Wild-type P53 Protein Research Articles

Abstract B5: Preclinical characterization of a novel orally-available MDM2 inhibitor DS-3032b: Anti-tumor profile and predictive biomarkers for sensitivity

Abstract The p53 protein exerts its tumor suppressor function by inducing cell cycle arrest, apoptosis, or senescence in cells under stress conditions. Inactivation of p53 by somatic mutations is a frequent tumorigenic event. In human tumors that retain wildtype p53 protein, its activity is often inhibited by interaction with the negative regulator MDM2. DS-3032b, a novel, specific, small-molecule inhibitor of MDM2, disrupts the MDM2-p53 interaction in tumor cells and is being developed as a cancer therapeutic based on a p53 reactivation mechanism. Cell-free study using recombinant MDM2 and p53 proteins demonstrated inhibition of MDM2-p53 binding by DS-3032b with an IC50 value of 5.57nM. Treatment with graded concentrations of DS-3032b resulted in a dose-dependent increase of p21 and PUMA mRNA levels in an osteosarcoma cell line (SJSA-1) harboring MDM2 gene-amplification. DS-3032b inhibited cell growth in p53 wildtype (GI50s: 0.043 - 0.276μM), but not p53 mutant or null cell lines (GI50s: >10μM). Studies in athymic nude mice inoculated with SJSA-1 xenografts showed tumor regression following treatment with DS-3032b given once daily for 10 days at a dose of 50 mg/kg. To identify biomarkers predictive of sensitivity to MDM2 inhibitors, we treated a panel of 240 molecularly-annotated cancer cell lines (OncoPanel, Eurofins Panlabs) with the prototypic MDM2 inhibitor DS-5272 structurally unrelated to DS-3032b. Cells were dichotomized as sensitive or resistant to DS-5272, based on IC50 cut-off value of <1.5μM and >4 μM, respectively. As expected, TP53 mutation was identified as a strong predictor for resistance. In addition, a 175-gene signature was identified as an indicator for sensitivity. This gene signature predicts for response to 3 different MDM2 inhibitors (DS-5272, nutlin3a, and DS-3032a (free form of DS-3032b)). A combination of TP53 genotype and gene signature showed further enrichment of sensitive cell lines compared to using either of the biomarker alone. In vivo studies using patients derived tumor xenografts have further validated the predictive value of the gene signature. Bioinformatic reduction of the complexity of the gene signature to a clinically applicable level is on-going. Mapping of this signature to genomic data from 15,000+ clinical samples in the OncoMine database has revealed that only a subset of TP53 wildtype tumors contain high expression of the signature genes. The top-ranked genes in the signature were well-known p53-inducible genes, suggesting that the gene signature might be a surrogate for functional p53. Consistent with this postulate, we showed low signature gene expression and resistance to MDM2 inhibitors in cervical cancers in which p53 function is inactivated by the E6 viral oncoprotein. Cancer subtypes enriched for a low incidence of TP53 mutation and high signature gene expression are melanoma, lymphoma, myeloma, renal cell cancer, and leukemia. In summary, DS-3032b is a specific and potent MDM2 inhibitor showing promising anti-tumor activity in vitro and in vivo. The sensitivity gene signature might serve as a pharmacologically defined surrogate for p53 functionality that is critical for predicting clinical benefits from DS-3032b treatment. Citation Format: Kenji Nakamaru, Takahiko Seki, Koichi Tazaki, Archie Tse. Preclinical characterization of a novel orally-available MDM2 inhibitor DS-3032b: Anti-tumor profile and predictive biomarkers for sensitivity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B5.

