Mutant Forms Of P53 Research Articles

Salvation of the fallen angel: Reactivating mutant p53.

The transcription factor p53 is known as the guardian of the genome for its powerful anti-tumour capacity. However, mutations of p53 that undermine their protein structure, resulting in loss of tumour suppressor function and gain of oncogenic function, have been implicated in more than half of human cancers. The crucial role of mutant forms of p53 in cancer makes it an attractive therapeutic target. A large number of candidates, including low MW compounds, peptides, and nucleic acids, have been identified or designed to rescue p53 mutants and reactivate their anti-tumour capacity through a variety of mechanisms. In this review, we summarize the progress made in the reactivation of mutant forms of p53, focusing on the pharmacological mechanisms of the reactivators of p53 mutants.

Fam83F induces p53 stabilisation and promotes its activity.

p53 is one of the most important tumour suppressor proteins currently known. It is activated in response to DNA damage and this activation leads to proliferation arrest and cell death. The abundance and activity of p53 are tightly controlled and reductions in p53's activity can contribute to the development of cancer. Here, we show that Fam83F increases p53 protein levels by protein stabilisation. Fam83F interacts with p53 and decreases its ubiquitination and degradation. Fam83F is induced in response to DNA damage and its overexpression also increases p53 activity in cell culture experiments and in zebrafish embryos. Downregulation of Fam83F decreases transcription of p53 target genes in response to DNA damage and increases cell proliferation, identifying Fam83F as an important regulator of the DNA damage response. Overexpression of Fam83F also enhances migration of cells harbouring mutant p53 demonstrating that it can also activate mutant forms of p53.

Wild-type and cancer-related p53 proteins are preferentially degraded by MDM2 as dimers rather than tetramers.

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.

Friend or Foe: MicroRNAs in the p53 network

The critical tumor suppressor gene TP53 is either lost or mutated in more than half of human cancers. As an important transcriptional regulator, p53 modulates the expression of many microRNAs. While wild-type p53 uses microRNAs to suppress cancer development, microRNAs that are activated by gain-of-function mutant p53 confer oncogenic properties. On the other hand, the expression of p53 is tightly controlled by a fine-tune machinery including microRNAs. MicroRNAs can target the TP53 gene directly or other factors in the p53 network so that expression and function of either the wild-type or the mutant forms of p53 is downregulated. Therefore, depending on the wild-type or mutant p53 context, microRNAs contribute substantially to suppress or exacerbate tumor development.

Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

TP53, which encodes the tumour-suppressor protein p53, is the most frequently mutated gene across all cancer types. The presence of mutant p53 predisposes to cancer development, promotes the survival of cancer cells, and is associated with ineffective therapeutic responses and unfavourable prognoses. Despite these effects, no drug that abrogates the oncogenic functions of mutant p53 has yet been approved for the treatment of cancer. Current investigational therapeutic strategies are mostly aimed at restoring the wild-type activity of mutant p53, based on the assumption that all p53 mutants are functionally equal. Our increasing knowledge of mutant forms of p53, however, supports the antithetical hypothesis that not all p53 mutants have equivalent cellular effects; hence, a judicious approach to therapeutic targeting of mutant p53 is required. In this Review, we propose a categorization of the major classes of p53 mutants based on their functionality in tumour suppression and response to therapy. The emerging picture is that the mutations across TP53 form a 'rainbow of mutants', with varying degrees of functionality and different pathobiological consequences, necessitating the use of diverse therapeutic strategies to selectively target specific classes of mutation. The utility of this knowledge of TP53 mutations in developing selective therapeutic options, and in facilitating clinical decision-making is discussed.

From the Literature

From the Literature

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: (i) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and (ii) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Effect of p53β on human gastric cancer cells treated with recombinant mutated human TNF and cisplatin.

