Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D

p53 is the most commonly mutated gene in human cancer. Although the loss of tumor suppressor functions for p53 in tumorigenesis is well characterized, gain-of-function p53 mutations observed in most cancers are not as widely appreciated. The human breast cancer cell line MDA-MB-231, which has high levels of a mutant p53, has high levels of phospholipase D (PLD) activity, which provides a survival signal in these cells when deprived of serum growth factors. We report here that the mutant p53 in MDA-MB-231 cells is stabilized by the elevated PLD activity in these cells. Surprisingly, the survival of MDA-MB-231 cells deprived of serum was dependent on the mutant p53. These data indicate that a mutant p53, stabilized by elevated PLD activity, can contribute to the suppression of apoptosis in a human breast cancer cell line and suggest a rationale for the selection of p53 mutations early in tumorigenesis to suppress apoptosis in an emerging tumor.     

Elevated expression of p53 gain-of-function mutation R175H in endometrial cancer cells can increase the invasive phenotypes by activation of the EGFR/PI3K/AKT pathway

Background  

p53 is the most commonly mutated tumor suppressor gene in human cancers. In addition to the loss of tumor suppression function and exertion of dominant-negative effects over the remaining wild-type protein, several p53 mutants can gain novel oncogenic functions (gain-of-function, GOF) that actively regulate cancer development and progression. In human endometrial cancer, p53 mutation is more often associated with aggressive nonendometrioid cancer. However, it was unknown if p53 mutants contributed to endometrial cancer progression through the GOF properties.     

Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas

Centrosomes play critical roles in processes that ensure proper segregation of chromosomes and maintain the genetic stability of human cells. They contribute to mitotic spindle organization and regulate aspects of cytokinesis and cell cycle progression. We and others have shown that centrosomes are abnormal in most aggressive carcinomas. Moreover, centrosome defects have been implicated in chromosome instability and loss of cell cycle control in invasive carcinoma. Others have suggested that centrosome defects only occur late in tumorigenesis and may not contribute to early steps of tumor development. To address this issue, we examined pre-invasive human carcinoma in situ lesions for centrosome defects and chromosome instability. We found that a significant fraction of precursor lesions to some of the most common human cancers had centrosome defects, including in situ carcinomas of the uterine cervix, prostate, and female breast. Moreover, centrosome defects occurred together with mitotic spindle defects, chromosome instability, and high cytologic grade. Because most pre-invasive lesions are not uniformly mutant for p53, the development of centrosome defects does not appear to require abrogation of p53 function. Our findings demonstrate that centrosome defects occur concurrently with chromosome instability and cytologic changes in the earliest identifiable step in human cancer. Our results suggest that centrosome defects may contribute to the earliest stages of cancer development through the generation of chromosome instability. This, together with ongoing structural changes in chromosomes, could accelerate accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing wild-type tumor suppressor genes and thus accelerate the genomic changes characteristic of carcinoma, the most prevalent human cancer.     

The interaction of p53 and DNA repair gene mutations and their impact on tumor mutation burden and immune response in human malignancies

P53 suppresses tumorigenesis through multiple cellular functions/mechanisms, including genomic stability surveillance. Recently, it has also be reported for its role in cancer immune response modulation. Deficiency in DNA repair pathways lead to the accumulation of genomic alterations and tumor mutation burden and in consequence resulting in the activation of immune response. We investigated the interaction of p53 and DNA repair gene mutations and their impact on tumor mutation burden and immune response in human malignancies by mining cBioPortal data of a range of human cancers. We found that in the majority of human cancers, p53 mutations are equally distributed between DNA repair gene mutation positive and negative cases and in a number of human cancers, p53 and DNA repair gene mutations have a tendency of co-occurrence. Only in colorectal cancer, there is a tendency of ‘mutual exclusivity’ of mutations in p53 and DNA repair genes. In most tumors, p53 and DNA repair gene mutations have synergistic/additive effect in increasing tumor mutation burden, but not in colorectal cancer where they are mutually exclusive. The impact of p53 and DNA repair gene mutations and their interaction on tumor microenvironment immune cells are complex and tumor type specific and not always correlated with tumor mutation burden. In colorectal cancers, these two types of mutations resulted in similar immune cell subpopulation changes and in tumors where the mutations have a tendency of co-occurrence, p53 showed dominant roles on immune response, although they can also counter-act each other for their effect on certain immune cell subtypes.     

Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles

Evidence supporting a broad role for the inactivation of the p53 gene in human tumorigenesis has been provided by studies showing that the p53 gene is mutated in many human cancers. In this study, we report on the mutational status of the p53 gene in prostate cancer cells and provide functional evidence that the wild-type p53 gene may have a role in suppressing prostatic tumorigenesis. Sequence analysis of exons 5-8 of the p53 gene reveals that three of five prostate cancer cell lines (TSUPr-1, PC3, DU145) contain mutations which alter the amino acid sequence of this most highly conserved portion of the gene. One of two primary prostatic cancer specimens examined also contained a mutation in this region. Transfection of the wild-type p53 gene versus a mutated p53 gene into two cell lines with p53 mutations results in reduced colony formation. Wild-type p53 gene expression is apparently incompatible with continued growth of these tumor cells inasmuch as none of the colonies which formed after wild-type transfections retain the transfected p53 sequences. Immunocytochemical data indicate that prostate carcinoma cells expressing the transfected wild-type p53 gene are growth arrested because they exhibit a reduced level of thymidine incorporation into DNA. This study is the first report of p53 gene mutations in prostate cancer cells and suggests a functional role for the p53 gene in suppressing prostatic tumorigenesis.     

ENU administration causes genomic instability along with single nucleotide polymorphisms in p53 during gliomagenesis: T11TS administration demonstrated in vivo apoptosis of these genetically altered tumor cells

Advancement of molecular analysis of neoplastic cells demonstrated that multiple genetic changes are associated with the development of tumors. Cancer cell must exhibit a mutator phenotype, which is likely to be responsible for the genomic instability found in cancer tissues. The mutator phenotype, such as defective mismatch repair, is known to cause microsatellite instability, which is associated with certain cases of sporadic cancer. Previously many studies have been carried out to determine the relationship between microsatellite instability and human brain tumors. However information on genomic instability in the animal model of brain tumor is still very limited. In the present course of investigations we genetically characterized our ENU induced brain tumor animal model by using PCR based randomly amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP) with three microsatellite probes. ENU induced tumors demonstrated genetic instability, including some microsatellite instability. As single nucleotide polymorphisms of the tumor suppressor gene p53 were associated with diverse types of human cancer, we examined the p53 gene of the tumor cells isolated from ENU induced brain tumor animal model, by PCR based RFLP method in p53 exon-2, -3 and -4. In these studies we showed that the restriction site of p53 exon-3 and 4 were mutated in ENU induced brain tumor indicating a genetic defect associated with ENU induced tumorigenesis. In the therapeutic part, we confirmed the anti-tumor property of T11TS/S-LFA-3 in the ENU induced genetically altered cells. Histological evidences, cytotoxic study, PI-FACS cell-cycle analysis and TUNEL assay confirmed the apoptotic death of glioma cells by T11TS treatment in which p53 is mutated. From the present study we can conclude that ENU administration causes genomic instability along with mutations in p53 during the process of gliomagenesis. Whereas, T11TS/S-LFA3 demonstrated the potential to induce apoptosis of these tumor cells even when p53 is mutated and thus showed its immense potential to be an anti-neoplastic probe against p53 mutated diverse types of tumors.     

p53 and SCFFbw7 cooperatively restrain cyclin E-associated genome instability

Cancers often exhibit high levels of cyclin E expression, and aberrant cyclin E activity causes genomic instability and increased tumorigenesis. Two tumor suppressor pathways protect cells against cyclin E deregulation. The p53 pathway is induced by excess cyclin E in primary cells and opposes cyclin E activity through induction of p21Cip1. In contrast, the Fbw7 pathway targets cyclin E for degradation, and Fbw7 mutations occur commonly in cancers. We investigated the cooperativity of these two pathways in countering cyclin E-induced genomic instability in primary human cells. We find that loss of p53 and Fbw7 synergistically unmasks cyclin E-induced instability. In normal cells, impaired cyclin E degradation produces genome instability, but this is rapidly mitigated by induction of p53 and p21. In contrast, p53 loss allows the high level of cyclin E kinase activity that results from Fbw7 loss to persist and continuously drive genome instability. Moreover, p21 plays a critical role in suppressing cyclin E when Fbw7 is disabled, and in the absence of p21, sustained cyclin E activity induces rapid cell death via apoptosis. These data directly demonstrate the cooperative roles of these Fbw7 and p53 pathways in restraining cyclin E activity and its associated genome instability.     