Computational identification of novel piperidine derivatives as potential HDM2 inhibitors designed by fragment-based QSAR, molecular docking and molecular dynamics simulations

Tumor suppressor protein p53 maintains integrity of genome and regulates the genes responsible for DNA repair mechanism, apoptosis as well as cell cycle and growth arrest. As with murine double minute 2 (MDM2), the human homolog HDM2 is a principal cellular antagonist of p53. In unstressed cells, cellular levels of p53 and HDM2 are maintained in an autoregulatory manner in which both mutually control cellular levels of each other. About half of the human cancers express wild-type p53 protein that is antagonized by over-expressed HDM2. Restoring p53 function via HDM2 antagonists is a leading therapeutic approach for treating a variety of tumors. In this study, we have developed a novel statistically sound group-based QSAR (GQSAR) model using piperidine-derived compounds that have been validated experimentally to inhibit p53–HDM2 interaction. On the basis of developed GQSAR model, a combinatorial library of molecules was prepared and its activity was predicted. These molecules were then docked to HDM2, and two top-scoring molecules possessing a binding energy of −6.639 and −6.305 kcal/mol were selected for further study. These molecules and their binding poses were analyzed further via molecular dynamic simulations. In this study, we report two lead compounds as potent HDM2 inhibitors and also provide an insight into mechanism of interaction of the lead compounds to HDM2 target.

Nontoxic virus nanofibers improve the detection sensitivity for the anti-p53 antibody, a biomarker in cancer patients.

The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-specific virus nanofiber that can be mass-produced by infecting host bacteria in an error-free manner, and genetically engineered it to display a peptide capable of recognizing and capturing anti-p53 antibody on its side wall. We employed the resultant phage nanofibers as a capture probe to develop a modified version of the enzyme-linked immunosorbent assay (ELISA) method, termed phage-ELISA. We compared it to the traditional ELISA method for the detection of anti-p53 antibody, p53-ELISA, which uses recombinant wild-type p53 protein to capture anti-p53 antibody. We applied phage-ELISA to detect anti-p53 antibody in an experimental group of 316 patients with various types of malignant tumors. We found that a detection rate of 17.7% (56 positive cases) was achieved by phage-ELISA, which was comparable to the detection rate of 20.6% for p53-ELISA (65 positive cases). However, when both phage and p53 were combined to form antibody-capturing probes for phage/p53-ELISA, a detection rate of 30.4% (96 positive cases) was achieved. Our work showed that owing to the combined capture of the anti-p53 antibody by both phage nanofibers and p53, the phage/p53-ELISA achieved the highest diagnostic accuracy and detection efficiency for the anti-p53 antibody in patients with various types of cancers. Our work suggests that a combination of nanofibers and antigens, both of which capture antibody, could lead to increased detection sensitivity, which is useful for applications in the life sciences, clinical medicine, and environmental sciences.

Sulfonoquinovosyl diacylglyceride selectively targets acute lymphoblastic leukemia cells and exerts potent anti-leukemic effects in vivo.

DNA topoisomerase II inhibitors e.g. doxorubicin and etoposide are currently used in the chemotherapy for acute lymphoblastic leukemia (ALL). These inhibitors have serious side effects during the chemotherapy e.g. cardiotoxicity and secondary malignancies. In this study we show that sulfonoquinovosyl diacylglyceride (SQDG) isolated from Azadirachta indica exerts potent anti-ALL activity both in vitro and in vivo in nude mice and it synergizes with doxorubicin and etoposide. SQDG selectively targets ALL MOLT-4 cells by inhibiting catalytic activity of topoisomerase I enzyme and inducing p53 dependent apoptotic pathway. SQDG treatment induces recruitment of ATR at chromatin and arrests the cells in S-phase. Down-regulation of topoisomerase I or p53 renders the cells less sensitive for SQDG, while ectopic expression of wild type p53 protein in p53 deficient K562 cells results in chemosensitization of the cells for SQDG. We also show that constant ratio combinations of SQDG and etoposide or SDQG and doxorubicin exert synergistic effects on MOLT-4 cell killing. This study suggests that doses of etoposide/doxorubicin can be substantially reduced by combining SQDG with these agents during ALL chemotherapy and side effects caused can be minimized. Thus dual targeting of topoisomerase I and II enzymes is a promising strategy for improving ALL chemotherapy.

Germ cell tumors overexpress the candidate therapeutic target cyclin B1 independently of p53 function.

