Tissue-specific expression of SV40 in tumors associated with the Li-Fraumeni syndrome

Inactivation of wild-type p53 tumor suppressor function is the primary mechanism of tumor initiation in Li-Fraumeni syndrome (LFS) individuals with germline p53 mutations. Tumors derived from LFS patients frequently retain the normal p53 allele, suggesting that alternative mechanisms in addition to gene deletion must be involved in inactivating wild-type p53 protein. DNA tumor viruses, such as SV40, target p53 for inactivation through the action of viral oncoproteins. We studied the probands from two unrelated LFS families, each of whom presented with multiple malignant neoplasms. Patient 1 developed an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), while patient 2 developed a CPC and subsequently presented with both an osteosarcoma (OS) and renal cell carcinoma (RCC). We utilized DNA sequence analysis and immunohistochemistry to determine p53 gene status in the germline and tumors, as well as evidence for SV40 T-antigen oncoprotein expression. Each patient harbored a heterozygous germline p53 mutation at codons 175 and 273, respectively. In patient 1, the normal p53 gene was lost while the mutant p53 allele was reduced to homozygosity in the RMS. Both normal and mutant genes were maintained in the CPC. In patient 2, normal and mutant p53 alleles were retained in both the CPC and RCC. Both specific PCR and immunostaining detected SV40 T-antigen in both CPCs and the RCC. In addition to chromosomal alterations, epigenetic mechanisms may disrupt p53 function during tumorigenesis. In two LFS patients, we found SV40 DNA sequences and viral T-antigen expression that could account for inactivation of the normal p53 protein. Inactivation of p53 or other tumor suppressors by viral proteins may contribute to tumor formation in specific tissues of genetically susceptible individuals.     

Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development

Chronic exposure to ultraviolet (UV) radiation causes skin cancer in humans and mice. We have previously shown that in hairless SKH-hr1 mice, UVB-induced p53 mutations arise very early, well before tumor development. In this study, we investigated whether discontinuation of UVB exposure before the onset of skin tumors results in the disappearance of p53 mutations in the skin of hairless SKH-hr1 mice. Irradiation of mice at a dose of 2.5 kJ/m2 three times a week for 8 weeks induced p53 mutations in the epidermal keratinocytes of 100% of the mice. UVB irradiation was discontinued after 8 weeks, but p53 mutations at most hotspot codons were still present even 22 weeks later. During that period, the percent of mice carrying p53(V154A/R155C), p53(H175H/H176Y), and p53R275C mutant alleles remained at or near 100%, whereas the percentage of mice with p53R270C mutation decreased by 45%. As expected, discontinuation of UVB after 8 weeks resulted in a delay in tumor development. A 100% of tumors carried p53(V154A/R155C) mutant alleles, 76% carried p53(H175H/H176Y) mutants, and 24 and 19% carried p53R270C and p53R275C mutants, respectively. These results suggest that different UVB-induced p53 mutants may provide different survival advantages to keratinocytes in the absence of further UVB exposure and that skin cancer development can be delayed but not prevented by avoidance of further exposure to UVB radiation.     

DN‐R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells: Role of E‐cadherin

One of the hallmarks of carcinomas is epithelial disorganization, linked to overexpression of matrix metalloproteases (MMP) like MMP‐9, loss of intercellular E‐cadherin and activation of epidermal growth receptor (EGFR/erbB1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or defective wt p53 signaling, we now investigated in human wt p53 breast carcinoma MCF‐7 cells, whether single treatment with the p53 transactivation pharmacological inhibitor pifithrin‐α, transient p53 siRNA interference or stable insertion of a dominant‐negative (DN) R175H p53 mutant increase: (i) EGFR/erbB1 activation, (ii) MMP‐9 expression and (iii) loss of surface E‐cadherin. Transient abrogation of wt p53 function increased phosphorylation of EGFR/erbB1 and MMP‐9 expression. However, all these effects were much more pronounced in cells stably transduced with the dominant negative–Arg‐175His mutant p53 (DN‐R175H mutant p53), which also showed loss of epithelial cytoarchitecture and extensive E‐cadherin downregulation. Collectively, these results support the notion that the DN‐R175H mutant p53 exerts a gain of oncogenic function by promoting disruption of E‐cadherin intercellular contacts and activation of proliferation signals. Our data suggests that epithelial shape and growth control are unequally affected depending on how wt p53 function is impaired and whether partial or full tumor suppressor activity is lost. © 2009 UICC     