The present study aimed to investigate the role of tumour protein 53 isoform b (p53β) on human gastric cancer (GC) cell lines treated with recombinant mutated human tumour necrosis factor (rmhTNF) and cisplatin. The Cell Counting Kit‑8 assay was used to assess growth in the GC cell lines MKN45 and SGC7901, following treatment with rmhTNF in the presence or absence of cisplatin. Levels of p53β and bcl‑2 apoptosis regulator (bcl‑2) mRNA were assessed using reverse transcription‑polymerase chain reaction. The results demonstrated that growth was significantly inhibited by either cisplatin or rmhTNF treatments alone in MKN45 cells, and combination treatment with cisplatin and rmhTNF had a synergistic effect on growth inhibition of MKN45 cells. Notably, these observations were not evident in SGC7901 cells, where a mutant form of p53 is present. Treatment of MKN45 cells with rmhTNF did not affect bcl‑2 or p53β mRNA expression levels. However, treatment of MKN45 cells with cisplatin induced upregulation of p53β and downregulation of bcl-2 mRNA expression levels, and these effects were enhanced by combination treatment with rmhTNF. Pearson correlation analysis revealed a negative correlation between the expression of p53β and bcl‑2 mRNA, and a negative correlation between bcl-2 mRNA expression and the inhibition of cell growth. In conclusion, the inhibitory effect of cisplatin on the growth of MKN45 GC cells was enhanced by rmhTNF via unknown mechanisms that involved p53β, indicating that p53β may be an appropriate therapeutic target for the treatment of GC.

Transcription factor p53 and skin aging

The review is dedicated to one of the molecular factors of skin aging essential for cosmetic medicine for the elderly. Cell renewal processes slow down with aging, and the proliferation–apoptosis equilibrium shifts towards cell death. One of the pivotal apoptotic markers is transcription factor p53. Its expression in skin keratinocytes increases under the influence of ultraviolet radiation. Mutant forms of p53 have been revealed in 70% of keratinocytes of skin exposed to ultraviolet radiation. On the one hand, suppression of p53 expression decreases apoptosis in skin cells, thereby decelerating senescence. On the other hand, it promotes the development of neoplasms in the skin. Thus, support of the physiological balance of p53 expression in skin cells is important for theoretical and practical gerontocosmetology. In addition, p53 can be used as a marker for skin cell functionality in response to anti-aging cosmetic products and to instrumental cosmetology.

When the guardian sleeps: Reactivation of the p53 pathway in cancer.

The p53 tumor suppressor is inactivated in most cancers, thus suggesting that loss of p53 is a prerequisite for tumor growth. Therefore, its reintroduction through different means bears great clinical potential. After a brief introduction to current knowledge of p53 and its regulation by the ubiquitin-ligases MDM2/MDMX and post-translational modifications, we will discuss small molecules that are able to reactivate specific, frequently observed mutant forms of p53 and their applicability for clinical purposes. Many malignancies display amplification of MDM genes encoding negative regulators of p53 and therefore much effort to date has concentrated on the development of molecules that inhibit MDM2, the most advanced of which are being tested in clinical trials for sarcoma, glioblastoma, bladder cancer and lung adenocarcinoma. These will be discussed as will recent findings of MDMX inhibitors: these are of special importance as it has been shown that cancers that become resistant to MDM2 inhibitors often amplify MDM4. Finally, we will also touch on gene therapy and vaccination approaches; the former of which aims to replace mutated TP53 and the latter whose goal is to activate the body's immune system toward mutant p53 expressing cells. Besides the obvious importance of MDM2 and MDMX expression for regulation of p53, other regulatory factors should not be underestimated and are also described. Despite the beauty of the concept, the past years have shown that many obstacles have to be overcome to bring p53 reactivation to the clinic on a broad scale, and it is likely that in most cases it will be part of a combined therapeutic approach. However, improving current p53 targeted molecules and finding the best therapy partners will clearly impact the future of cancer therapy.

Transcriptional Regulation by Wild-Type and Cancer-Related Mutant Forms of p53.

TP53 missense mutations produce a mutant p53 protein that cannot activate the p53 tumor suppressive transcriptional response, which is the primary selective pressure for TP53 mutation. Specific codons of TP53, termed hotspot mutants, are mutated at elevated frequency. Hotspot forms of mutant p53 possess oncogenic properties in addition to being deficient in tumor suppression. Such p53 mutants accumulate to high levels in the cells they inhabit, causing transcriptional alterations that produce pro-oncogenic activities, such as increased pro-growth signaling, invasiveness, and metastases. These forms of mutant p53 very likely use features of wild-type p53, such as interactions with the transcriptional machinery, to produce oncogenic effects. In this review, we discuss commonalities between wild-type and mutant p53 proteins with an emphasis on transcriptional processes.

