Cancer-derived p53 mutants suppress p53-target gene expression--potential mechanism for gain of function of mutant p53

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.     

Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337H-beta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt beta-strands, followed by unfolding of the alpha-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degrees C) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degrees C over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.     

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.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

The role of p53 and pRB in apoptosis and cancer.

Loss of function of both the p53 pathway and the retinoblastoma protein (pRB) pathway plays a significant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis. In the past two years, mouse genetics and gene expression profiling have led to major advances in our understanding of how the pRB and p53 pathways regulate apoptosis and thus the development of tumours.     

The role of p53 in treatment responses of lung cancer

Resistance to radio- and chemotherapy is a major problem in treatment responses of lung cancer. In this disease, biological markers, that can be predictive of response to treatment for guiding clinical practice, still need to be validated. Radiotherapy and most chemotherapeutic agents directly target DNA and in response to such therapies, p53 functions as a coordinator of the DNA repair process, cell cycle arrest, and apoptosis. In fact, it participates in the main DNA repair systems operative in cells, including NHEJ, HRR, NER, BER, and MMR. Given the high p53 mutation frequency in lung cancer which likely impairs some of the p53-mediated functions, a role of p53 as a predictive marker for treatment responses has been suggested. In this review, we summarize the conflicting results coming from preclinical and clinical studies on the role of p53 as a predictive marker of responses to chemotherapy or radiotherapy in lung cancer.     

Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis

Mutant p53 proteins not only lose their tumor-suppressor function but some acquire oncogenic gain of function (GOF). The published mutp53 knock-in (KI) alleles (R172H, R270H, R248W) manifest GOF by broader tumor spectrum and more metastasis compared with the p53-null allele, but do not shorten survival. However, whether GOF also occurs with other mutations and whether they are all biologically equal is unknown. To answer this, we created novel humanized mutp53 KI mice harboring the hot spot alleles R248Q and G245S. Intriguingly, their impact was very different. Compared with p53-null mice, R248Q/− mice had accelerated onset of all tumor types and shorter survival, thus unprecedented strong GOF. In contrast, G245S/− mice were similar to null mice in tumor latency and survival. This was associated with a twofold higher T-lymphoma proliferation in R248Q/− mice compared with G245S/− and null mice. Moreover, R248Q/− hematopoietic and mesenchymal stem cells were expanded relative to G245S/− and null mice, the first indication that GOF also acts by perturbing pretumorous progenitor pools. Importantly, these models closely mirror Li–Fraumeni patients who show higher tumor numbers, accelerated onset and shorter tumor-free survival by 10.5 years when harboring codon R248Q mutations as compared with Li–Fraumeni patients with codon G245S mutations or p53 deletions/loss. Conversely, both KI alleles caused a modest broadening of tumor spectrum with enhanced Akt signaling compared with null mice. These models are the first in vivo proof for differential oncogenic strength among p53 GOF alleles, with genotype–phenotype correlations borne out in humans.     

p53: The barrier to cancer stem cell formation

The role of p53 as the “guardian of the genome” in differentiated somatic cells, triggering various biological processes, is well established. Recent studies in the stem cell field have highlighted a profound role of p53 in stem cell biology as well. These studies, combined with basic data obtained 20 years ago, provide insight into how p53 governs the quantity and quality of various stem cells, ensuring a sufficient repertoire of normal stem cells to enable proper development, tissue regeneration and a cancer free life. In this review we address the role of p53 in genomically stable embryonic stem cells, a unique predisposed cancer stem cell model and adult stem cells, its role in the generation of induced pluripotent stem cells, as well as its role as the barrier to cancer stem cell formation.     

Human breast cancer: frequent p53 allele loss and protein overexpression

A sample of 114 primary breast tumors and corresponding constitutional DNA were tested for loss of heterozygosity (LOH) of the YNZ22 and p53 genes, both located in the 17p13 region. Loss of the p53 allele was found in 28 of 44 primary breast carcinomas (64%). In contrast LOH in only 26 of 61 tumors (43%) was detected with the variable number of tandem repeats (VNTR) probe YNZ22 mapping at 17p13.3 close to the p53 locus at 17p13.1. Among 19 tumors informative for both probes allele loss at 17p13.3 never occurred without p53 involvement. These data suggest, that p53 is the target of 17p13 allelic deletions in human breast cancer. Immunohistochemistry showed overexpression of the p53 protein in 25 of 50 cases (50%) presumably reflecting activating point mutations. Overexpression was not correlated with allele loss but seemed to be closely related to the presence of point mutations in this study. No homozygous deletions or rearrangements of the p53 gene were detected. This would argue for an important role of heterozygous p53 mutations in human breast cancer.     

A gain of function p53 mutant promotes both genomic instability and cell survival in a novel p53-null mammary epithelial cell model.

Approximately 40% of human breast cancers contain alterations in the tumor suppressor p53. The p53 172R-H gain-of-function mutant (equivalent to the common 175R-H human breast cancer mutant) has been shown to promote aneuploidy and tumorigenesis in the mammary gland in transgenic mice and may affect genomic stability in part by causing centrosome abnormalities. The precise mechanism of action of these gain-of-function mutants is not well understood, and has been studied primarily in fibroblast cell lines. A novel p53-null mouse mammary epithelial cell line developed from p53-null mice has been used in adenovirus-mediated transient transfection experiments to study the properties of this p53 mutant. Marked centrosome amplification and an increased frequency of aberrant mitoses were observed within 72 h of introduction of p53 172R-H. However, few cells with aberrant centrosome numbers were observed in cells stably expressing the p53 172R-H mutant. Furthermore, stable expression of this p53 mutant reduced both basal and DNA damage-induced apoptosis. This result may be mediated in part through abrogation of p73 function. The p53 172R-H mutant, therefore, appears to influence tumorigenesis at the molecular level in two distinct ways: promoting the development of aneuploidy in cells while also altering their apoptotic response after DNA damage.     

Regulation of p53 stability and function in HCT116 colon cancer cells

We have used a lentiviral vector to stably express p53 at a physiological level in p53 knockout HCT116 cells. Cells transduced with wild type p53 responded to genotoxic stress by stabilizing p53 and expressing p53 target genes. The reconstituted cells underwent G(1) arrest or apoptosis appropriately depending on the type of stress, albeit less efficiently than parental wild type cells. Compared with cells expressing exogenous wild type p53, the apoptotic response to 5-fluorouracil (5FU) was >50% reduced in cells expressing S15A or S20A mutant p53, and even more reduced by combined mutation of serines 6, 9, 15, 20, 33, and 37 (N6A). Among a panel of p53 target genes tested by quantitative PCR, the gene showing the largest defect in induction by 5FU was BBC3 (PUMA), which was induced 4-fold by wild type p53 and 2-fold by the N6A mutant. Mutation of N-terminal phosphorylation sites did not prevent p53 stabilization by doxorubicin or 5FU. MDM2 silencing by RNA interference activated p53 target gene expression in normal fibroblasts but not in HCT116 cells, and exogenous p53 could be stabilized in HCT116 knockout cells despite combined mutation of p53 phosphorylation sites and silencing of MDM2 expression. The MDM2 feedback loop is thus defective, and other mechanisms must exist to regulate p53 stability and function in this widely used tumor cell line.