Inactivation Of Wild-type P53 Research Articles

Mutant P53 induces MELK expression by release of wild-type P53-dependent suppression of FOXM1

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.

MDM2 antagonists as a novel treatment option for acute myeloid leukemia: perspectives on the therapeutic potential of idasanutlin (RG7388).

Acute myeloid leukemia (AML) is a clonal heterogenous malignancy of the myeloid cells with a poor prognosis lending itself to novel treatment strategies. TP53 is a critical tumor suppressor and plays an essential role in leukemogenesis. Although TP53 is relatively unusual in de novo AML, inactivation of wild-type p53 (WT-p53) is a common event. Murine double minute 2 (MDM2) is a key negative regulator of p53 and its expression; inhibition of MDM2 is postulated to reactivate WT-p53 and its tumor suppressor functions. Nutlins were the first small molecule inhibitors that bind to MDM2 and target its interaction with p53. RG7388 (idasanutlin), a second-generation nutlin, was developed to improve upon the potency and toxicity profile of earlier nutlins. Preliminary data from early phase trials and ongoing studies suggest clinical response with RG7388 (idasanutlin) both in monotherapy and combination strategies in AML. We herein briefly discuss currently approved therapies in AML and review the clinical data for RG7388 (idasanutlin) and MDM2 inhibition as novel treatment strategies in AML. We further describe efficacy and toxicity profile data from completed and ongoing trials of RG7388 (idasanutlin) and other MDM2-p53 inhibitors in development. Many targeted therapies have been approved recently in AML, with a focus on the older and unfit population for intensive induction therapy and in relapsed/refractory disease. The “nutlins”, including RG7388 (idasanutlin), merit continued investigation in such settings.

An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas

BackgroundInactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.MethodsWe analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.Results50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.ConclusionsThe results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.

Alterations in gene expression and sensitivity to genotoxic stress following HdmX or Hdm2 knockdown in human tumor cells harboring wild-type p53

While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth.Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown.Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.

Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis along with reduced endoreduplication in acute myelogenous leukemia

AbstractAberrant expression of Aurora kinases and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML), and preclinical data for inhibition of Aurora kinases or Mdm2 are promising. However, it remains largely unknown whether the viability of cells exposed to Aurora kinase inhibitors depends on the p53 status. We investigated the interaction of Aurora kinases and p53 pathways after their simultaneous blockades using a small-molecule pan-Aurora kinase inhibitor, MK-0457, and a selective small-molecule antagonist of Mdm2, Nutlin-3. We found that MK-0457, which itself activates p53 signaling, acts synergistically with Nutlin-3 to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13 but not in p53-null HL-60 cells. MK-0457 and Nutlin-3 showed synergism in inducing p53, conformational change of Bax and Δψm loss, suggesting an involvement of p53-mediated mitochondrial apoptosis. Nutlin-3 constrained endoreduplication after Aurora inhibition via activation of a p53-dependent postmitotic checkpoint and p21 induction in pseudo-G1 cells. Our findings provide the molecular rationale for concomitant targeting of Aurora kinases and Mdm2 in AML where TP53 mutations are rare and downstream p53 signaling is mostly intact.

The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2 inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML.

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

Disruption of estrogen receptor α-p53 interaction in breast tumors: a novel mechanism underlying the anti-tumor effect of radiation therapy

Inactivation of tumor suppressor p53 is one of the most frequent events in cancer. Unlike many other cancers, however, p53 gene mutations are infrequent in breast cancers, as about 80% of breast tumors contain wild type p53. The mechanisms underlying functional inactivation of wild type p53 in breast cancer have remained elusive. Besides, how p53 gets activated in breast tumors subjected to radiation therapy remains unknown. We recently reported that in MCF-7 breast cancer cells, estrogen receptor alpha (ERalpha) directly binds to p53 and represses its function. Furthermore, the ERalpha-p53 interaction was disrupted by ionizing radiation. These observations have important translational implications especially as there are no reliable cellular or molecular criteria for rational radiotherapy for breast cancer. Here we report our studies towards addressing this important issue, using an MCF-7 breast cancer xenograft model in mice. Radiation effectively inhibits growth of these tumors and stabilizes p53, but has no observable effect on ERalpha protein level. Importantly, chromatin immunoprecipitation (ChIP) assays demonstrated that ERalpha interacts with p53 bound to endogenous target gene promoters in tumors in vivo, and this interaction is considerably reduced in response to radiotherapy although p53 level is increased. Concomitant with its effect on ERalpha-p53 interaction, radiation increases p53-mediated transcriptional activation of several target genes and increases p53-mediated transcriptional repression of survivin. Our studies show that disruption of ERalpha-p53 interaction in vivo resulting in restoration of functional p53 is a cellular response to radiation. Radiation could be affecting ERalpha and/or p53 directly or it could be influencing other proteins associated with the ERalpha-p53 complex. To the best of our knowledge, this is the first report on analysis of DNA-protein-protein interaction occurring on endogenous gene promoters in vivo in breast tumor tissues. These findings suggest that alleviating the inhibitory effect of ERalpha on p53 could be one of the molecular mechanisms underlying activation of p53 by radiation in breast tumors, and therefore, could be exploited to develop more effective ways of combining radiation therapy with systemic therapies such as hormonal therapy and chemotherapy.