P53 and HIC1 in Brain Cancer Gliomas

Hypermethylated in Cancer 1 (HIC1) is a novel tumor suppressor gene (TSG) which acts as a transcriptional enhancer and growth obliterator, identified by Baylin et al. more than 10 years back. HIC1 is found to be methylated in varieties of human cancers as seen in ductal carcinoma of the breast wherein promoter methylation and allelic loss of 17p13.3 chromosomal locus has been reported without any significant mutation in p53 gene. To determine the targets, Zhang et al. reported that Ephrin-A1 is one potential target for the TSG role of HIC1 in breast cancer cells. The region 17p13.3 consists of Variable Number of Tandem Repeats (VNTR) microsatellite markers such as YNZ22/D17S5/D17S30 and show intermittent DNA hypermethylation in neural cancer, colon cancer and lung cancer. Epigenetic silencing also is very commonly observed in liver cancer, breast cancer, gastric cancer, prostate cancer and is also seen in human male nonseminomatous germ cell tumors. The central coding domain of HIC1 exhibits elevated and eminent level of methylation in acute myeloid leukemia. Our laboratory had showed that 17p13.3 was preferentially affected in high grade primary human gliomas and the same was associated with increased p53 immunopositivity and proliferation within a particular tumor grade. Both HIC1 and p53 are catalogued as tumor repressors, HIC1 practically comply with p53 in cells cultures in laboratory as well as in genetically modified transgenic animals to repress/supress tumors and have binding sites on their promoters. Since P53 has binding site onto the promoter of HIC1 it transcriptionally up-regulates its expression. In corroboration, our result did showed an increased expression of HIC1 with wild type p53 containing U87MG cells whereas less or no expression with mutated cell lines (U373MG) or null p53 (SaOS-2) (Sanjay et al.). The ambiguity was previously reported that wild type P53 continuously being formed and degraded by ubiquitination and proteasomal mediated pathway in absence of stress signal. On contrary, mutant forms of P53 are usually stable and escape degradation from ubiquitination pathway. We observed almost no expression of p53 in U87MG and SaOS-2 cells whereas U373MG showed high level of mutated p53. Conventionally HIC1 is a tumour suppressor gene (TSG), hence the over expression of this gene leads to arrest of cell cycle. Since HIC1 plays the role of TSG, the activities like cell cycle regulation and p53 dependency is not known. We have done a research on the activity of HIC1 in cell cycle as well as during glioma cell line (U87MG) proliferation, that had the wild type p53 gene in both serum containing and serum deprived medium. Reduction in number of cells and cell proliferation was revealed during HIC1 knocking as compared to control siRNA of cells without any treatment with serum containing medium and serum free medium. Flow cytometry and FacsDiva software verified the arrest of the cell cycle at the G2-M phase without any significant increase in the number of cell death i.e., cell apoptosis in both the medium. An increase in the expression of p53 gene associated with the knockdown of the HIC1 was seen. Moreover, we observed an increase in p21, p27 along with p53 gene when compared to the controls. The expression level of another cell cycle regulatory protein p27 found to be very significantly high comparing to controls. This could be the cause of arresting the cell cycle even after knocking down a tumor suppressor gene like HIC1, which was quite unexpected and beyond the convention. The results revealed the significance of HIC1 in the normal progression of cell cycle and also its activity the at molecular level, affecting the homeostasis of p53 gene as well as a number of genes, which are directly or indirectly related to p53, taking part in the cell cycle process.

Low Expression of p53 Protein in Bone Marrow after Hematopoietic Stem Cell Transplantation Indicates Risk of Relapse in Pediatric Patients with Acute Lymphoblastic Leukemia

