically enhances nutlin-induced cytotoxic responses.
Mol Cancer Ther. 2010; 9(11):3041-51 [PubMed
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Mutations or deletions of p53 are relatively rare in multiple myeloma (MM), at least in newly diagnosed patients. Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of murine double minute 2 (MDM2) is a promising and applicable therapeutic strategy. RITA and nutlin are two new classes of small molecule MDM2 inhibitors that prevent the p53-MDM2 interaction. Earlier reports showed p53-dependent activity of RITA in solid tumors as well as in leukemias. We and others recently described nutlin-induced apoptosis in MM cells, but it remains unclear whether RITA exerts antimyeloma activity. Here, we found that RITA activates the p53 pathway and induces apoptosis in MM cell lines and primary MM samples, preferentially killing myeloma cells. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including upregulation of a proapoptotic protein (NOXA), downregulation of an antiapoptotic protein, Mcl-1, and activation of caspases through extrinsic pathways. Moreover, a number of key p53-mediated apoptotic target genes were identified by gene expression profiling and further validated by quantitative real-time PCR. Importantly, the combination of RITA with nutlin displayed a strong synergism on growth inhibition with the combination index ranging from 0.56 to 0.82 in MM cells. Our data support further clinical evaluation of RITA as a potential novel therapeutic intervention in MM.
Hedström E, Eriksson S, Zawacka-Pankau J, et al.p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA.
Cell Cycle. 2009; 8(21):3584-91 [PubMed
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Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel approximately 130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the approximately 130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA did not inhibit TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.
One in nine women will develop breast cancer in her lifetime (Canadian Cancer Society, 2007). Hereditary breast cancer accounts for only five to 10 per cent of all breast cancers. However, women carrying a single high-penetrance gene mutation have a 40% to 80% chance of developing breast cancer (Fackenthal & Olopade, 2007). Most of these breast cancers occur in women under the age of 50. The BRCA 1 gene mutation was first reported in 1994, and the BRCA 2 gene mutation in 1995. The BRCA 2 gene mutation is often carried in males, and accounts for approximately six per cent of male breast cancer. Women with this gene mutation have a lifetime risk of developing breast cancer of between 50% and 85%, a second breast cancer of between 30% and 50%, and ovarian cancer between 10% and 20%. Each parent with the BRCA 2 gene mutation has a 50% chance of passing this gene mutation to their children (National Cancer Institute, 2006). The emotional impact of receiving cancer risk information such as this is difficult to predict. When presented with information about risk-reduction surgery, chemoprevention, risk avoidance and increased screening, how does one make decisions? Walk with me, as I share how my family discovered we carry the Icelandic founder gene mutation, the steps we took together during the testing process, and the decision-making by the family members who tested positive. We'll focus on my sister Rita--ordinary days, an extraordinary woman.
Di Cintio A, Di Gennaro E, Budillon ARestoring p53 function in cancer: novel therapeutic approaches for applying the brakes to tumorigenesis.
Recent Pat Anticancer Drug Discov. 2010; 5(1):1-13 [PubMed
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p53 tumor suppressor gene encodes for a critical cellular protein that regulate the integrity of the cell and can induce cell cycle arrest and/or apoptosis upon cellular stresses of several origins, including chemotherapeutics. Loss of p53 function occurs in an estimated 50% of all cancers by mutations and deletions while in the presence of wild-type p53 alleles other mechanisms may affect the expression and activity of p53. Alternate mechanisms include methylation of the promoter of p53, deletion or epigenetic inactivation of the p53-positive regulator p14/ARF, elevated expression of the p53 regulators murine double minute 2 (MDM2) and MDMX, or alteration of upstream regulators of p53 such as the kinase ATM. MDM2 is a p53 E3 ubiquitin ligase that mediates the ubiquitin-dependent degradation of p53 while p14/ARF is a small MDM2-binding protein that controls the activity of MDM2 by displacing p53 and preventing its degradation. MDMX antagonize p53-dependent transcriptional control by interfering with p53 transactivation function. The understanding of the key role of p53 inactivation in cancer development generated considerable interest in developing compounds that are capable of restoring the p53 functions. Several patents have been issued on such compounds. Adenovirus-based p53 gene therapy as well as small molecules such as PRIMA that can restore the transcriptional transactivation function to mutant p53, or NUTLIN and RITA that interfere with MDM2-directed p53 degradation, have tested in a preclinical setting and some of these approaches are currently in clinical development.
Grinkevich VV, Nikulenkov F, Shi Y, et al.Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis.