Germ cell tumors (GCTs) generally express wild-type p53 protein. Rare p53 mutations may be associated with cisplatin resistance. There is growing interest in the role of cyclins as targets for GCTs. Cyclin B1 is involved in G2/M transition and its overexpression has been reported in tumors carrying nonfunctional p53. Conversely, cyclin B1-specific small interfering RNAs have been shown to dramatically reduce tumor proliferation. We investigated whether a subset of chemotherapy-resistant GCTs overexpressed cyclin B1 as a result of nonfunctional p53, as this would make cyclin B1 a potential therapeutic target. Our data showed that GCTs consistently overexpressed cyclin B1 independently of their responsiveness to chemotherapy or the presence of p53 mutations. Cyclin B1 was overexpressed by GCT cell lines carrying functional p53. Cyclin B1-specific small interfering RNAs only slightly reduced the proliferation of JAR and JEG-3 placental choriocarcinoma cells. Further research into targeting cyclin B1 could provide a novel intervention for GCTs.

Caspase Activation and Aberrant Cell Growth in a p53+/+ Cell Line from a Li-Fraumeni Syndrome Family

Wild-type p53 is well known to induce cell cycle arrest and apoptosis to block aberrant cell growth. However, p53's unique role in apoptosis and cell proliferation in Li-Fraumeni Syndrome (LFS) has not been well elucidated. The aim of this study is to characterize the activity of wild-type p53 protein in LFS family dominated by a germline negative mutant p53. As expected, etoposide-treated wild-type p53-containing cell lines, LFS 2852 and control Jurkat, showed a greater rate of caspase- and annexin V-induced apoptotic cell death compared to the p53-mutant LFS 2673 cell line although mitochondrial and nuclear assays could not detect apoptosis in these organelles. The most intriguing part of the observation was the abnormal proliferation rate of the wild-type p53-containing cell line, which grew twice as fast as 2673 and Jurkat cells. This is important because apoptosis inducers acting through the mitochondrial death pathway are emerging as promising drugs against tumors where the role of p53 is not only to target gene regulation but also to block cell proliferation. This study casts a long shadow on the possible dysregulation of p53 mediators that enable cell proliferation. The deregulation of proliferation pathways represents an important anticancer therapeutic strategy for patients with the LFS phenotype.

Amyloidogenicity of p53: A Hidden Link Between Protein Misfolding and Cancer

Pathogenic aggregation is closely associated with various protein misfolding diseases such as type 2 diabetes mellitusand Alzheimer's disease. Amyloidogenic proteins that have a propensity to assemble into amyloid oligomers and fibrils form the aggregates. The tumor suppressor p53, a transcription factor that regulates the cell cycle and apoptosis, is also amyloidogenic. In tumor models, both wild type and mutant p53 proteins show aggregation kinetics and morphology similar to those of classical amyloidogenic proteins, such as -amyloid peptide and -synuclein. Wild type p53 loses its anticancer activity when it aggregates, while p53 mutants with enhanced amyloidogenicity show accelerated aggregation. So far, amyloidogenic p53 mutations have been implicated in more than ten different types of cancer, suggesting a connection between p53 aggregation and cancer. Therefore, inhibition of both inherent and mutation induced p53 aggregation may stabilize p53 in a functional conformation and provide a novel approach to cancer prevention and treatment. Here, we summarize recent findings on carcinogenic aggregation of wild type p53 and its clinical mutants, structure-dependent amyloidogenesis of p53, and several promising strategies based on inhibition of p53 aggregation are also discussed.

Cell context dependent p53 genome-wide binding patterns and enrichment at repeats.

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.