Advances in treatment over the last decades have resulted in almost 90% 5-year survival of children with acute lymphoblastic leukemia (ALL). Nonetheless, a minor group with unfavorable prognosis becomes candidates for hematopoietic stem cell transplantation (HSCT). After HSCT, blast count, minimal residual disease (MRD) and chimerism analysis are used as markers for successful engraftment and risk of relapse. However, additional markers are needed to complement the existing ones, as 30 % of ALL-patients still experience relapse after HSCT. Mutations in the tumor suppressor gene TP53 are detected in 2% at diagnosis of pediatric ALLs, but in 11-28 % after relapse. The aim of this study was to evaluate if alterations in the expressions of the TP53 encoded protein p53, analyzed by immunohistochemistry, could act as a prognostic marker in pediatric ALL patients post HSCT.Paraffin-embedded bone marrow samples were collected retrospectively from 49 children diagnosed with pre-B ALL and, treated with HSCT at Karolinska University Hospital between 1997-2010. Patients samples were requested from six different times during the course of the disease; time of diagnosis, before HSCT and at routine controls after HSCT at 0-3, >3-6, >6-12 and >12-24 months. For no patient data was available from all time periods. In total 176 samples were collected. A control group consisted of fifty-five children, where a bone marrow biopsy had been performed due to suspected bone marrow disorder, but a malignant blood disease had been excluded.Tissue micro array (TMA) prepared microscope slides were stained for the p53 protein using a monoclonal mouse anti-human antibody (clone DO-7, Leica), which detects both the wild-type and mutant forms of p53. Protein expression was examined by two independent examiners in a light microscope at high magnification (40X). Cells positive for the p53-protein identified by brown nuclear staining, were counted and calculated as a percentage of total cells per sample for each antibody respectively. Mean number of cells counted per sample was 695. Some samples were lost prior to final analysis due to either; sample core lost during TMA (n=12) or, sample had <100 cells which was minimum number of cells for inclusion in analysis (n=10). The final analysis included 154 bone marrow samples from 49 patients and 55 control samples.Data was analyzed in Statsoft Statistica 12.7. Nonlinear logistic regression was used to evaluate predictive value of the protein. Data was analyzed independently at each of the following times; diagnosis, time of HSCT and at 0-3, >3-6, >6-12 and >12-24 months post HSCT. A p value < 0.05 was considered significant.Interestingly, we found that a low percentage of cells positive for p53 protein expression in the bone marrow at >3-6 months after HSCT was associated with increased risk of relapse in pediatric ALL patients, odds ratio 0.82 (confidence interval 0.68-0.99) p= 0.041. Figure I shows the range of p53 expression in the two groups individually at each time.At >3-6 months after HSCT, the cut off value was calculated to 3.3% p53 positive cells, and was correct for 16 of 20 non-relapse cases and for 9 of 14 relapse cases (overall 73.5% correct). All relapses occurred between 6-50 months after HSCT (mean=20, median=14).The lower amount of p53 positive cells in the relapsed patients compared to the non-relapsed patients could possibly be due to an underlying mutation in TP53 that has altered the ability of producing a protein. This would be in similarity with the literature where it is suggested that TP53 mutations are more common at relapse in pediatric ALL patients.In summary, a lower expression of p53 protein in the bone marrow at >3-6 months after HSCT was significantly associated with increased risk of relapse. Evaluation of p53 protein expression by immunohistochemistry, in bone marrow from pediatric ALL-patients that undergo HSCT may be a potential additional marker for predicting relapse. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 5324: Selective Inhibition of HDAC6 regulates preferential cytotoxicity in cancer cells by modulating p53 and Hsp90 stability

Abstract A HDAC6-selective inhibitor is a promising new therapeutic agent for cancer treatment, but its precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDAC6 inhibitor-induced effects has not been fully elucidated. Here, we show that a γ-lactam based HDAC inhibitor A452 selectively inhibits HDAC6 catalytic activity in vivo and in vitro. A452 causes cell death as well as growth inhibition of transformed cells not observed in normal cells. A452 exhibits differential cytotoxicity for wild-type (wt) and mutant p53 (mutp53) human colorectal cancer cells. Interestingly, A452 shows different mechanisms of action of modulating p53 in cancer cells. A452 increases wtp53 by destabilizing MDM2 and MDMX. In contrast, A452 causes mutp53 destabilization by inhibiting complex formation between Hsp90 and hyperstable mutp53, which is a hallmark of p53 mutation and cancer cell-specific. A452 also chemosensitizes cancer cells differing in p53 status for chemotherapy due to its ability to degrade mutp53 and stabilize wtp53. Intriguingly, acetylation of Hsp90 by A452 results in its degradation as well as inactivation of its chaperone activity. Furthermore, A452 effectively suppresses cell migration and invasion. These results demonstrate that A452 is a specific HDAC6 inhibitor and requires for p53 and Hsp90 for inducing anticancer action. Therefore, these results suggest that therapeutic targeting of HDAC6 may represent a new way to treat cancers expressing mutant forms of p53. Citation Format: So Hee Kwon. Selective Inhibition of HDAC6 regulates preferential cytotoxicity in cancer cells by modulating p53 and Hsp90 stability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5324. doi:10.1158/1538-7445.AM2015-5324

Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Abstract Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2215. doi:10.1158/1538-7445.AM2015-2215

Involvement of the insulin-like growth factor binding proteins in the cancer cell response to DNA damage.