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.     

p53 tetramerization domain mutations: germline R342X and R342P, and somatic R337G identified in pediatric patients with Li–Fraumeni syndrome and a child with adrenocortical carcinoma

Germline p53 mutations are associated with Li–Fraumeni syndrome (LFS) and other familial cancer phenotypes not fulfilling the definition for LFS. The majority of germline p53 mutations cluster in exons 5–8, corresponding to a DNA binding domain. We report the identification of two germline mutations and a somatic mutation in a tetramerization domain (TD), a rare site for mutations. The germline mutation, R342X (16915C>T), and the novel mutation, R342P (16916G>C), were found in a child with adrenocortical carcinoma and in a LFS pediatric patient with multiple primaries. The novel somatic mutation, R337G (16900C>G), was discovered in myelodysplastic syndrome with transformation to acute myeloblastic leukemia, developing as the third primary in the LFS child. These findings add further information on p53 TD mutations and TD contribution to tumorigenesis.     

Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactivate tumor p53 mutants.

Since highly expressed human p53 can inhibit human and yeast cell growth, we predicted that p53 mutants could be generated with increased growth inhibition of the yeast Saccharomyces cerevisiae and that these would be useful for characterizing p53 functions and tumor p53 mutants. A random mutagenesis screen led to the isolation of mutations in the DNA binding domain that result in p53 being lethal even at moderate expression levels in yeast. Three independent mutants had an alanine change at the evolutionary invariant V122 in the L1 loop. The other toxic mutations affected codons 277 (C277R, C277W) and 279 (G279R). This latter amino acid change was also reported in tumors, while all the other mutations are novel. A recently developed rheostatable GALI promoter system that provides graded increases in expression of p53 was used to examine the transactivation function of the toxic mutations when expression was greatly reduced and cells were viable. At low expression levels the toxic mutants lacked transactivation from a 3xRGC responsive element (RE). Surprisingly some exhibited enhanced transactivation with p21 and bax REs. The V122A mutant was able to re-activate transactivation of various p53 tumor mutants and retained growth inhibition when co-expressed with dominant-negative tumor mutations. Upon expression in human Saos-2 cells the V122A p53 mutant caused growth suppression, was capable of transactivation and exhibited higher than wild type activity with the bax promoter in luciferase assays. A non-functional p53 tumor mutant was partially reactivated by V122A for both transactivation and growth suppression. Thus, the screen for toxic p53 mutants in yeast can identify novel p53 variants that may be useful in dissecting p53 regulated cellular responses and in developing p53-based cancer therapies.     

The interaction of p53 with replication protein A mediates suppression of homologous recombination

The tumor suppressor protein p53 is emerging as a central regulator of homologous recombination (HR) processes and DNA replication. P53 may downregulate HR through multiple mechanisms including the reported associations with the Rad51 and Rad54 recombinases, and the BLM and WRN helicases. Here, we investigated whether the interaction of p53 with human replication protein A (RPA) is necessary for the regulation of HR. By employing a plasmid-based HR assay in p53-null H1299 lung carcinoma cells, we studied the HR-suppressing properties of a panel of p53 mutants, which varied in their ability to interact with RPA. Both wild-type p53 and a transactivation-deficient p53 mutant (L22Q/W23S) suppressed HR and prevented RPA binding to ssDNA in vitro and in vivo. Conversely, p53 mutations that specifically disrupt the RPA-binding domain, while not compromising p53 transactivation function (D48H/D49H and W53S/F54S), did not affect HR. Suppression of HR was also not seen with missense mutations in the p53 core domain (His175 and His273), which retained the ability to interact with RPA, suggesting that the disruption of additional binding interactions of p53, for example, with Rad51 or recombination intermediates, also impacts on HR. We hypothesize that sequestration of RPA by p53 at the sites of recombination is one means by which p53 can inhibit HR processes. Our data support and extend the previously formulated 'dual model' of p53's role as guardian of the genome.     

