Mutant Forms Of P53 Research Articles

Restoration of DNA‐binding and growth‐suppressive activity of mutant forms of p53 via a PCAF‐mediated acetylation pathway

Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53.

Abstract 66: Corruption of the activity of the mitochondria citrate transporter, CIC, by mutant forms of p53

Abstract Introduction. A distinctive feature of cancer cells is the implementation of the synthesis of fatty acids (FA), (lipogenesis), which is instead suppressed in most normal adult tissues. Because metabolites produced via lipogenesis provide redox potential and influence the activity of enzymes involved in transcription regulation, by turning on this pathway tumor cells might enable metabolic and transcriptional adjustments necessary for survival in various stress conditions. A still unanswered question is whether oncogenes expressed in human tumors directly favor the shift towards lipogenesis and, if so, whether this has consequences for proliferation or for therapy. The initial goal of this study was to identify metabolic gene targets regulated by tumor-derived mutant forms of the p53 gene product, whose role in metabolism is currently unknown. Results. We found that p53 mutants enhance the expression levels of the mitochondria citrate transporter, CIC. CIC is central to lipogenesis as it scaffolds citrate from the mitochondria to the cytosol, yielding acetyl-CoA, in turn necessary for fatty acid synthesis. Data extracted from gene expression databases, confirmed a strong association between high levels of CIC mRNA and p53 mutant-expressing human tumors, suggesting that up-regulation of CIC is a molecular signature for p53 mutations. CIC up-regulation is also a negative prognostic factor, and a predictor of tumor recurrence, thus mirroring the negative prognostic value of p53 mutant positive tumors. We show that the CIC promoter is transcriptionally up-regulated by mutant p53, via a unique mechanism that involves p53 mutant binding to FOXO-1- and to p53-consensus elements contained therein, as well as a physical interaction between these two proteins. Importantly, while in glucose-deprived cells CIC promoter activity is physiologically shut down, p53 mutants maintain higher levels of CIC transcription in these conditions. This is in contrast with wild-type p53 that suppresses CIC transcription. Furthermore, we demonstrate that inhibition of CIC with a siRNA, or blocking export of citrate to the cytoplasm with a CIC inhibitor, lowers fatty acid levels and impairs survival of p53-mutant tumors. This effect is not seen in pseudo-normal or tumor cells that have a wild-type p53 gene, again suggesting specificity for mutant p53. Additional experiments indicate that by promoting cytoplasmic export of citrate, elevated CIC levels might hamper mitochondrial pathways of energy production. Conclusions. We have identified the first metabolic signature gene regulated by p53 mutants, CIC, and shown that some tumors expressing these proteins depend upon CIC up-regulation and FA synthesis for survival especially in metabolic stress conditions. Furthermore, we have identified strategies for targeting CIC activity, thus opening new opportunities for the treatment of p53 positive tumors. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 66.

Abstract 4517: HDAC inhibitors suppress p53 expression through transcriptional repression: Implication for therapy against metastatic breast cancer

Abstract Cancer specific mutations of the p53 tumor suppressor gene are the most prevalent genetic lesions that underpin breast cancer pathogenesis and progression. In most cases, mutations result in a single amino acid substitution in the DNA-binding domain of p53. Such missense mutant forms of p53 accumulate at high levels in tumor cells. Recent advances demonstrated that missense p53 mutants have a critical role in driving cancer metastasis. Thus elimination of mutant p53 can potentially prevent metastatic progression of breast cancer. Inhibitors of histone deacetylases (HDACi) have proven anticancer effects and vorinostat, an FDA approved HDACi, has been in clinical use for treating certain malignancies. HDACi shapes epigenetic landscapes by restoring histone acetylation that is often suppressed in breast and other cancer types. One consequence of the restoration of histone acetylation is reactivation of tumor suppressors and other genes. Other effects of HDACi include modulation of posttranslational acetylation of non-histone proteins, which can influence their biochemical functions, resulting in a net effect of enhanced cell cycle arrest and apoptosis. Interestingly, HDACi profoundly suppresses the expression levels of p53. We found that this suppression occurs irrespectively of the mutational status of p53, but surprisingly only in certain types of cancer cells such as breast cancer. We further showed that the expression of exogenous p53 under a heterologous promoter was not affected by HDACi. These results suggest that HDACi might downregulate p53 through gene repression. To further test this idea, we assessed the effects of HDACi treatment and ectopic expression of classical HDACs (Classes I, II and IV) on luciferase reporter gene under the control of the p53 promoter. Among the 11 classical HDACs, ectopic expression of HDAC7 and HDAC10 seem to upregulate the reporter gene. We are currently investigating how these HDACs interact with the p53 promoter and what transcription factors and corepressors are involved in regulating p53 expression. Additionally, we are studying roles of depleting mutant forms of p53 by HDACi in suppressing migration, invasion and metastasis of breast cancer cells and assessing the efficacy of HDACi in conjunction with p53 gene therapy for treating breast cancer in a mouse model. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health, Stop! Children's Cancer, Inc. and the NIH.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4517.