Abstract 881: Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer

Abstract Introduction: Mutations in TP53 are common in early pancreatic and ovarian cancer development, and are detected in the majority of pancreatic cancers (75%) and serous ovarian cancers (97%). Mutations in TP53 induce autoantibody (AAb) immune responses, which may be biomarkers for the early detection of these cancers. The most common mutations in p53 occur in the DNA binding domain at residues that directly contact the DNA (Arg175, 248, and 273). In contrast, preliminary studies suggest that mutations in the N- and C- termini of p53 alter the thermodynamic stability of p53, leading to increased accumulation in tumors. It is not known if the location of the mutations impacts immunogenicity, or if p53-AAb are specific to mutant p53 proteins. Methods: To evaluate AAb to mutant p53 proteins, we generated custom programmable protein microarrays displaying 52 common p53 point mutations. cDNAs encoding full-length p53 with c-terminal GST tags were printed on arrays with anti-GST antibody, and transcribed and translated in situ using HeLa expression lysate. The microarrays were screened with sera from pancreatic cancer cases (Stage I-III, n=60), healthy and benign disease controls (n=63), ovarian cancer cases (n=19) and controls (n=17). Bound IgG was detected by fluorescence. For validation, four immunogenic mutant p53 proteins Arg248Met, Pro278Ser, Arg213Leu, Gly187Ser and wild-type p53 were individually displayed in 96-well format using a programmable ELISA. The ELISAs were re-screened with a subset of the sera from pancreatic cancer cases (n=49) and controls (n=47) and all of the ovarian cancer cases and controls. Cutoff values were established to determine if a patient is positive for all wild-type and mutant p53 AAb by measuring the mean antigen florescence intensity +2 standard deviations of the controls. Results: AAb were detected to all four mutant and wild-type p53 proteins in sera from 8/49 (16%) of pancreatic cases and 2/47 (4.2%) of pancreatic controls, but the results were not statistically significant. In comparison, AAb to all four mutant and wild-type p53 proteins were identified in sera from 8/19 (42%) of ovarian cases and 0/17 (0%) of healthy control women (p ≤ 0.005). All sera with AAb to mutant p53 also had measurable AAb to wild-type p53 protein. All patients with immune responses had evidence of broad-reactivity to at least at least two mutant p53 proteins, as well as to wild-type p53. Conclusion: Our results indicate that patients with serum AAb to wild type p53 protein also have AAb to multiple mutant p53 antigens, suggesting degeneracy of the epitope specificity. No patients have been identified with AAb specific to mutant-only p53. We observed inter-patient variation in the patterns of immune reactivity to p53 mutations that is of unclear significance. Correlation with tumor p53 mutation status is ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Garrick Wallstrom, Joshua LaBaer, Michael A. Hollingsworth, Daniel W. Cramer, Karen S. Anderson. Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 881. doi:10.1158/1538-7445.AM2014-881

Abstract 468: Cancer cells with G/G mdm2 SNP309 have compromised transcriptional elongation of p53 target genes

Abstract MDM2 protein is often found over-expressed in cancers with wild-type p53. A single nucleotide polymorphism (T to G) near the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression. This polymorphism is associated with accelerated tumor formation, decreased sensitivity to DNA damage treatment and compromised p53 transcriptional activity. Two G/G SNP309 cell lines MANCA (Burkitt lymphoma) and A875 (melanoma) express wild-type p53 protein not readily degraded by MDM2. This wild-type p53 does not activate target genes even after DNA damage. During the blocked transcription, MDM2 and p53 co-localize at p53 response elements on chromatin. Therefore, we hypothesized that MDM2 localized with p53 on the chromatin was the contributing factor for the compromised p53 transcriptional activity. We explored the mechanism for compromised p53 transcriptional activity in these G/G SNP309 cancer cell lines. When compared to fully activated wild-type p53 in ML-1 cells treated with etoposide, MANCA and A875 cell lines had comparable recruitment of total and initiated RNA pol II at transcription start sites (TSS) for p21 and puma genes. This indicated that G/G SNP309 cells had functional transcription initiation at p53 target genes after DNA damage treatment. We next assessed epigenetic modifications associated with active transcriptional elongation. Using H3K36 trimethylation (H3K36me3) as a hallmark for active elongation, we observed that ML-1 cells had a substantial increase in H3K36me3 after etoposide treatment at p21 and puma TSS that was not seen in MANCA or A875 cell lines. This lack of H3K36me3 suggested an inhibition in early transcriptional elongation. To address the participation of MDM2 in the elongation block we created a stable MDM2 knockdown MANCA cell line. Interestingly, MDM2 knockdown drastically increased MDMX protein levels and moderately increased activation of p53 target genes implying MDMX played a redundant role for MDM2. We are currently investigating the roles of MDM2 and MDMX in blocking transcriptional elongation. Interestingly, we have found that treatment of MANCA cells with low dose Actinomycin D reactivates the p53 pathway and studies are in progress to determine if this is through activation of transcriptional elongation. This work was supported by The Breast Cancer Research Foundation and The National Science Foundation (grant MCB-0744316) to J.B. M.R. was supported by the MBRS-RISE Program to Hunter College 3R25-GM060665. Citation Format: Melissa Rosso, Alla Polotskaia, Jill Bargonetti. Cancer cells with G/G mdm2 SNP309 have compromised transcriptional elongation of p53 target genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 468. doi:10.1158/1538-7445.AM2014-468

Role of p53 in the progression of gastric cancer.