p53蛋白及其突变体对RhoE基因转录调控的影响

目的:探索p53蛋白及其突变体对RhoE基因转录调控的影响。方法:构建pEGFP-wt-p53质粒,利用基因定点突变PCR技术构建p53单点和双点突变体质粒,构建pGL3-RhoE-promotor-Luc质粒。以P21基因的启动子序列(P21-promoter-luc)为阳性对照,EGFP-N1空载体为阴性对照,将野生型和突变型p53质粒瞬时转染PC3(p53-null)细胞,利用双荧光素酶报告基因和Westernblot方法检测p53蛋白及其突变体蛋白在转录水平和蛋白水平对RhoE基因表达的影响。结果:电泳及测序表明以上质粒均构建成功。双荧光素酶报告基因检测显示野生型p53蛋白可调控RhoE基因转录,但其突变体丧失对RhoE的转录调控作用(P0.05),且不同位点的p53突变体蛋白之间对RhoE转录调控作用的差别无统计学意义(P0.05)。Western blot结果与基因转录结果一致。结论:RhoE是受p53蛋白调控的基因之一,而p53突变体失去了在转录水平调控RhoE表达的作用。     

Clinical sensitivity of p53 mutation detection in matched bladder tumor, bladder wash, and voided urine specimens

BACKGROUND: Mutations in the p53 tumor suppressor gene may correlate with an increased risk of recurrence and disease progression in patients with bladder carcinoma. The ability to accurately and sensitively detect p53 mutations in cytology specimens may be of benefit in the treatment of bladder carcinoma patients with superficial, minimally invasive disease. METHODS: Genomic DNA was isolated from 49 cases, each of which was comprised of matched bladder tumor tissue, bladder wash, and voided urine specimens obtained concurrently at a single institution. The genomic DNA was analyzed for mutations in the p53 tumor suppressor gene using a p53 mutation detection assay. Automated dideoxy sequencing of mutant specimens also was performed. RESULTS: Of the 49 cases, 29 (59%) showed no evidence of p53 mutations in the tumor, bladder wash, or voided urine specimens. Of the remaining 20 cases, 19 showed evidence of mutations in the tumor. Of these 19 p53 mutant bladder tumors, 16 (84%) were detected in the matched bladder wash and 16 (84%) were detected in the matched voided urine specimens. One case resulted in the detection of mutant p53 in the voided urine and the bladder wash, but not in the tumor. Analysis of the results between tumor tissue and bladder wash or tumor and voided urine showed 84.2% sensitivity, 96.8% specificity, and 91.8% accuracy. Sequence analysis of the mutant cases showed that the mutations detected in the tumor tissue were the same mutations detected in the bladder wash and the voided urine specimens. CONCLUSIONS: Both voided urine and bladder wash specimens from patients with bladder carcinoma were found to provide a high rate of clinical accuracy for the determination of the p53 gene status in patients with bladder tumors.     

Induction of genetic instability by gain-of-function p53 cancer mutants

p53 plays critical roles in tumor suppression and the loss of its function is required for cancer progression. In this context, the p53 gene is the most commonly mutated tumor suppressor gene in human cancers. The majority of the p53 gene mutations in human cancers are missense mutations, leading to the expression of the full-length mutant p53 protein in cancer cells. In addition to the loss of tumor suppression activity, p53 cancer mutants gain new oncogenic activities to promote tumorigenesis and drug resistance. Recent studies have identified a novel gain-of-function of p53 cancer mutants in inducing genetic instability by inactivating critical tumor suppressors such as ATM. Genetic instability is a common mechanism by which cancer cells efficiently accumulate genetic mutations to promote their growth, survival and metastatic potential. Therefore, this gain-of-function of p53 cancer mutants could play important roles in tumorigenesis and drug resistance of various types of human cancers. In addition, because many cancer therapies such as radiation therapy suppress or kill cancer cells by activating ATM-dependent responses to DNA double-stranded break damage, elucidation of this gain-of-function of p53 cancer mutants will have important implications on cancer therapy.     

