P53 Hotspot Mutants Research Articles

The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

Heterogeneity of Li-Fraumeni Syndrome links to unequal gain-of-function effects of p53 mutations

Mutations of p53 cause not only loss of wild-type function but also gain of novel oncogenic functions (GOF). Accumulating evidence suggest that p53 hotspot mutations may confer different types and magnitudes of GOF. Here we add support to the heterogeneity of mutant p53 GOF by showing their unequal association with early tumor onset and spectrum of tumor types. We stratified Li-Fraumeni syndrome (LFS) patients according to carried p53 mutations using data from the updated p53 germline mutation database. When compared to loss-of-function nonsense mutations, the R282 GOF mutation associated with significantly earlier onset, while the G245 mutation displayed later onset. The R175, Y220, R248, R282 and nonsense mutations showed preferential distribution in certain cancer types, which varied in the age of onset. Multivariate COX regression model adjusting for cancer types and patient sex suggested that nonsense and G245 mutations had lower risk than R248 for early onset, suggesting unequal strengths of mutant GOF effects. Our results suggest that Li-Fraumeni syndrome can be subdivided into subtypes linking to unequal GOF effects of p53 mutations. These findings have potential implications in the prevention, early detection and targeted treatment of LFS tumors.

P53 Pathway Alterations Influence Survival and Treatment Response In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy, with survival rates that range from months to decades. Numerous genetic markers have been identifying that predict for poor survival outcomes. One of the most significant prognostic risk factors is loss of integrity of the p53 pathway. It is well known that loss of the short arm of chromosome 17 (17p-), containing the p53 locus, is a major predictor poor outcome in CLL, however, there is a lack of clarity how specific p53 mutations influence cancer progression. It is our contention that understanding the impact of p53 mutations in CLL progression will facilitate the development of better treatments options. We are using a mouse model of B-CLL (Eµ-TCL1) that also carry a p53 hot-spot mutation p53R172H, corresponding to R175H in humans, to study the impact of p53 mutation on treatment response and survival. Cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ were treated with fludarabine at 35mg/kg per day for 5 days each month. We have observed significant changes in survival and selective pressure on the p53 locus following fludarabine treatment, leading to a refractory population of clones. Mice in the Eu-TCL1 cohort received a median of 1 treatment cycle (Range; 1-3), while the mice in the Eµ-TCL1;p53R172H/+ cohort received a median of 3 treatment cycles (Range;1-4), suggesting that the mutant cohort are likely to benefit initially with treatment only to succumb to a refractory clone of CLL. Mice in the Eµ-TCL1 cohort had an overall survival of 373 days (range; 341-464 days) in comparison to 322 days (range; 270-382days) in the Eµ-TCL1;p53R172H/+ cohort, indicating a trend toward inferior survival in the Eµ-TCL1;p53R172H/+ cohort. Interestingly, mice in the Eµ-TCL1 cohort had significant splenomegaly (40%) as compared to the Eµ-TCL1;p53R172H/+ cohort (0%). However, we observed that all Eµ-TCL1;p53R172H/+ had extensive extranodal disease with mice having abdominal and/or cervical mass, which is suggestive of Richter's transformation. Together, these data indicate that fludarabine based therapies may have a profound negative transformational impact on CLL progression when used as a treatment strategy in the context of p53 mutations. These results would suggest that less cytotoxic therapies may benefit patients harboring p53 mutations. To this end, we are evaluating the effect of Ibrutinib therapy in the context of p53 mutations. We are currently treating cohorts of Eµ-TCL1 and Eµ-TCL1;p53R172H/+ mice with Ibrutinib 25mg/kg/day. Our preliminary results suggest Ibrutinib's innovative mechanism may lessen some of these selective pressures on the p53 pathway and may indicate Ibrutinib is a superior treatment option compared to fludarabine when p53 mutations are present. In conjunction with these mouse studies, we are also determining the impact of p53 mutation in human patients by examining changes in CLL patient samples and cell lines following fludaribine and Ibrutinib treatment. This dual mouse/human strategy represents an ideal system in which to study genetic anomalies in the p53 pathway and examine the dynamic molecular pressures exerted by both fludarabine and Ibrutinib in CLL. Disclosures:No relevant conflicts of interest to declare.

Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity

Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53-MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53-MDMX interaction, SJ-172550, the efficacy of a simplified yeast-based screening assay to search for inhibitors of p53-MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild-type (wt) p53-induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53-induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin-α, nutlin-3a and SJ-172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.

Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions.

A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.

Not all p53 gain-of-function mutants are created equal

Not all p53 gain-of-function mutants are created equal

Abstract LB-53: Prodigiosin rescues mutant p53 signaling and antitumor effects via p73 upregulation and disruption of mutant p53:p73 interaction.

Abstract Rescue of mutant p53 function is an attractive strategy for cancer therapy development. Using high throughput drug screening, we identified prodigiosin, which can reactivate p53-dependent transcriptional activity in p53 mutant cancer cells. We find that prodigiosin has the therapeutic potential of p53 signaling restoration in a panel of cancer cells with p53 hotspot mutants. Our study has indicated that prodigiosin can up-regulate the expression of p73. However, the function of the increased p73 could be inhibited by interacting with the overexpressed mutant p53 in cancer cells. The immunoprecipitation assay indicates that prodigiosin treatment disrupts the interaction of mutant p53 and p73 in mutant p53-containing SW480 cells. Prodigiosin has been reported to be able to bind to mTOR and inhibit mTOR activity. In order to determine whether prodigiosin disrupts the interaction of mutant p53/p73 complex through mTOR inhibition, we treated SW480 cells with the mTOR inhibitor rapamycin to assess whether mTOR inhibition can disrupt the interaction. The results indicate that prodigiosin disrupts the physical interaction of mutant p53 and p73, but rapamycin does not disrupt this interaction. Western blot confirms that both prodigiosin and rapamycin inhibit the mTOR pathway as indicated by the decrease of phospho-mTOR, phospho-p70 S6 Kinase and phospho-4E-BP1 when SW480 cells are treated with either prodigiosin or rapamycin. The result suggests that prodigiosin disrupts the interaction of mutant p53/p73 complex independent of mTOR inhibition. Furthermore, we examine the role of mutant p53 in the mechanism of p53 restoration by prodigiosin. By the knock-down of mutant p53 by siRNA treatment, we find that prodigiosin-induced p53 activity is increased in mutant p53 knock-down SW480 cells and mutant p53 knock-down SW480 cells are more sensitive to prodigiosin-induced cell death. The result suggests that mutant p53 has the dominant-negative function. Our study implies that the only p73 up-regulation may not have a potent anti-tumor effect in p53 mutant cancers, as overexpressed mutant p53 would be expected to inhibit p73 function by protein-protein interaction. Our study indicates that prodigiosin not only up-regulates p73, but also prevents p73 from interacting with mutant p53. Therefore, the dual effects (up-regulation of p73 and disruption of the interaction between mutant p53 and p73) of prodigiosin make it a very promising candidate in anti-cancer drug development. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: Bo Hong, Antonius Van Den Heuvel, Levy Kopelovich, Wafik S. El-Deiry. Prodigiosin rescues mutant p53 signaling and antitumor effects via p73 upregulation and disruption of mutant p53:p73 interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-53. doi:10.1158/1538-7445.AM2013-LB-53

Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis.

Mutant p53 proteins not only lose their tumor-suppressor function but some acquire oncogenic gain of function (GOF). The published mutp53 knock-in (KI) alleles (R172H, R270H, R248W) manifest GOF by broader tumor spectrum and more metastasis compared with the p53-null allele, but do not shorten survival. However, whether GOF also occurs with other mutations and whether they are all biologically equal is unknown. To answer this, we created novel humanized mutp53 KI mice harboring the hot spot alleles R248Q and G245S. Intriguingly, their impact was very different. Compared with p53-null mice, R248Q/- mice had accelerated onset of all tumor types and shorter survival, thus unprecedented strong GOF. In contrast, G245S/- mice were similar to null mice in tumor latency and survival. This was associated with a twofold higher T-lymphoma proliferation in R248Q/- mice compared with G245S/- and null mice. Moreover, R248Q/- hematopoietic and mesenchymal stem cells were expanded relative to G245S/- and null mice, the first indication that GOF also acts by perturbing pretumorous progenitor pools. Importantly, these models closely mirror Li-Fraumeni patients who show higher tumor numbers, accelerated onset and shorter tumor-free survival by 10.5 years when harboring codon R248Q mutations as compared with Li-Fraumeni patients with codon G245S mutations or p53 deletions/loss. Conversely, both KI alleles caused a modest broadening of tumor spectrum with enhanced Akt signaling compared with null mice. These models are the first in vivo proof for differential oncogenic strength among p53 GOF alleles, with genotype-phenotype correlations borne out in humans.