The complex mechanisms that cells have evolved to meet the challenge of constant exposure to DNA-damaging stimuli, also serve to protect cancer cells from the cytotoxic effects of chemo- and radiotherapy. IGFBPs appear to be involved, directly or indirectly, in some of these protective mechanisms. Activation of p53 is an early response to genotoxic stress, and all six human IGFBP genes have predicted p53 response elements in their promoter and/or intronic regions, at least some of which are functional. IGFBP3 has been extensively characterized as a p53-inducible gene, but in some cases it is suppressed by mutant p53 forms. DNA double-strand breaks (DSBs), induced by radiotherapy and some chemotherapies, potentially lead to apoptotic cell death, senescence, or repair and recovery. DSB damage can be repaired by homologous recombination or non-homologous end-joining (NHEJ), depending on the cell cycle stage, availability of key repair proteins, and other factors. The epidermal growth factor receptor (EGFR) has been implicated in the NHEJ pathway, and EGFR inhibition may inhibit repair, promoting apoptosis and thus improving sensitivity to chemotherapy or radiotherapy. Both IGFBP-3 and IGFBP-6 interact with components of the NHEJ pathway, and IGFBP-3 can facilitate this process through direct interaction with both EGFR and the catalytic subunit of DNA-PK. Cell fate after DNA damage may in part be regulated by the balance between the sphingolipids ceramide and sphingosine-1-phosphate, and IGFBPs can influence the production of both lipids. A better understanding of the involvement of IGFBPs in the DNA damage response in cancer cells may lead to improved methods of sensitizing cancers to DNA-damaging therapies.

Proteasome inhibitors suppress the protein expression of mutant p53

Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

Abstract PL01-02: The two faces of p53: Tumor suppressor and oncogene

Abstract The p53 protein is a sequence-specific transcriptional regulator of multiple genes that control processes associated with tumor suppression such as cell cycle arrest, cell death, senescence, and metabolic homeostasis. It is held in check by two closely related proteins, Mdm2 and MdmX, that work in concert to repress p53 as a transcriptional activator and also to degrade p53 in unstressed cells. While wild-type p53 is an extensively studied and validated tumor suppressor, epidemiological studies, mouse models and cell-based assays support the likelihood that the mutant forms of p53 found in many tumors contribute to oncogenic characteristics such as malignant morphology, motility, invasion and metastasis. Our recent studies have revealed novel pathways in which p53 operates as well as new regulators of p53 and Mdm2. In each case datasets from cancer patients have supported the potential clinical relevance of our basic research findings. First, we discovered that mutant p53 regulates the malignant phenotype of breast cancer cell lines grown in 3D culture conditions (Freed-Pastor et al., Cell 2012). Expression levels of over 1000 genes are significantly altered when levels of mutant p53 are experimentally manipulated in this setting. Among the gene sets to be affected by mutant p53 is the mevalonate (MVA) pathway that is responsible for biosynthesis of cholesterol as well as key intermediates required for protein prenylation. The ability of mutant p53 to regulate the malignant morphology of breast cancer cells in 3D cultures likely requires the MVA pathway. A p53 mutant can cooperate with the SREBP2 transcription factor required for transcription of MVA genes. Breast cancer datasets indicate a correlation between mutant p53 and increased expression of some MVA genes. We are currently examining the role of wild-type p53 in regulating MVA genes and have also identified another highly cancer relevant pathway that is regulated by mutant p53. Second, we showed that TAB 1 (TGFβ-activated kinase 1 (TAK1)-binding protein 1) can bind and inhibit the E3 ligase activity of Mdm2 towards both p53 and MdmX (Zhu et al. Genes Dev 2013). We discovered that cisplatin is unique among diverse chemotherapeutic agents that were tested in requiring full levels of TAB1 to produce p53-dependent apoptosis. Two separate but possibly interrelated cell death pathways are regulated by TAB1 in cisplatin treated cells. TAB1 is required for both phosphorylation of p53 by p38 and the ability of MdmX to facilitate apoptosis in cisplatin-treated cells. TAB1 levels are reduced in a significant fraction of ovarian tumors indicating its possible role in tumor suppression. TAB1 therefore serves as a functional link between p53-MDM2 circuitry and a key MAPK signaling pathway. Elucidating the mechanism by which TAB1 regulates Mdm2 and MdmX in a cisplatin-specific manner is an ongoing focus of our research as is examining TAB1 as a potential therapeutic target. Third, we found that Nup98, a component of the nuclear pore complex is required for selective expression of a small subset of tested p53 target genes (eg. p21 and 14-3-3-sigma) in liver cancer cell lines (Singer et al., Mol. Cell 2012). Interestingly, Nup98 plays a post transcriptional role that involves stabilization of p21 mRNA via complex formation with its 3’UTR, thereby protecting it from exosomal degradation. Depending on the setting, depletion of Nup98 leads to decreased senescence or increased cell death. Nup98 expression is significantly lower than normal in hepatocellular carcinoma (HCC) tumors in mice and in a fraction of human HCCs, suggesting its role as a potential tumor suppressor. We are currently examining the roles and activities of Nup98 fusion proteins that occur in some forms of leukemia. Citation Format: Carol L. Prives. The two faces of p53: Tumor suppressor and oncogene. [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 PL01-02. doi:10.1158/1538-7445.AM2014-PL01-02

