Altered mammary gland development in the p53+/m mouse, a model of accelerated aging

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.     

Human cancer-associated mutations in the Aα subunit of protein phosphatase 2A increase lung cancer incidence in Aα knock-in and knockout mice

Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B' subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.     

The p53R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53^R175H (corresponding to p53^R172H in mice) is a hotspot for mutation that demonstrates “prometastatic” gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53^R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53^R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53^flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53^R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53^R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53^R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.     

Mutations in the Trp53 gene of UV-irradiated Xpc mutant mice suggest a novel Xpc-dependent DNA repair process

Mutational hot spots in the human p53 gene are well established in tumors in the human population and are frequently negative prognosticators of the clinical outcome. We previously developed a mouse model of skin cancer with mutations in the xeroderma pigmentosum group C gene (Xpc). UVB radiation-induced skin cancer is significantly enhanced in these mice when they also carry a mutation in one copy of the Trp53 gene (Xpc-/-Trp53+/-). Skin tumors in these mice often contain inactivating mutations in the remaining Trp53 allele and we have previously reported a novel mutational hot spot at a non-dipyrimidine site (ACG) in codon 122 of the Trp53 gene in the tumors. Here we show that this mutation is not a hot spot in Xpa or Csa mutant mice. Furthermore, the mutation in codon T122 can be identified in mouse skin DNA from (Xpc-/-Trp53+/-) mice as early as 2 weeks after exposure to UVB radiation, well before histological evidence of dysplastic or neoplastic changes. Since this mutational hot spot is not at a dipyrimidine site and is apparently Xpc-specific, we suggest that some form of non-dipyrimidine base damage is normally repaired in a manner that is distinct from conventional nucleotide excision repair, but that requires XPC protein.     

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.     

ROS-mediated p53 induction of Lpin1 regulates fatty acid oxidation in response to nutritional stress

The p53 protein is activated by stress signals and exhibits both protective and death-promoting functions that are considered important for its tumor suppressor function. Emerging evidence points toward an additional role for p53 in metabolism. Here, we identify Lpin1 as a p53-responsive gene that is induced in response to DNA damage and glucose deprivation. Lpin1 is essential for adipocyte development and fat metabolism, and mutation in this gene is responsible for the lypodystrophy phenotype in fld mice. We show that p53 and Lpin1 regulate fatty acid oxidation in mouse C2C12 myoblasts. p53 phosphorylation on Ser18 in response to low glucose is ROS and ATM dependent. Lpin1 expression in response to nutritional stress is controlled through the ROS-ATM-p53 pathway and is conserved in human cells. Lpin1 provides a critical link between p53 and metabolism that may be an important component in mediating the tumor suppressor function of p53.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.     

neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.

Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of the nontransgenic mice or the p53-172H transgenic mice developed tumors within this time period. Tumors arising in the p53-172H/neu bitransgenic mice were anaplastic and aneuploid and exhibited increased apoptosis, in distinction to tumors arising in p53-null mice, in which apoptosis is diminished. Further experiments address potential mechanisms of cooperativity between the two transgenes. In these bitransgenic mice, we have recapitulated two common genetic lesions that occur in human breast cancer and have shown that p53 mutation is an important cooperating event in neu-mediated oncogenesis.     

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.     

Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.     

A conformation hypothesis for the suppressor and promoter functions of p53 in cell growth control and in cancer.

Cancer is a genetic disease caused by defective control of cell proliferation. As cancer cells divide, the genetic defect is inherited by each daughter cell, leading to tumour development with possible progression to malignancy. The identification of those genes linked with cancer is essential for our understanding of the regulation of cell proliferation and for the therapeutic management of cancer cell growth. Recent studies have revealed that p53 is the most commonly affected gene in human cancer. It is a single copy gene and functions in the regulation of cell proliferation. Mutation of p53 is linked with tumour development, and this may involve abnormal functioning of mutant p53 protein. A mutant allele of p53 is functionally temperature-sensitive and can promote or suppress cell proliferation. The tertiary structure of the mutant protein is also sensitive to temperature and adopts promoter and suppressor forms of p53. A conformation model for the functioning of p53 proposes that wild-type p53 is induced to change from suppressor to promoter form during the cell growth response. This model predicts that any mutation that deregulates the normal control of p53 conformation may lead to cancer.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.     

The diversity of p53 mutations among human brain tumors and their functional consequences

The p53 tumor suppressor is implicated in cell cycle control, DNA repair, replicative senescence and programmed cell death. Inactivation of the p53 contributes to the wide range of human tumors, including glial neoplasms. In this review, we describe the regulation and biochemical properties of p53 protein that may explain its ability to activate various genetic programs underlying cellular responses to stress conditions. The overall spectrum of p53 mutations is rather shared between tumor types indicating that these mutations are not tumor type-specific. However, there is one example of germ-line mutation of p53 gene (the deletion of the codon 236) that is associated with a familiar brain tumor syndrome. We compare the frequency and type of most common mutations among various brain tumours (focusing on glioblastomas) and their consequences on protein functions. Furthermore, we discuss the most promising approaches of potential brain tumor therapy, including an adenovirus-mediated p53 gene transfer. Human glioblastomas are highly sensitive to the effects of p53 activity when the wild-type p53 is introduced ectopically. It suggests that the genetic or pharmacological modulation of the p53 pathway is potentially important strategy in the treatment of human cancers.     

靶向突变p53蛋白的抗肿瘤药物

p53蛋白是人体内十分重要的肿瘤抑制因子,通过调节细胞周期阻滞、诱导细胞凋亡等作用发挥肿瘤抑制功能。突变后的p53蛋白不仅具有显性负性效应（dominant negative effect,DN）抑制野生型p53蛋白功能,而且还通过功能获得性效应（gain of function,GOF）调节细胞代谢、侵袭、迁移等方式促进肿瘤的发生。p53蛋白在超过50%的肿瘤组织中发生突变,是肿瘤细胞区别于正常细胞的一个特异性药物靶点。因此,针对突变p53蛋白开发新型抗癌药物一直是研究的热点。长期以来,由于突变p53蛋白表面较为光滑,缺乏药物结合口袋,使其被认为是一个不可成药的靶点。随着高通量筛选技术的发展以及对突变p53蛋白结构的深入了解,许多靶向突变p53蛋白的小分子化合物被报道并在体外展现出较好的抗肿瘤活性,多款基于突变p53蛋白研发的化合物已经进入临床试验阶段。本文就靶向p53蛋白治疗肿瘤的直接和间接策略进行综述,重点针对突变p53蛋白重激活剂与降解突变p53蛋白的小分子化合物作用机制进行梳理,以期为后续开发靶向突变p53蛋白药物的创新提供帮助。     

突变体p53研究进展

抑癌基因突变是癌症发生过程中一个极为关键的事件。p53作为体内最重要的抑癌基因之一, 在人类癌症中发生突变的频率高达50%。同时, p53突变也是人类遗传病Li-Fraumeni综合征的主要病因。p53最常见的突变形式是错义突变, 所形成的突变体p53不但失去了野生型p53的抑癌功能, 而且还获得了一系列类似于癌基因的功能, 促进了肿瘤的进程。文章拟对突变体p53的结构功能改变, 获得癌基因活性的分子机制, 以及近年来对封闭突变体p53活性所进行的探索等研究方向所取得的进展做一综述。