Cancer Cell. 2009; 15(5):441-53 [PubMed
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Targeting "oncogene addiction" is a promising strategy for anticancer therapy. We report a potent inhibition of crucial oncogenes by p53 upon reactivation by small-molecule RITA in vitro and in vivo. RITA-activated p53 unleashes the transcriptional repression of antiapoptotic proteins Mcl-1, Bcl-2, MAP4, and survivin; blocks the Akt pathway on several levels; and downregulates c-Myc, cyclin E, and beta-catenin. p53 ablates c-Myc expression via several mechanisms at the transcriptional and posttranscriptional level. We show that the threshold for p53-mediated transrepression of survival genes is higher than for transactivation of proapoptotic targets. Inhibition of oncogenes by p53 reduces the cell's ability to buffer proapoptotic signals and elicits robust apoptosis. Our study highlights the role of transcriptional repression for p53-mediated tumor suppression.
Nahi H, Selivanova G, Lehmann S, et al.Mutated and non-mutated TP53 as targets in the treatment of leukaemia.
Br J Haematol. 2008; 141(4):445-53 [PubMed
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TP53 is mutated in 10-20% of cases of chronic lymphocytic leukaemia (CLL) and 3-8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) restores the wild-type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA-1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with -17, which are more resistant to daunorubicin and cytarabine compared with samples without -17, were effectively killed by PRIMA-1. RITA, which stabilizes the function of wild-type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA-1, AML patient samples without -17 were significantly more sensitive to RITA. Similarly, RITA exerted dose-dependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug-resistant leukaemia.
Serda RE, Bisoffi M, Thompson TA, et al.1alpha,25-Dihydroxyvitamin D3 down-regulates expression of prostate specific membrane antigen in prostate cancer cells.
Prostate. 2008; 68(7):773-83 [PubMed
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BACKGROUND: Prostate specific membrane antigen (PSMA) expression correlates with prostate cancer grade and is increased in hormone-refractory prostate cancer. The increased expression of PSMA following androgen deprivation therapy may be a consequence of the down-regulation of PSMA expression by androgen. Moreover, 1alpha,25-dihydroxyvitamin D3 (1,25-VD) has been shown to suppress prostate cancer progression as well as cell motility and invasion. Since PSMA is positively correlated with both of these characteristics, we hypothesized that 1,25-VD would regulate PSMA expression.
METHODS: LNCaP prostate cancer cells were treated with 1,25-VD, followed by analysis of cell surface PSMA expression. The PSMA enhancer, located within the third intron of the PSMA gene, was cloned into a reporter vector and regulation by 1,25-VD was investigated. The role of the androgen receptor (AR) in 1,25-VD mediated suppression of PSMA expression was examined using Casodex and AR specific siRNA.
RESULTS: Surface expression of PSMA was significantly decreased in a dose-dependent manner by 10 nM 1,25-VD or greater. Regulation by 1,25-VD occurred at the level of the PSMA enhancer. Over-expression of the vitamin D receptor (VDR) also decreased expression of PSMA. Additionally, suppression of AR translation using siRNA technology blocked the suppressive effect of 1,25-VD on PSMA expression, however inhibition of PSMA expression by 1,25-VD occurred in the absence of androgens.
CONCLUSIONS: Suppression of PSMA by 1,25-VD occurs at the level of the PSMA enhancer and is elevated by over-expression of the VDR. This regulation involves the AR, but is not dependent on the presence of androgens.
Li G, Yin SProgress of epidemiological and molecular epidemiological studies on benzene in China.
Ann N Y Acad Sci. 2006; 1076:800-9 [PubMed
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Benzene is an organic solvent that has been used in industry for about 100 years throughout the world. Since 1973, a series of toxicological and molecular epidemiological studies on benzene were conducted by researchers at the Chinese Academy of Preventive Medicine (CAPM) (1973-1986) and subsequently by a collaboration between the CAPM and the National Cancer Institute (NCI) in the United States that began in 1986, which was joined by investigators from the University of California at Berkeley, the University of North Carolina at Chapel Hill, and New York University. The findings demonstrated that the risk of leukemia and lymphoma among benzene-exposed workers was significantly increased, with elevated risks for leukemia present not only at higher exposure but also among workers exposed to under 10 ppm. Therefore, the benzene permissible level was decreased to 1.8 ppm (6 mg/m(3)) and benzene-induced leukemia is treated as an occupational cancer in China. The benzene permissible level is 1.0 in the United States and in several other developed countries and it has been suggested to be decreased to 0.5 ppm (ACGIH). A number of potential biomarkers are related to benzene exposure and poisoning. Some of these are benzene oxide-protein adducts, chromosome aberration of lymphocytes, and GPA mutations in erythrocytes, a decrease in B cell and CD4(-)T cell counts in peripheral blood, and altered expression of CXCL16, ZNF331, JUN, and PF4 in lymphocytes. Variation in multiple benzene metabolizing genes may be associated with risk of benzene hematotoxicity, including CYP2E1, MPO, NQO1, and GSTT1.