Intestinal metaplasia (IM) is a premalignant lesion associated with gastric cancer (GC) but is poorly described in terms of molecular changes. Here, we explored the role of TP53, a commonly mutated gene in GC, to determine if p53 protein expression and/or the presence of somatic mutations in TP53 can be used as a predictive marker for patients at risk of progressing to GC from IM. Immunohistochemistry and high resolution melting were used to determine p53 protein expression and TP53 mutation status respectively in normal gastric mucosa, IM without concurrent GC (IM-GC), IM with concurrent GC (IM+GC) and GC. This comparative study revealed an incremental increase in p53 expression levels with progression of disease from normal mucosa, via an IM intermediate to GC. TP53 mutations however, were not detected in IM but occurred frequently in GC. Further, we identified increased protein expression of Mdm2/x, both powerful regulators of p53, in 100% of the IM+GC cohort with these samples also exhibiting high levels of wild-type p53 protein. Our data suggests that TP53 mutations occur late in gastric carcinogenesis contributing to the final transition to cancer. We also demonstrated involvement of Mdmx in GC.

IL-1β microenvironment promotes proliferation, migration, and invasion of human glioma cells.

Among the primary brain tumors, glioblastoma is the most common and severe. Glioblastoma have poor prognosis because of their highly diffusive growth pattern and invasion into surrounding brain tissue. Human glioblastoma cells overexpress interleukin-1β (IL-1β) and also the levels of IL-1β in U87MG glioma cells are significantly higher than in U373, T98G, A172 glioma cell lines. Malignant tumors are characterized by unlimited proliferation, migration and invasion. This study examines the effect of IL-1β microenvironment on proliferation, migration and invasion in human glioma cell line U87MG that expresses wild-type p53 protein, and U251MG which expresses mutant p53. Proliferation was investigated by MTT assay, migration by wound-healing migration assay and invasion by in vitro transwell Matrigel invasion assay. An IL-1β microenvironment significantly increased migration and invasion of both wild-type and mutant p53 expressing glioma cells, but significantly increased proliferation only in U87MG glioma cells. These effects were inhibited by IL-1 receptor antagonist (IL-1Ra), thus giving evidence that an IL-1β milieu promotes glioma cell migration, invasion, and proliferation.

Senescence induction in renal carcinoma cells by Nutlin-3: a potential therapeutic strategy based on MDM2 antagonism

Although the role of p53 as a tumour suppressor in renal cell carcinoma (RCC) is unclear, our recent analysis suggests that increased wild-type p53 protein expression is associated with poor outcome. A growing body of evidence also suggests that p53 expression and increased co-expression of MDM2 are linked with poor prognosis in RCC. We have therefore examined whether an MDM2 antagonist; Nutlin-3, might rescue/increase p53 expression and induce growth inhibition or apoptosis in RCC cells that retain wild-type p53. We show that inhibition of p53 suppression by MDM2 in RCC cells promotes growth arrest and p53-dependent senescence – phenotypes known to mediate p53 tumour suppression in vivo. We propose that future investigations of therapeutic strategies for RCC should incorporate MDM2 antagonism as part of strategies aimed at rescuing/augmenting p53 tumour suppressor function.

Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells.