Abstract 4158: The p53 gain-of-function mutation p53R172H promotes oral cancer progression in response to DNA damage

Abstract Oral cancer is one of the most common cancers worldwide, presenting high mortality rates and a severe impact on the quality of life of patients and survivors. Oral cancers arise from the accumulation of genetic alterations. Some of the most prevalent mutations found in patients with oral cancer involve mutations in Ras and p53 genes, although the mutation rates exhibit geographical variation, most likely as a result of different exposure to carcinogens. Most p53 mutations result in the expression of mutant forms of p53, rather than inactivation of the p53 gene. To determine the role of these mutations during oral cancer development we generated mice in which the endogenous K-rasG12D allele and p53 gain-of-function mutation p53R172H could be activated exclusively in the oral epithelium. We found that the p53R172H mutation, but not deletion of p53, cooperates with K-rasG12D to initiate oral tumor formation, accelerates oral tumor growth and promotes progression to squamous cell carcinoma. Expression profiling of the tumors that developed in these mice revealed that cyclin B1 was overexpressed in the oral tumors that expressed p53R172H compared to tumors with no p53. To expand the functional studies, we generated cell lines from these mouse oral tumors. Similar to the primary tumors, cell lines derived from the tumors expressing p53R172H generated tumors that grew faster upon injection in nude mice than cell lines that lacked p53. Using these cell lines we found that the expression of cyclin B1 was dramatically induced in the p53R172H cells in response to DNA damage, suggesting that cyclin B1 may contribute to the oncogenic properties p53R172H in oral tumor cells in response to DNA damage. Notably, we observed DNA damage in the oral tumors, as determined by staining for γ-H2AX. Since these tumors were initiated by oncogenic K-ras, we speculated that oncogene activation induced DNA damage in the tumors, thereby allowing the induction of cyclin B1 expression. Consistent with this hypothesis we found that suppressing K-ras in cells that express p53R172H resulted in significant reduction in cyclin B1 expression, indicating that p53R172H can induce the expression of cyclin B1 in response to oncogenic K-ras. In addition, we found increased DNA damage in the oral tumors that expressed p53R172H compared to tumors that lack p53, suggesting that p53R172H may modulate the DNA damage response. Together, these findings indicate that p53R172H may promote oral tumor progression in response to DNA damage, and suggest that attenuation of the DNA damage response may contribute to the oncogenic properties of mutant p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4158.

Abstract 5041: Aspirin acetylates and activates p53 in HT-29 and HCT-116 colon cancer cells

Abstract Increasing evidences from human epidemiological studies, animal models and in vitro experiments suggest that regular use of aspirin may reduce the risk of cancer of the colon; however, the mechanisms are not well understood. Since aspirin's protective effect is mainly observed in epithelial cell types which are more resistant to chemotherapy, an urgent need exists to identify the primary targets and cancer preventive pathways affected by aspirin. We hypothesized that aspirin's anti-cancer effect may involve acetylation of the tumor suppressor protein p53 and modulation of target gene expression. In the present study, we demonstrate that exposure of HT-29 and HCT-116 colon cancer cells to aspirin caused acetylation of p53 at lysine 382 (K382). HT-29 cells harbor a mutant form of p53 (R273H), whereas HCT-116 contain wild type p53. Acetylated p53 was mainly localized to the nucleus. In both cell types aspirin induced acetylation of p53 in a concentration dependent manner, and this was associated with increased p53 DNA binding activity. Consistent with this, we also observed induction of p21, a protein involved in cell cycle arrest as well as Bax, a mitochondrial proapoptotic protein. Aspirin also caused acetylation of bacterially expressed recombinant p53 in vitro, suggesting that it is a non-enzymatic reaction. Our observation that aspirin acetylates p53 leading to induction of target gene expression suggest that its anti-cancer effect may involve activation of the p53 pathway. Since p53 is mutated in nearly 50% of all tumors which leads to loss of its function, our observation also suggests that aspirin may find use in chemotherapy to reactivate mutant p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5041.