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.

Effect of CpG methylation at different sequence context on acrolein- and BPDE-DNA binding and mutagenesis

Acrolein (Acr), an α,β-unsaturated aldehyde, is abundant in tobacco smoke and cooking and exhaust fumes. Acr induces mutagenic α- and γ- hydroxy-1,N(2)-cyclic propano-deoxyguanosine adducts in normal human bronchial epithelial cells. Our earlier work has found that Acr-induced DNA damage preferentially occurs at lung cancer p53 mutational hotspots that contain CpG sites and that methylation at CpG sites enhances Acr-DNA binding at these sites. Based on these results, we hypothesized that this enhancement of Acr-DNA binding leads to p53 mutational hotspots in lung cancer. In this study, using a shuttle vector supF system, we tested this hypothesis by determining the effect of CpG methylation on Acr-DNA binding and the mutations in human lung fibroblasts. We found that CpG methylation enhances Acr-induced mutations significantly. Although CpG methylation enhances Acr-DNA binging at all CpG sites, it enhances mutations at selective--TCGA--sites. Similarly, we found that CpG methylation enhances benzo(a)pyrene diol epoxide binding at all -CpG- sites. However, the methylated CpG sequences in which benzo(a)pyrene diol epoxide-induced mutations are enhanced are different from the CpG sequences in which Acr-induced mutations are enhanced. CpG methylation greatly increases Acr-induced G to T and G to A mutation frequency to levels similar to these types of mutations found in the CpG sites in the p53 gene in tobacco smoke-related lung cancer. These results indicate that both CpG sequence context and the chemical nature of the carcinogens are crucial factors for determining the effect of CpG methylation on mutagenesis.

Structural Basis for ASPP2 Recognition by the Tumor Suppressor p73

Tumor suppressors p53, p63 and p73 comprise a family of stress-responsive transcription factors with distinct functions in development and tumor suppression. Most human cancers lose p53 function, yet all three proteins are capable of inducing apoptosis or cellular senescence. Mechanisms are therefore under investigation to activate p73-dependent apoptosis in p53-deficient cancer cells. Significantly, the DNA-binding domain (DBD) of p73 escapes viral oncoproteins and displays an enhanced thermal stability. To further understand the variant features of p73, we solved the high‐resolution crystal structure of the p73 DBD as well as its complex with the ankyrin repeat and SH3 domains of the pro-apoptotic factor ASPP2. The p73 structure exhibits the same conserved architecture as p53 but displays a divergent L2 loop, a known site of protein–protein interaction. The loop in p73 is changed by a two-residue insertion that also induces repacking around the site of the p53 mutational hotspot R175. Importantly, the binding of ASPP2 is preserved by conformational changes in both the ankyrin repeat and SH3 domains. These results further highlight the structural variation that impacts p53 family interactions within the p53 interactome.

MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function

p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21(waf1) transcription. p21(waf1) protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance.

TopBP1 Mediates Mutant p53 Gain of Function through NF-Y and p63/p73

Nearly half of human cancers harbor p53 mutations, which can promote cancerous growth, metastasis, and resistance to therapy. The gain of function of mutant p53 is partly mediated by its ability to form a complex with NF-Y or p63/p73. Here, we demonstrate that TopBP1 mediates these activities in cancer, and we provide both in vitro and in vivo evidence to support its role. We show that TopBP1 interacts with p53 hot spot mutants and NF-YA and promotes mutant p53 and p300 recruitment to NF-Y target gene promoters. TopBP1 also facilitates mutant p53 interaction with and inhibition of the transcriptional activities of p63/p73. Depletion of TopBP1 in mutant p53 cancer cells leads to downregulation of NF-Y target genes cyclin A and Cdk1 and upregulation of p63/p73 target genes such as Bax and Noxa. Mutant p53-mediated resistance to chemotherapeutic agents depends on TopBP1. The growth-promoting activity of mutant p53 in a xenograft model also requires TopBP1. Thus, TopBP1 mediates mutant p53 gain of function in cancer. Since TopBP1 is often overexpressed in cancer cells and is recruited to cooperate with mutant p53 for tumor progression, TopBP1/mutant p53 interaction may be a new therapeutic target in cancer.