Background:Transforming growth factor-beta (TGF-β) induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Mechanisms involved in TGF-β-mediated EMT and cell motility are unclear. Recent studies showed that p53 affects TGF-β/SMAD3-mediated signalling, cell migration, and tumorigenesis. We previously demonstrated that Nox4, a Nox family NADPH oxidase, is a TGF-β/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our present study investigates the involvement of p53 in TGF-β-regulated Nox4 expression and cell migration.Methods:We investigated the effect of wild-type p53 (WT-p53) and mutant p53 proteins on TGF-β-regulated Nox4 expression and cell migration. Nox4 mRNA and protein, ROS production, cell migration, and focal adhesion kinase (FAK) activation were examined in three different cell models based on their p53 mutational status. H1299, a p53-null lung epithelial cell line, was used for heterologous expression of WT-p53 or mutant p53. In contrast, functional studies using siRNA-mediated knockdown of endogenous p53 were conducted in MDA-MB-231 metastatic breast epithelial cells that express p53-R280K and MCF-10A normal breast cells that have WT-p53.Results:We found that WT-p53 is a potent suppressor of TGF-β-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells. In contrast, tumour-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-β-dependent and TGF-β-independent pathways. Moreover, knockdown of endogenous mutant p53 (R280K) in TGF-β-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-β-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. Remarkably, immunofluorescence analysis revealed MCF-10A cells expressing p53-R280K mutant showed an upregulation of Nox4 in both confluent and migrating cells.Conclusions:Collectively, our findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signalling in TGF-β-mediated cell motility.

P53 N-terminal binding and stabilisation by PIAS3 inhibits MDM2-induced p53 ubiquitination and regulates cell growth

Mouse double minute 2 homolog (MDM2) binds to p53 through the 1-52 amino acid region of p53, in order to ubiquitinate p53. MDM2 thus destabilises p53 and inhibits the effect of p53 on cell cycle arrest and apoptosis. In the present study, the 1-52 amino acid region of the wild-type (wt) p53 protein was stably expressed in H1299 cells. The lysate of the transfected cells was then analysed using co-immunoprecipitation. A specific fraction of the precipitate was subjected to mass spectrometry analysis, which revealed that p53 bound to protein inhibitor of activated STAT3 (PIAS3). To the best of our knowledge, the present study is the first to report that the interaction of PIAS3 with p53 occurs through the 1-52 amino acid region of p53. Overexpression of PIAS3 in the A549 wt p53-expressing cell line was found to significantly increase the half‑life of p53 in the presence of cycloheximide, an inhibitor of protein synthesis. However, PIAS3 overexpression did not affect p53 mRNA levels. Furthermore, PIAS3 overexpression was observed to decrease p53 ubiquitination. Protein-protein interaction analysis revealed that PIAS3 binds to the 1-52 amino acid region of p53, thus disrupts the formation of the p53‑MDM2 complex. In addition, overexpression of PIAS3 in A549 cells was found to enhance the transcription of the p53‑transactivated target p21/waf1, due to p53 accumulation, which led to an increase in p53 binding to the p21 gene promoter. These findings indicate that this newly identified p53‑PIAS3 interaction through the 1-52 amino acid region of p53, reduces p53‑MDM2 complex formation, which not only increases the half-life of p53, but also its transactivation of target genes.