Abstract 2499: Single agent activity of checkpoint kinase inhibitor PF-477736, in a MYC-driven lymphoma model

Abstract CHK1 and CHK2 are protein kinases that function as effectors of cell cycle checkpoint arrest following DNA damage. Small molecule inhibitors with specificity for CHK1 such as PF-477736 (Blasina et al, 2008 Mol Cancer Ther 7:2394) and others with specificity for CHK1 and CHK2, are currently in phase 1 clinical evaluation in combination with chemotherapeutic agents known to induce DNA damage. We therefore hypothesized that inhibitors of CHK1/CHK2 may have single agent activity in tumours with oncogene-induced DNA damage. Cell lines derived from Eμ-MYC driven murine lymphomas display intrinsic DNA damage detected by γH2AX foci staining. Within 2 hours of 500nM PF-477736 treatment, a dramatic increase in the intensity of γH2AX staining and proportion of cells with γH2AX staining, was observed in all Eμ-MYC lines. An increase in mitotic cells detected by expression of phospho-Histone3, was also observed in all Eμ-MYC lines with a high proportion displaying features of aberrant mitosis, demonstrating inhibition of the S and/or G2/M checkpoints resulting in premature entry to mitosis. Early apoptosis was detected in 6 separate Eμ-MYC lines expressing functional p53, following treatment in vitro (>50% Annexin V positive cells 4 hrs after 500nM PF-477736). Subsequent cell death assessed as the proportion of PI positive cells, was observed in all cell lines expressing functional p53 (IC50 200-500nM at 16 hrs). Enforced expression of anti-apoptotic protein Bcl-2 by retroviral transduction abrogated cell death at up to 3μM PF-477736. Two separate Eμ-MYC lines expressing spontaneously arising mutant forms of p53, and three separate Eμ-MYC lines derived on a p53 null background, were relatively insensitive to PF-477736 (IC50>1μM at 16hrs), indicating that functional p53 is necessary to mediate apoptosis/death following accumulation of DNA damage due to CHK1 inhibition. Treatment with AZD7762 a CHK1/2 inhibitor, also resulted in death of Eμ-MYC lines in vitro, (IC50 200nM-2μM at 16 hrs), however the variation in sensitivity could not be attributed to p53 status. Treatment of C57Bl/6 mice transplanted with an Eμ-MYC line expressing functional p53, resulted in a dramatic increase in the proportion of apoptotic/dead cells in the established lymphomas within 6 hours following 20mg/kg PF-477736 i.p. A significant decrease in the wbc of 5 mice, from an average 48.2×106 wbc/ml pre-treatment to 31.8×106 at 16 hours post PF-477736 (P=0.0053), compared to vehicle treated mice (51.1×106 pre-treatment and 60.4×106 post-treatment), together with a lower average spleen weight of the PF-477736 treated mice (0.52g) compared to vehicle treated mice (0.68g), was observed. We propose that checkpoint kinase inhibitors will be beneficial as therapeutic agents able to specifically exploit tumours containing intrinsic DNA damage that are reliant on checkpoints for maintenance of genomic integrity, such as in “MYC-driven cancers”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2499.

