Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome

The p53 tumor suppressor gene is commonly altered in human tumors, predominantly through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of-function properties to p53. To ascertain the physiological effects of p53 point mutation, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are models of Li-Fraumeni Syndrome; they developed allele-specific tumor spectra distinct from p53+/- mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including a variety of carcinomas and more frequent endothelial tumors. Dominant effects that varied by allele and function were observed in primary cells derived from p53R270H/+ and p53R172H/+ mice. These results demonstrate that point mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss of p53 function.     

A Novel p53 Mutant Found in Iatrogenic Urothelial Cancers Is Dysfunctional and Can Be Rescued by a Second-site Global Suppressor Mutation

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.     

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.     

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

A novel p53 rescue compound induces p53-dependent growth arrest and sensitises glioma cells to Apo2L/TRAIL-induced apoptosis

Reactivation of mutant p53 in tumours is a promising strategy for cancer therapy. Here we characterise the novel p53 rescue compound P53R3 that restores sequence-specific DNA binding of the endogenously expressed p53(R175H) and p53(R273H) mutants in gel-shift assays. Overexpression of the paradigmatic p53 mutants p53(R175H), p53(R248W) and p53(R273H) in the p53 null glioma cell line LN-308 reveals that P53R3 induces p53-dependent antiproliferative effects with much higher specificity and over a wider range of concentrations than the previously described p53 rescue drug p53 reactivation and induction of massive apoptosis (PRIMA-1). Furthermore, P53R3 enhances recruitment of endogenous p53 to several target promoters in glioma cells bearing mutant (T98G) and wild-type (LNT-229) p53 and induces mRNA expression of numerous p53 target genes in a p53-dependent manner. Interestingly, P53R3 strongly enhances the mRNA, total protein and cell surface expression of the death receptor death receptor 5 (DR5) whereas CD95 and TNF receptor 1 levels are unaffected. Accordingly, P53R3 does not sensitise for CD95 ligand- or tumour necrosis factor alpha-induced cell death, but displays synergy with Apo2L.0 in 9 of 12 glioma cell lines. Both DR5 surface induction and synergy with Apo2L.0 are sensitive to siRNA-mediated downregulation of p53. Thus this new p53 rescue compound may open up novel perspectives for the treatment of cancers currently considered resistant to the therapeutic induction of apoptosis.     

Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion

The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Mutant p53 disrupts MCF-10A cell polarity in three-dimensional culture via epithelial-to-mesenchymal transitions

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.     

Localization of the steroid-binding site of the human sex steroid-binding protein of plasma (SBP or SHBG) by site-directed mutagenesis.

The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.     

Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study

The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H) in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R). In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R) can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis) to treat human cancer.     

Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA-binding domain, is one of the major hallmarks of cancer. The R273 position is a DNA-contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant variants of R273 in p53 have different survival rates, indicating that the DNA-contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild-type (WT) p53: [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical, and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but they also show distinct structural stability, aggregation, and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulfide mediated, leading to cross-β, thioflavin-T-positive aggregates, whereas hydrophobic interactions dominate self-assembly in [R273L]p53, leading to a mixture of amyloid and amorphous aggregates. Molecular dynamics simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process-specific protein aggregation as a target.     

Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes

The majority of the p53 genes derived from human colorectal carcinomas contain point mutations. A significant number of these mutations occur in or around amino acids 143, 175, 273, or 281. Experiments presented here demonstrate for the first time that p53 DNA clones containing any one of these mutations cooperate with the activated ras oncogene to transform primary rat embryo cells in culture. These transformed cells produce elevated levels of the human p53 protein, which has extended half-lives (1.5-7 h), as compared to the wild-type human p53 protein (20-30 min). The p53 mutant with an alteration at residue 175 (p53-175H) binds tightly to the cellular heat shock protein, hsc70. In contrast, the p53 mutants possessing mutations at either residue 273 or 281 (p53-273H/281G) do not bind detectably to this heat shock protein and generally are less efficient at forming transformed foci in culture. The transformed cell lines are tumorigenic in nude mice. Thus, two classes of p53 mutant proteins can be distinguished: p53-175H, which cooperates with ras efficiently and binds to hsc70, and p53-273H/281G, which has a reduced efficiency of transformed foci formation and does not bind hsc70. This demonstrates that complex formation between mutant p53 and hsc70 is not required for p53-mediated transformation, but rather it facilitates this function, perhaps by ensuring sequestration of the endogenous wild-type p53 protein. The positive effect on cell proliferation by these mutant p53 proteins is consistent with a role for activated p53 mutants in the genesis of colorectal carcinomas.     

Stabilization of the MDM2 oncoprotein by mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.     

In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis.

Several clinical trials have revealed that individuals who were given beta-carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150-1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145-1149]. Using differential display techniques, an initial survey using rats showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency. These findings prompted us to evaluate the expression of c-Jun, p53 and p21WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21WAF1/CIF1. Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A supplementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis.     

Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells

Stimulation of growth factor signaling has been implicated in the development of invasive phenotypes and the activation of p21-activated kinase (Pak1) in human breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238-28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000) J. Biol. Chem. 275, 12041-12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft agar in a preferential mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of Pak1 kinase activity and base-line invasiveness of human breast cancer cells and breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.