Mitogen-Activated Protein Kinase Kinase Inhibition Enhances Nuclear Proapoptotic Function of p53 in Acute Myelogenous Leukemia Cells

Activation of the Raf/MEK/ERK pathway and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML). We investigated the interaction of Raf/MEK/ERK and p53 pathways after their simultaneous blockades using a selective small-molecule antagonist of Mdm2, Nutlin-3a, and a pharmacologic MEK-specific inhibitor, PD98059. We found that PD98059, which itself has minimal apoptogenic activity, acts synergistically with Nutlin-3a to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13. Interestingly, PD98059 enhanced nuclear proapototic function of p53 in these cells. In accordance with the activation of transcription-dependent apoptosis, PD98059 treatment promoted the translocation of p53 from the cytoplasm to the nucleus in OCI-AML-3 cells, in which p53 primarily initiates transcription-independent apoptosis when cells are treated with Nutlin-3a alone. The critical role of p53 localization in cells with increased p53 levels was supported by enhanced apoptosis induction in cells cotreated with Nutlin-3a and the nuclear export inhibitor leptomycin B. PD98059 prevented p53-mediated induction of p21 at the transcriptional level. The repressed expression of antiapototic p21 also seemed to contribute to synergism between PD98059 and Nutlin-3a because (a) the synergistic apoptogenic effect was preserved in G(1) cells, (b) p53-mediated induction of p21 was preferentially seen in G(1) cells, (c) PD98059 strongly antagonized p21 induction by Nutlin-3a, and (d) cells with high p21 levels were resistant to apoptosis. This is the first report showing that the Raf/MEK/ERK pathway regulates the subcellular localization of p53 and the relative contribution of transcription-dependent and transcription-independent pathways in p53-mediated apoptosis.

5-lipoxygenase antagonizes genotoxic stress-induced apoptosis by altering p53 nuclear trafficking.

5-lipoxygenase (5-LO) promotes cancer cell proliferation and survival by unclear mechanisms. Here, we show that 5-LO expression and activity were induced by genotoxic agents in a p53-independent manner and antagonized p53- or genotoxic drug-induced apoptosis in a variety of cancer cells. 5-LO inhibited p53-governed transactivation of the pro-apoptotic genes bax and pig3 but not of p21(WAF1/CIP1) or mdm2. This may be explained by 5-LO capability to inhibit the binding of p53 to promyelocytic leukemia protein (PML) and p53 subnuclear relocalization into PML-nuclear bodies in response to genotoxic stress. Interestingly, 5-LO activity appears to be involved in nuclear retention and inactivation of wild-type p53 in malignant mesothelioma cells. In these cells, genetic or pharmacological inhibition of 5-LO enabled suppression of in vitro tumorigenicity by low doses of chemotherapeutic drugs. Together, these results uncover novel functions of 5-LO and contribute to the understanding of 5-LO involvement in tumor progression. Moreover, they provide a rationale to the therapeutic use of 5-LO inhibitors to enhance cancer chemosensitivity in selected tumors.

Inactivation of wild-type p53 by a dominant negative mutant renders MCF-7 cells resistant to tubulin-binding agent cytotoxicity.