Abstract 4833: Glucose restriction induces degradation of p53 mutants via a selective autophagy-mediated pathway

Abstract Introduction. More than 50% of human tumors harbor missense mutations of the p53 gene product. In its wild-type conformation p53 is degraded via the proteasome, mainly through the action of the MDM2 E3 ubiquitin ligase. Unlike the wild-type protein, mutant forms of p53 accumulate at high levels and elude proteolysis, in part due to an altered modality of interaction with MDM2. High levels of p53 mutants in tumors appear to correlate with resistance to radio- and chemo-therapy, and are proposed to promote tumor progression. Furthermore, therapeutic interventions aimed at reducing p53 mutant levels have shown anti-tumor activity in vitro and in vivo. The mechanisms that regulate the stability of wild-type p53 are well understood during the DNA damage response. However, p53 can also be stabilized during adaptive metabolic stress conditions, such as during glucose deprivation. The goal of this work was to determine the effects of glucose availability on the oncogenic activity of mutant p53. Methods. We investigated the effects of glucose availability in tumor cells expressing p53 mutants, in transgenic animals harboring p53 mutant alleles, and in xenograft models of breast cancer. The read-outs of these experiments were p53 stability, cellular proliferation and response to therapeutic agents. Results. We found that p53 mutant protein levels are exquisitely sensitive to glucose availability: high glucose levels stabilize p53 mutants, while glucose restriction (GR) leads to their MDM2- and proteasome-independent degradation. This degradation requires instead the participation of the autophagic machinery and of the autophagic protein Beclin-1, which forms a complex with p53 in GR-treated cells. Importantly, in breast cancer cell lines GR alone does not lead to a net increase in autophagic flux, thus differing from autophagy activated during serum- or amino acid- depletion. Rather, GR uses the autophagic machinery to degrade selective targets, such as mutant p53. We further show that tumor cells where p53 mutant levels are lower due to GR, have reduced proliferation potential compared to tumors expressing native p53, and can be easily chemo-sensitized, significantly reducing the IC50 of all drugs examined. We then asked whether dietary restriction of glucose affects p53 mutant stability in living organisms. Preliminary results indicate that p53 mutant levels are higher in tissues of animals fed with high glucose-diet, relative to animals fed with a low glucose diet. We are currently investigating whether this change in p53 levels affects proliferation rates. Conclusions. We propose that GR uses the autophagic machinery to induce degradation of oncogenic mutant p53, and thus interrupts their proliferative signals. Our findings further imply that dietary restriction of glucose would keep p53 mutant activity in check. Finally, GR overcomes the well-known chemo-resistance conveyed by p53 mutations. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4833.

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Mutation at Ser392 specifically sensitizes mutant p53H175 to mdm2-mediated degradation

Mdm2 is one of the main E3 ubiquitin ligases, which targets both wild type and mutant p53 for degradation. The ability of post-translational modifications, such as phosphorylation, to modulate the function and stability of wild type p53 has been extensively studied. However, their ability to modulate the functions and stability of mutant forms of p53 remains poorly documented. Here we show, for the first time, that the stability of mutant p53 can be regulated by phosphorylation. Mutation of serine 392 to alanine shortens the half life of p53H175, and renders p53H175A392 more sensitive to mdm2-mediated degradation than p53H175. This effect of Ser392 phosphorylation specifically affects p53H175, a misfolded mutant, and does not affect p53W248 which maintains a native conformation. Detailed analysis subsequently showed that the reduced stability of p53H175A392 is not due to an increase in mdm2/p300 binding or polyubiquitin chain formation, uncoupling the extent of polyubiquitin chain formation and the stability of mutant p53. This is supported by the observation that Ser392 mutation enhances polyubiquitin chain formation on p53W248, without reducing its stability. These results suggest that the inhibition of phosphorylation at Ser392 of p53, together with the use of an mdm2-enhancing agent such as nutlin, could present a new therapeutic strategy with which to treat tumors expressing mutant p53H175.

ZBTB5 is a potential tumor suppressor in the absence of functional p53

Previously, we isolated and characterized a novel POZ domain Krüppel like zinc finger transcription repressor, ZBTB5 (Zinc finger and BTB domain containing 5). SAGE analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Our data suggested that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto‐oncogene stimulating cell proliferation.Interestingly, in the HCT116 p53 null cells lacking functional tumor suppressor p53, expression of ZBTB5 was greatly induced prior to induction of cell cycle arrest gene p21 when cells were expose to DNA damaging agent, etoposide. ZBTB5 potently activated transcription of p21 gene in the absence of p53. ZBTB5 and Sp1 synergistically activates transcription by acting on the p21 promoter lacking distal p53 binding elements both in the p53 null and p53 positive cells. Furthermore, ZBTB5 increased transcription of p21 in the presence of dominant negative mutant form of p53. Our data suggest that ZBTB5 has functional duality in the regulation of cell proliferation and can acts either as oncogene or tumor suppressor depending on presence or absence of functional p53.

Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of tumor epithelial cell AP might provide a target for 2aG4 and contribute to the increased effectiveness of such combination therapy in vivo. We conclude that the combined targeting of mtp53 and the tumor vasculature is a novel effective strategy for combating advanced breast tumors.

Interference with the p53 family network contributes to the gain of oncogenic function of mutant p53 in hepatocellular carcinoma

Whereas the hallmark of wild-type p53 is its tumor suppressor activity, tumor-associated mutant p53 proteins can exert novel anti-apoptotic gain-of-function activities, which confer a selective advantage upon tumor cells harboring such mutations. We investigated the molecular mechanisms of mutant p53 gain-of-function in hepatocellular carcinoma with special emphasis on the interaction of mutant p53 gain-of-function proteins with the p53 family members p63 and p73. Mutant forms of p53, namely the hot-spot mutants p53R143A, p53R175D, p53R175H, p53R248W, and p53R273H, acquire anti-apoptotic gain-of-function in hepatocellular carcinoma by repressing the activity of genes regulating both, the extrinsic apoptosis pathway initiated by ligation of death receptors and the intrinsic/mitochondrial apoptosis pathway. In the presence of mutated p53, the CD95L-CD95 apoptotic pathway is markedly attenuated. This is due to repression of CD95 gene transcription by mutant p53. In addition, these mutants repress the expression of the Bax gene and attenuate mitochondria-mediated apoptosis signaling. Furthermore, and of clinical relevance, these gain-of-function mutants are anti-apoptotic due to their inhibitory interaction with the pro-apoptotic p53 family members TAp63 and TAp73. p53 gain-of-function mutants significantly decrease activation of pro-apoptotic target genes by wild-type p53, TAp63, and TAp73. This contributes to the ability of cancer cells to withstand DNA damage-induced apoptosis. Interference with the interaction of p53 gain-of-function mutants with TAp63 or TAp73 may thus sensitize hepatocellular carcinoma to elimination by therapy.

Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer

TP53 mutation occurs in 50-75% of human pancreatic ductal adenocarcinomas (PDAC) following an initiating activating mutation in the KRAS gene. These p53 mutations frequently result in expression of a stable protein, p53(R175H), rather than complete loss of protein expression. In this study we elucidate the functions of mutant p53 (Trp53(R172H)), compared to knockout p53 (Trp53(fl)), in a mouse model of PDAC. First we find that although Kras(G12D) is one of the major oncogenic drivers of PDAC, most Kras(G12D)-expressing pancreatic cells are selectively lost from the tissue, and those that remain form premalignant lesions. Loss, or mutation, of Trp53 allows retention of the Kras(G12D)-expressing cells and drives rapid progression of these premalignant lesions to PDAC. This progression is consistent with failed growth arrest and/or senescence of premalignant lesions, since a mutant of p53, p53(R172P), which can still induce p21 and cell cycle arrest, is resistant to PDAC formation. Second, we find that despite similar kinetics of primary tumor formation, mutant p53(R172H), as compared with genetic loss of p53, specifically promotes metastasis. Moreover, only mutant p53(R172H)-expressing tumor cells exhibit invasive activity in an in vitro assay. Importantly, in human PDAC, p53 accumulation significantly correlates with lymph node metastasis. In summary, by using 'knock-in' mutations of Trp53 we have identified two critical acquired functions of a stably expressed mutant form of p53 that drive PDAC; first, an escape from Kras(G12D)-induced senescence/growth arrest and second, the promotion of metastasis.