Abstract 240: Functional impact of p53 hotspot mutantions on tumor development and therapy responses in the Eµ-myc mouse lymphoma model

Abstract The tumor suppressor p53 is mutated in the majority of human cancers, and inactivation of p53 correlates with more aggressive clinical courses and resistance to therapy. Loss of p53 may result in pleiotropic cellular effects such as cell-cycle deregulation, increased survival and aneuploidy. Utilizing the Eµ-myc transgenic mouse as a model system, we previously identified apoptosis as the prime tumor suppressor function of p53 during Myc-driven lymphoma development. However, increasing evidence supports premature senescence as another p53-dependent tumor suppressor program. When exposed to therapy, apoptosis-blocked lymphomas selected against p53, because p53 not only controls apoptosis but also senescence in response to DNA damage. Most of the naturally occurring p53 mutations are missense mutations, preferentially at some ‘hotspots’, which result in expressed mutant proteins. Moreover, different entities show distinct hotspot preferences, implying that individual p53 mutants may have different impact on p53-effector programs, or may even present with novel, oncogenic functions. Revealing the functional impact of mutant p53 on cancer cell biology and subsequent response to treatment is of great interest in the field, and numerous studies including individual mutant knock-in mouse models have been conducted. Nevertheless, no comprehensive approach to elucidate the specific role of the cancer-derived mutations in tumor formation and therapy has been undertaken yet. Our study focuses on the functions of p53 hotspot mutants in a mouse model that faithfully recapitulates human B-cell lymphomagenesis. Mutant p53 constructs were introduced into Eµ-myc transgenic hematopoietic progenitor cells to generate primary malignancies that express individual. Tumor development under different wild-type/mutant p53 combinations was compared and the resulting lymphomas were assayed with particular emphasis on apoptosis and senescence as the key tumor suppressor mechanisms. Most mutants supported rapid tumor development in the presence of wild-type p53, while only some promoted lymphoma formation in p53+/- and p53-null backgrounds. These mutants are selected for since they inhibit p53-dependent apoptosis as the main cause of accelerated lymphoma development. Interestingly, these mutants did not compromise the cellular ability to enter senescence. We found senescence driven by mutant and wild-type p53 not to be strictly dependent on the p53 downstream target p21. The precise mechanism how mutant p53 induces senescence is currently under investigation. Importantly, p53 mutant-specific alterations of pro-apoptotic, pro-senescent and metabolic p53 functions have profound implications for the outcome to cancer therapy in vitro and in vivo, as we will present at the meeting. Our goal is to develop personalized treatment strategies against tumors with defined p53 lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 240. doi:10.1158/1538-7445.AM2011-240

A High-Frequency Regulatory Polymorphism in the p53 Pathway Accelerates Tumor Development

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele.

Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA

Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA that binds p53 and induces a conformational change which prevents the binding to p53 of several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests the generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.

Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells

Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.

Abstract B54: Identification of the PI3K-mTOR pathway as regulator of mutant p53 turnover in human cancer cells