Interaction between p53 and MDM2 in Human Lung Cancer

The oncoprotein p53 protein induces cell growth arrest (apoptosis) in response to endo or exogenous stimuli. Mutation of TP53 (gene encoding the p53 protein) is common in human malignancies and alters the conformation of p53. The result is a more stable protein which accumulates in nuclei of tumor cells with loss of function. Mutant p53 is stabilized, and it is possible to detect this form very clearly by immunohistochemistry (IHC). Expression of the MDM2 protein is used as a potential marker of p53 function. P53 levels in normal cells are highly determined by the MDM2 protein (murine double minute 2) - mediated degradation of p53. MDM2 overexpression represents at least one mechanism by which p53 function can be abrogated during tumorigenesis. Lung carcinoma samples were obtained from patients, who underwent radical resection (lobectomy or pulmonectomy and lymphadectomy). Pathological dia-gnosis was based on the WHO criteria. In our study, we investigated the expression of p53 and MDM2 protein that might improve IHC as a marker for p53 status. Proteins were IHC detected in 136 samples of primary lung carcinoma. Immunostaining results of p53 positive samples were compared to IHC expression of MDM2 positive and MDM2 negative samples. Strong brown nuclear staining was visible in p53 and MDM2 positive cells. The most p53 positive cases were samples of squamocellular carcinoma (55%), then samples of large cell carcinoma (53%) and 26% adenocarcinoma samples showed the p53 immunoreactivity. No one sample of different types was p53 positive. When we compared the p53 expression and grade of tumor, we found that the p53 expression increased with the grade of tumor. For statistical evaluation, the chi square test was used. The relationship between p53 expression and type of tumor, also the p53 expression and grade of tumor was statistically significant (p = 0.000425; p = 0.00157). Regarding p53 and MDM2 expression, only nine samples (7%) were simultaneously p53 and MDM2 positive. In 46 (34%) cases, elevation of p53 was combined with MDM2 negative expression. Other tumor samples were either negative for both proteins (71/ 52%), or p53 negative and MDM2 positive in 10 (7%) tumor samples. Absence of p53 staining in most studies indicates absence of p53 mutation, and on the contrary, positive expression of p53 is a sign of p53 mutations with loss of function. In our study, 34% of cases with extensively high level of p53 without increased level of MDM2 were identified. We suppose that these are tumors with inactivating mutations that stabilize p53. On the other hand, tumors with high level of stabilized wildtype p53 protein and simultaneously with increased MDM2 staining (9 samples/7%) represent group with functional p53. In this group of patients, we could expect better prognosis with regard to function of p53 protein.

Oncomorphic TP53 Mutations in Gynecologic Cancers Lose the Normal Protein:Protein Interactions with the microRNA Microprocessing Complex.

Mutations in the tumor suppressor TP53 occur in almost all advanced ovarian cancers and in many advanced serous endometrial cancers. Mutations in TP53 can alter the function of the p53 protein, and some mutations result in a mutated protein with oncogenic activity. Previously referred to as gain of function (GOF) p53 proteins, we now term these “oncomorphic” mutations to better describe their function as oncogenes. We reviewed the data from The Cancer Genome Atlas (TCGA) and demonstrate that of the patients diagnosed with endometrial cancer that harbor TP53 mutations, approximately 30% of these mutations are oncomorphic. In ovarian cancer, approximately 20% are oncomorphic. The wild type (WT) p53 protein transactivates genes and micro- RNAs (miRNAs) necessary in the response to cellular stress, which turn off growth and induce apoptosis. In addition to direct transcriptional activation, WT p53 also acts through protein:protein interactions with Drosha and the miRNA processing complex to mediate rapid, enhanced processing of a subset of anti-growth miRNAs. We validated the interaction of WT p53 with the Drosha complex in the cell line UCI-107. We observed that miRNAs that inhibit the expression of oncogenes were induced. Specifically, some miRNAs were induced very rapidly over minutes, consistent with enhanced processing, while others required hours, consistent with transcriptional activation. In contrast, the most common oncomorphic TP53 mutations failed to interact with the Drosha complex and lost the ability to rapidly induce the miRNAs which inhibit oncogene expression. These studies highlight one mechanism underlying the oncomorphic properties of specific TP53 mutations: loss of the enhanced processing of anti-proliferative miRNAs.

P53 mutation in the genesis of metastasis.

Development of metastatic cancer is a complex series of events that includes genesis of tumor-related vascular and lymphatic systems, enhanced cellular motility, and the capacity to invade and survive at distant sites, as well as evasion of host defences. The wild-type p53 protein plays key roles in controlling these facets of tumor progression, and loss of normal p53 function can be sufficient to predispose tumor cells to gain metastatic properties. In contrast, dominant p53 mutants that have gained oncogenic functions can actively drive metastasis through a variety of mechanisms. This chapter aims to highlight these processes.