Drosophila melanogaster p53 has developmental stage-specific and sex-specific effects on adult life span indicative of sexual antagonistic pleiotropy

Truncated and mutant forms ofp53 affect life span in Drosophila, nematodes and mice, however the role of wild-type p53 in aging remains unclear. Here conditional over-expression of both wild-type and mutant p53 transgenes indicated that, in adult flies, p53 limits life span in females but favors life span in males. In contrast, during larval development, moderate over-expression of p53 produced both male and female adults with increased life span. Mutations of the endogenous p53 gene also had sex-specific effects on life span under control and stress conditions: null mutation of p53 increased life span in females, and had smaller, more variable effects in males. These developmental stage-specific and sex-specific effects of p53 on adult life span are consistent with a sexual antagonistic pleiotropy model.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

A novel regulator of the p53-mediated mitochondrial apoptotic pathway

AbstractThe p53 tumor suppressor protein induces apoptosis in response to genotoxic and environmental stress. Recent studies have revealed the existence of a transcription-independent mitochondrial p53 apoptosis pathway, however the mechanism regulating p53 translocation to mitochondria and subsequent initiation of apoptosis was not known. Here, we show that Tid1, also known as mtHsp40 or Dnaja3, interacts with p53 and directs its translocation to mitochondria in cells exposed to hypoxia. Overexpression of Tid1 in tumor cells promoted mitochondrial localization of both wildtype and mutant forms of p53 and was able to restore the pro-apoptotic activity of mutant p53 proteins that were otherwise unable to induce apoptosis. Tid1's mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria. Our findings establish Tid1 as a novel regulator of p53 localization and apoptotic function.

Does control of mutant p53 by Mdm2 complicate cancer therapy?: Figure 1.

Missense mutant forms of p53 are expressed at high levels in some human cancers and may contribute to oncogenesis. In this issue of Genes & Development, Terzian and colleagues (pp. 1337-1344) describe a mutant p53 knock-in mouse in which normal tissues and some tumors have low levels of mutant p53 protein unless Mdm2 or p16(INK4A) are absent. Once stabilized, mutant p53 promotes metastasis. Therefore, therapies that release p53 from Mdm2 might have unwanted consequences when cells have sustained a mutation in p53.

Oncogenomic Approaches in Exploring Gain of Function of Mutant p53

Cancer is caused by the spatial and temporal accumulation of alterations in the genome of a given cell. This leads to the deregulation of key signalling pathways that play a pivotal role in the control of cell proliferation and cell fate. The p53 tumor suppressor gene is the most frequent target in genetic alterations in human cancers. The primary selective advantage of such mutations is the elimination of cellular wild type p53 activity. In addition, many evidences in vitro and in vivo have demonstrated that at least certain mutant forms of p53 may possess a gain of function, whereby they contribute positively to cancer progression. The fine mapping and deciphering of specific cancer phenotypes is taking advantage of molecular-profiling studies based on genome-wide approaches. Currently, high-throughput methods such as array-based comparative genomic hybridization (CGH array), single nucleotide polymorphism array (SNP array), expression arrays and ChIP-on-chip arrays are available to study mutant p53-associated alterations in human cancers. Here we will mainly focus on the integration of the results raised through oncogenomic platforms that aim to shed light on the molecular mechanisms underlying mutant p53 gain of function activities and to provide useful information on the molecular stratification of tumor patients.

CP-31398 restores mutant p53 tumor suppressor function and inhibits UVB-induced skin carcinogenesis in mice.

Mutations in the tumor suppressor p53 are detectable in over 50% of all human malignancies. Mutant p53 protein is incapable of transactivating its downstream target genes that are required for DNA repair and apoptosis. Chronic exposure to UVB induces p53 mutations and is carcinogenic in both murine and human skin. CP-31398, a styrylquinazoline compound, restores the tumor suppressor functions of mutant forms of p53 in tumor cells. However, its effectiveness in vivo remains unclear. Here, we demonstrate that CP-31398 blocked UVB-induced skin carcinogenesis and was associated with increases in p53, p21, and BclXs. CP-31398 downregulated Bcl2, proliferating nuclear cell antigen, and cyclin D1. Activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase also occurred in both tumor and perilesional skin following treatment. CP-31398 induced the expression of p53-dependent target proteins, and this was followed by apoptosis in UVB-irradiated wild-type mice but not in their p53-deficient littermates. Similar effects were observed in human skin carcinoma A431 cells expressing mutant p53. In addition, CP-31398 induced mitochondrial translocation of p53, leading to changes in mitochondrial membrane permeability pore transition (MPT) and consequent cytochrome c release in these cells. Blocking MPT diminished p53 translocation and apoptosis. These studies indicate that reconstituting p53 tumor suppressor functions in vivo by small molecular weight compounds may block the pathogenesis and progression of skin cancer.

Ubiquitination and Degradation of Mutant p53

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.