Abstract The tumor suppressor p53 plays a crucial role in our cells to induce cell cycle arrest or apoptosis in response to different kinds of genotoxic stresses. In 50% of all solid tumors, p53 is mutated by a single point mutation within the DNA-binding domain and thereby loses its activity as a tumor suppressor. However, at the same time, mutant p53 actively contributes to chemoresistance and tumor progression through a gain of function (Dittmer et al., 1993). It was previously shown that mutant p53 highly accumulates in tumors of mice with targeted germ line mutations of the p53 gene (knock-in), whereas normal cells of these mice express it at low levels (Lang et al., 2004; Terzian et al., 2008). This argues that the deregulation of tumor-specific signaling cascades leads to an accumulation of mutant p53 and subsequently to tumor progression. Our study aims at identifying these signaling pathways that contribute to the regulation of mutant p53 expression, and to describe their mechanisms. We performed quantitative analysis of mutant p53 protein levels in a cancer cell line after the individual knock-down of all known human kinases using automated microscopy (high content cell screening). The bladder carcinoma derived cell line 5637 (carrying the p53 R280T mutation) was transfected with three different synthetic siRNAs targeting each known kinase or kinase regulator in a 96-well format, and p53 was detected by automated immunofluorescence microscopy. In each of the resulting 2400 cell populations the p53 expression levels were determined for at least 500 individual cells. Alterations in the distribution of p53 staining intensities were assessed for each kinase relative to non-targeting control siRNAs. The results of the kinases, that lead to the strongest up- or down-regulations of mutant p53 in the screen, were confirmed in different cell lines (U251 and U373, both derived from glioma, with the p53 hot spot mutation R273H), by immunofluorescence and immunoblot analysis. We identified a group of kinases currently known to act in the PI3-kinase → mTOR pathway as potential regulators of mutant p53. The knock-down of these kinases resulted in a decrease of mutant p53 expression. Studying the mechanism of mutant p53 regulation, we performed quantitative real-time PCR after siRNA transfection and found no significant impairment of the p53 transcription. On the contrary, posttranslational modifications (phosphorylation and acetylation) of mutant p53 were decreased after knock-down of the identified kinases, even more strongly than the overall levels of the protein. These observations support the idea that the degradation of mutant p53 is decreased in cancer cells through alterations of PI3K-mTOR pathway associated kinases, resulting in p53 accumulation and oncogenic gain of function. Citation Information: Cancer Res 2009;69(23 Suppl):B54.

Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme‐Linked Electrochemical Assay

AbstractAn enzyme‐linked electrochemical technique for single nucleotide polymorphism (SNP) typing in the p53 tumor suppressor gene is presented. The technique is based on a DNA polymerase‐catalyzed extension of a primer hybridized to a target DNA strand upstream (5′→3′) to the SNP site by one nucleotide bearing a biotin tag. Under optimized conditions, efficient incorporation of the biotinylated nucleotide occurs only in the case of complementarity between the first nucleotide in single‐stranded 5′‐overhang of the target strand. The introduced biotin tag is detected after capture of the primer extension products at magnetic beads bearing oligoT strands via oligoA adaptors at 5′‐ends of the primer, binding of streptavidin‐alkaline phosphatase conjugate and enzymatic conversion of 1‐naphthyl phosphate into 1‐naphthol which is determined electrochemically at carbon electrodes. In addition to model studies with synthetic oligonucleotides, we report on detection of mutant p53 expression in human cell lines using reverse transcription‐PCR technique combined with amplified primer extension and the magnetic beads‐based electrochemical assay.

Mutagenicity and Sequence Specificity of Acrolein-DNA Adducts

Acrolein (Acr) is a major toxicant in cigarette smoke (CS); it can interact with DNA forming two major adduct isomers: alpha-OH-Acr-dG and gamma-OH-Acr-dG. Previously, we found that the Acr-DNA binding pattern in the human p53 gene coincides with the p53 mutational pattern in CS-related lung cancer; hence, we proposed that Acr is a major lung cancer etiological agent [ Feng , Z. , Hu , W. , Hu , Y. , and Tang , M.-s. ( 2006 ) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair . Proc. Natl. Acad. Sci. U.S.A. 103 , 15404 - 15409 ]. This hypothesis has been brought into question with recent work that failed to detect Acr-induced mutations in the pSP189 system [ Kim , S. I. , Pfeifer , G. P. , and Besaratinia , A. ( 2007 ) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells . Cancer Res. 67 , 11640 - 116472 ]. To resolve this controversy, we determined the level and the type of Acr-dG formation, and the mutagenicity of Acr-dG adducts in the same pSP189 system. We also mapped the Acr-dG adduct distribution at the nucleotide level and the Acr-dG-induced mutational spectrum in this system. We found that (1) gamma-OH-Acr-dG is the major adduct formed in Acr-modified DNA based on the LC-ESI-MS/MS analysis; (2) the mutation frequency is proportional to the extent of Acr modifications, the majority of which are G:C to T:A and G:C to A:T mutations; and (3) sequences with a run of Gs are the mutational hotspots. Using the UvrABC nuclease incision method to map the Acr-dG distribution in the supF gene sequence, we confirmed that Acr-DNA adducts preferentially form in guanine-rich sequences that are also mutational hotspots. These results reaffirm that Acr-dG adducts are mutagenic and support our hypothesis that Acr is a major etiological agent for CS and cooking fume-related lung cancer.