The induction of heme oxygenase-1 suppresses heat shock protein 90 and the proliferation of human breast cancer cells through its byproduct carbon monoxide

Heme oxygenase (HO)-1 is an oxidative stress-response enzyme which catalyzes the degradation of heme into bilirubin, ferric ion, and carbon monoxide (CO). Induction of HO-1 was reported to have antitumor activity; the inhibitory mechanism, however, is still unclear. In the present study, we found that treatment with [Ru(CO)3Cl2]2 (RuCO), a CO-releasing compound, reduced the growth of human MCF7 and MDA-MB-231 breast cancer cells. Analysis of growth-related proteins showed that treatment with RuCO down-regulated cyclinD1, CDK4, and hTERT protein expressions. Interestingly, RuCO treatment resulted in opposite effects on wild-type and mutant p53 proteins. These results were similar to those of cells treated with geldanamycin (a heat shock protein (HSP)90 inhibitor), suggesting that RuCO might affect HSP90 activity. Moreover, RuCO induced mutant p53 protein destabilization accompanied by promotion of ubiquitination and proteasome degradation. The induction of HO-1 by cobalt protoporphyrin IX (CoPP) showed consistent results, while the addition of tin protoporphyrin IX (SnPP), an HO-1 enzymatic inhibitor, diminished the RuCO-mediated effect. RuCO induction of HO-1 expression was reduced by a p38 mitogen-activated protein kinase inhibitor (SB203580). Additionally, treatment with a chemopreventive compound, curcumin, induced HO-1 expression accompanied with reduction of HSP90 client protein expression. The induction of HO-1 by curcumin inhibited 12-O-tetradecanoyl-13-acetate (TPA)-elicited matrix metalloproteinase-9 expression and tumor invasion. In conclusion, we provide novel evidence underlying HO-1's antitumor mechanism. CO, a byproduct of HO-1, suppresses HSP90 protein activity, and the induction of HO-1 may possess potential as a cancer therapeutic.

Abstract C299: Functional differences of mutant p53's expressed in MCF10A cells and their contribution to breast carcinogenesis.

Abstract Recent high-throughput tumor sequencing has confirmed a striking prevalence of somatic TP53 mutations in breast cancer (∼35% overall), usually associated with basal-like tumors. Patients with these aggressive tumors have worst clinical outcome, fewer treatment options and respond poorly to current therapies. Successful treatment of basal cancer patients may require a combined approach that addresses known cancer protein pathways, e.g, p53, as well as modifier pathways that collaborate with these driver pathways to cause cancer. For this, we are developing a functional screen to analyze the importance of co-driver mutations and find alternative targets that may offer target therapy for these breast cancer patients. Mutations in TP53 can induce dominant negative effects on wild-type (WT) p53 protein, including enhanced tumor growth, increased cell migration, invasion and metastasis. Dominant mutations do not necessarily lead to a null phenotype. Different p53 mutations result in gain-of-function phenotypes such as distinct patterns of growth, tumorigenicity and protein interactions, which suggest that individual TP53 mutations result in distinct cellular programs. We hypothesized that unique TP53 mutations would have distinct functional effects on the hallmarks of cancer. To study this, we generated MCF10A and HMEC-htert stable cells expressing the ten most frequent breast cancer mutations located in the DNA binding domain of TP53. Ectopic expression of p53 in stable cells has been confirmed by qRT-PCR and we are currently investigating the phenotypic changes associated with these mutations in several hallmarks of cancer such as 1) epithelial to mesenchymal transition (EMT), 2) migration 3) invasion 4) escape from apoptosis and 5) proliferation. Compared with wt-p53, the p53G245S, p53R248Q, p53Y234C and p53H179R mutants displayed a mesenchymal phenotype characterized by the presence of slug and a disrupted B-catenin and E-cadherin staining. Interestingly these same mutants were more invasive. Indeed, p53R248Q was one of the most resistant to apoptosis; follow by p53Y234C, which may suggest that these two mutations are the most aggressive. This also suggests that different mutants have different downstream effects like p53G245S and p53H179R, which are the most sensitive to apoptosis. In conjunction, these preliminary data suggest that different TP53 mutations result in distinct cellular programs. As soon as we complete the assay development we will set up the screen to determine the role of the co-drivers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C299. Citation Format: Laura G. Gonzalez-Malerva, Seron Eaton, Anasuya Pal, Donald Chow, Mayra Guzman, Eva Amouzougan, Hongwei Yin, Jin Park, Karen Anderson, Joshua LaBaer. Functional differences of mutant p53's expressed in MCF10A cells and their contribution to breast carcinogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C299.