Mutant p53 protein expression and antioxidant status deficiency in breast cancer.

It is well recognized that cancers develop and grow as a result of disordered function of tumor suppressor genes and oncogenes, which may be exploited for screening purposes. Extensive evidence indicated tumor suppressor protein p53 as candidate marker for mutation identification. We have investigated mutant p53 protein expression in human breast tumors in relation to antioxidant status deficiency. The study included 100 breast cancer patients. p53 protein expression was evaluated by Western blot assay and immunostaining using a CM-1, DO-7 and Pab240 antibodies. Antioxidant parameters and lipid peroxidation were estimated by biochemical analyses. Western blotting with epitopespecific monoclonal antibody Pab240 strongly suggests that nuclear extracts from breast cancer cells express mutant forms of p53. It is of interest that the mutant forms of p53 overexpression in conjunction with the appearance of nuclear bodies are observed in highly aggressive carcinomas. Expression of isoform Δp53 (45 kDa) and isoform of ~ 29 kDa were more common in cases with LN metastasis. These studies point out the molecular consequences of oxidative stress (lipid peroxides, LP, p<0.001) and antioxidant status deficiency (copper, zinc superoxid dismutase, SOD, p<0.001; catalase, CAT, p<0.01; glutathione reductase, GR, p<0.001; glutathione, GSH, p<0.05) and indicate the importance of p53 mutation as the commonest genetic alteration detected in breast cancer cells. The expression of mutant p53 is correlated to increased lipid peroxides (0.346, p<0.05 ) and lowered antioxidant activity of CAT (- 0.437, p<0.01) in the breast cancer patients.

Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased susceptibility of wild-type p53 cancer cells to oxidative stress and therapeutic agents

Sirtuin 1 (Sirt1) plays an important role in cellular redox balance and resistance to oxidative stress. Sirt1 exhibits oncogenic properties in wild-type p53 cancer cells, whereas it acts as a tumor suppressor in p53-mutated cancer cells. Here, we investigated the effects of metformin on Sirt1 expression in several cancer cell lines. Using human cancer cell lines that exhibit differential expression of p53, we found that metformin reduced Sirt1 protein levels in cancer cells bearing wild-type p53, but did not affect Sirt1 protein levels in cancer cell lines harboring mutant forms of p53. Metformin-induced p53 protein levels in wild-type p53 cancer cells resulted in upregulation of microRNA (miR)-34a. The use of a miR-34a inhibitor confirmed that metformin-induced miR-34a was required for Sirt1 downregulation. Metformin suppressed peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (Pgc-1α) expression and its downstream target Nrf2 in MCF-7 cells. Genetic tools demonstrated that the reduction of Sirt1 and Pgc-1α by metformin caused Nrf2 downregulation via suppression of PPARγ transcriptional activity. Metformin reduced heme oxygenase-1 and superoxide dismutase 2 but upregulated catalase expression in MCF-7 cells. Metformin-treated MCF-7 cells had no increase in basal levels of reactive oxygen species but were more susceptible to oxidative stress. Furthermore, upregulation of death receptor 5 by metformin-mediated Sirt1 downregulation enhanced the sensitivity of wild-type p53 cancer cells to TRAIL-induced apoptosis. Our results demonstrated that metformin induces miR-34a to suppress the Sirt1/Pgc-1α/Nrf2 pathway and increases susceptibility of wild-type p53 cancer cells to oxidative stress and TRAIL-induced apoptosis.