The present study was performed to gain insight into the role of p53 on the cytotoxicity of tubulin-binding agents (TBA) on cancer cells. Drug sensitivity, cell cycle distribution and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the MN-1 cell line containing wild-type p53 (wt-p53) and the MDD2 line, containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to the cytotoxic effects of vinblastine and paclitaxel than the MN1 cell line. MN1 cells, but not MDD2 cells, displayed wt-p53 protein accumulation as well as p21/WAF1 and cyclin G1 induction after exposure to TBA. Both cell lines arrested at G2/M after drug treatment. However exposure of MN1 cells to TBA resulted in a stronger variation in mitochondrial membrane potential, associated with cleavage of PARP, and more apoptosis, as measured by annexin V expression. After exposure to vinblastine, Raf 1 kinase activity was reduced in MDD2 cells but not in MN1 cells. Addition of flavopiridol to vinblastine- and paclitaxel-treated cells reversed the MDD2-resistant phenotype by inducing G1 cell cycle arrest and inhibiting endoreduplication. We conclude that the p53 status of cancer cells influences their sensitivity to TBA cytotoxicity. This effect is likely to involve differences in the apoptotic cascade. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells.

Numerous studies have indicated that inactivation of p53 is one of the essential requirements for the unrestrained growth of tumoral cells. When the status of the p53 gene was examined in various types of lymphoid malignancies, mutations in p53 have been predominantly detected in Burkitt's lymphoma (BL) cells, therefore suggesting that alteration of p53 could specifically contribute to the malignant phenotype of these tumoral cells. In addition to mutations, functional inactivation of p53 can also occur through interaction of the wild-type gene product with various viral or cellular proteins. The cellular MDM2 protein, for example, is able to inhibit p53 tumor suppressor function by concealing its transactivation domain. Mdm2 gene amplification has been described in several types of sarcomas, resulting in overexpression of the MDM2 protein. In this study, we have examined the status of MDM2 and p53 in 20 BL cell lines. Four were found to contain wild-type p53 and to overexpress MDM2 protein. Within these BL cells, both molecules are physically associated since they can be co-precipitated and p53 is inactivated as cells neither arrest in G1 nor enter apoptosis following gamma-radiation. We also report that the high level of the MDM2 protein in BL cells is neither associated with an amplification of the mdm2 gene nor with an elevated level of RNA or an increased protein stability, but is rather due to an enhanced translation ability of the mdm2 RNA. These results indicate that in certain BL cells, overexpression of MDM2 protein regulated at the posttranscriptional level, induces an escape from p53-controlled cell growth.

Loss of heterozygosity at 17p13 in gastric cancer and colorectal cancer

Southern hybridization was done on DNA samples of 22 gastric tumors and corresponding normal tissues, 14 colorectal tumors and corresponding normal tissues by probe php53B mapping at 17p13.1 and probe pYNZ22 mapping at 17p13.3 which were purchased from the American Type Culture Collection. RFLP heterozygosity was observed in 12 normal tissues of gastric cancers and 10 normal tissues of colorectal cancers. Among these informative tumors, 6(50%) cases of gastric cancers and 6(60%) cases of eolorectal cancers showed the loss of heterozygosity at 17p13. Our results demonstrated that the inactivation of wild type p53 might be involved in the carcinogenesis of gastric cancers and colorectal cancers. Furthermore, the mode of inactivation of p53 was in accord with the two hits hypothesis by knudson. The significance was discussed regarding the presence of LOH detected by probe pYNZ22 mapping at 17p13.3.

DIFFERENT SUBNUCLEAR LOCALIZATION OF WILD-TYPE AND MUTANT P53 IN HUMAN PROSTATE-CANCER CELLS

The growth suppressor protein p53 is abnormally expressed in a variety of different human tumor cells. We have analyzed the expression of p53 in cell cultures derived from tissues of radical prostatectomies and in the permanent prostate carcinoma cell line PC-3 using two different p53 specific monoclonal antibodies. With the wild-type specific monoclonal antibody PAb1620 we found p53 localized in nucleoli whereas only a few cells were positively stained in the nucleus with the mutant specific monoclonal antibody PAb240. Control experiments with p53 from SV80 cells which express wild-type p53 and HT29 cells expressing mutant p53 documented the specificity of the monoclonal antibodies. The specificity of the antibodies in recognizing indeed p53 was demonstrated further by immunoprecipitation analysis of p53 from the same cell cultures. Since p53 is usually localized to the nucleus our results may represent a specific feature of the wild-type phenotype of p53 in prostate carcinoma cells. The localization of p53 in nucleoli may be another mechanism of the inactivation of wild-type p53.

Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.