Prognostic significance of mutations to different structural and functional regions of the p53 gene in breast cancer.

Alteration to the p53 tumor suppressor gene is associated with more aggressive disease in breast cancer, as evidenced by the shortened survival of patients with mutation. Data obtained from in vitro experiments suggest that mutations to different structural and functional domains of p53 may give rise to different effects on its biological activities, notably transactivational and apoptotic properties. We evaluated the prognostic significance of various types of p53 mutation in a series of 178 tumors identified by PCR-single-strand conformational polymorphism screening as containing a mutant gene. Mutations within exon 4 were associated with particularly poor prognosis, possibly relating to the importance of this region in apoptosis. Mutations that caused denaturation of the protein structure were also associated with poor survival, again perhaps because of effects on apoptosis. In contrast, patients with mutations in the DNA contact region showed similar survival to that of patients with normal p53, suggesting a less important role for p53-mediated transactivation in determining tumor aggressiveness. Other mutation groups associated with poor prognosis were single-base substitutions and transversion mutations. Mutations in exon 6, exon 7, or the "hotspot" codons (175, 245, 248, 273) were associated with only a small reduction in patient survival compared with normal p53. These results allow some insight to be gained into the functional importance of various p53 domains in terms of their influence on overall patient survival. Further work is required to determine whether these domains are also important in influencing the response of breast tumors to adjuvant therapies.     

Analysis of Li-Fraumeni syndrome and Li-Fraumeni-like families for germline mutations in Bcl10

The Li-Fraumeni syndrome (LFS) is a dominant disease whose hallmark is an increased risk of breast cancers, brain tumours, sarcomas, leukaemia and adrenal carcinoma. Some, but not all LFS and Li-Fraumeni-like (LFL) families are caused by TP53 mutations. Bcl10 is a recently identified tumour suppressor reported to be commonly mutated in a wide range of cancers. To investigate the possibility that Bcl10 is a susceptibility gene for LFS and LFL we have analysed 27 LFS/LFL families. No mutations were observed. This indicates that Bcl10 is unlikely to act as a susceptibility gene for LFS and LFL.     

Metabolic utilization of exogenous pyruvate by mutant p53 (R175H) human melanoma cells promotes survival under glucose depletion

Dominant-negative (DN) p53 mutations in the tumor suppressor p53 gene partly contribute to human cancer progression by inactivating the remaining wild type allele. Since tumor cells face glucose and growth factor shortage when growing distant from sites of vascularization, we used genetically-matched human C8161 melanoma harbouring wt p53 or a tumor-associated (DN) mutant p53 (R175H), to investigate whether this mutation influences survival under metabolic stress. Metabolic restriction (18 hours in glucose-free medium plus 2% serum) induced apoptosis-associated PARP cleavage in wt p53 melanoma, even when supplemented with 2.77 mM pyruvate or lactate. Mutant p53 melanoma were resistant to a comparable metabolic restriction, only showing PARP fragmentation when glucose depletion was accompanied by treatment with diphenylene iodonium (DPI), a NADPH oxidase inhibitor of superoxide (O2*-) generation. DPI-mediated apoptosis in mutant p53 cells was counteracted by 2.77 mM glucose or pyruvate, but not by lactate supplementation. Metabolic utilization and survival under glucose depletion was increased by pyruvate in mutant p53 (R175H) cells. Our results show for the first time that melanoma cells harbouring a p53 (R175H) mutation increase: a) survival under glucose depletion, counteracted by NADPH-oxidase modulators like DPI; b) resistance to DPI when supplemented with exogenous pyruvate.