A naturally occurring p73 mutation in a p73-p53 double-mutant lung cancer cell line encodes p73 alpha protein with a dominant-negative function

p73, a close homolog of p53 tumor suppressor, induces growth arrest and apoptosis. However, its role in cancers is controversial because of the rarity of p73 mutations, lack of tumors in p73-knockout mice, and the presence of multiple isotypes, among which ΔN isotypes inhibit the function of TA isotypes. We analyzed three naturally occurring p73 mutants found in lung cancer cell lines, NCI-H1155, DMS 92 and A427. NCI-H1155 is a cell line that has a p73 mutation [p73(G264W)] in the DNA-binding domain, as well as a p53 mutation [p53(R273H)], which is frequently found in human cancers and has a "gain-of-function" characteristic. p73α(G264W) not only lacks transactivation activity itself, but also suppressed the transactivation activity of the wild-type p73α in a dose-dependent manner, indicating that p73α(G264W) is a dominant-negative mutant. p73α(G264W) failed to suppress colony formation. We tested two other mutations, p73(Del418) in DMS 92 and p73(Del603) in A427. Both mutants retained similar levels of transactivation activity and suppression of colony formation to those of wild-type p73. The biological significance of these two mutations is unclear. In NCI-H1155 cells the coexistence of mutations that abrogate the normal functions of p73 and p53 may indicate that each mutation confers an additive growth advantage upon the cells.     

Role of the tumor suppressor gene Brca1 in genetic stability and mammary gland tumor formation

Germline mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. Current evidence demonstrates that mutations in BRCA1 do not directly result in tumor formation, but instead cause genetic instability, subjecting cells to high risks of malignant transformation. In an animal model in which Brca1 is mutated specifically in mammary epithelium, tumorigenesis occurs in mutant glands at low frequency after a long latency. Notably, introduction of a p53-null allele significantly enhanced mammary gland tumor formation in Brca1 conditional mutant mice. These results are consistent with a model that Brca1 is a caretaker gene, whose absence causes genetic instability and triggers further alterations, including inactivation of tumor suppressor genes and/or activation of oncogenes, leading to tumor formation.     

TIP30 mutant derived from hepatocellular carcinoma specimens promotes growth of HepG2 cells through up-regulation of N-cadherin

TIP30 is a tumor suppressor whose expression is altered in human liver, prostate, lung, colon, and breast cancers. Mice lacking TIP30 spontaneously developed hepatocellular carcinomas (HCC) and other tumors at a higher incidence than wild-type mice. Somatic missense mutations in the TIP30 gene were identified in human HCC tissue specimens, which resulted in instability or abnormal cellular distribution of TIP30 protein in cells. Here, we show that TIP30 mutants are able to promote cell growth and invasion and inhibit cisplatin-induced apoptosis in the HCC cell line HepG2 negative for endogenous TIP30. Moreover, one of the TIP30 mutants can dramatically accelerate tumor formation in immunodeficient mice. Analysis of gene expression in HepG2 cells, ectopically expressing either wild-type TIP30 or mutant TIP30, by Affymetrix GeneChip array, real-time quantitative PCR, and Western blotting assays reveals that TIP30 mutants can alter expression of genes involved in the regulation of tumorigenesis. This includes up-regulation of expression of N-cadherin and c-MYC and down-regulation of expression of p53 and E-cadherin. N-cadherin knockdown with small interfering RNA in HepG2 cells expressing mutant TIP30 resulted in a profound reduction in cell viability. Taken together, our data indicate that somatic mutations in the TIP30 gene may abolish its native tumor-suppressor activity and gain oncogenic activities partially through up-regulation of N-cadherin, thereby potentiating the pathogenesis of HCC in patients.     

The cancer connection: BRCA1 and BRCA2 tumor suppression in mice and humans

BRCA1 and BRCA2 mutations are estimated to be responsible for the great majority of familial breast and ovarian cancers. Much progress has been made toward the understanding of the function of these proteins through genetic, biochemical, and structural studies. The embryonic lethality encountered in the knockout mouse initially hindered the development of mouse models aimed at studying tumor suppression. However, mice that harbor hypomorphic Brca1 and Brca2 alleles and cre-mediated tissue-specific deletions for Brca1 and Brca2 have been generated. Mice deficient for either Brca1 or Brca2 sustain a wide range of carcinoma and mammary epithelium deleted for Brca1 or Brca2 is highly susceptible to mammary tumorigenesis. Mammary (and other) tumors occur at long latency as compared to oncogene-induced mouse tumors. p53 deficiency is highly cooperative with both Brca1 and Brca2 in promoting tumorigenesis. Analysis of Brca1-associated mammary tumors reveals significant similarities to BRCA1-associated breast cancer in regard to high tumor grade, hormone receptor negativity, a high incidence of p53 mutations and genetic instability.     

Targeting loss-of-function mutations in tumor-suppressor genes as a strategy for development of cancer therapeutic agents

Two types of genetic mutations, gain-of-function in oncogenes and loss-of-function in tumor-suppressor genes, are important molecular bases of tumorigenesis of human cancers. Target-based drug discovery is the main stream of contemporary cancer therapeutic development but largely focuses on gain-of-function mutations in oncogenes. Loss-of-function mutations in tumor-suppressor genes are often neglected as therapeutic targets. In this review, we discuss the feasibility of targeting loss-of-function mutations in tumor-suppressor genes for the identification of cancer-specific therapeutic agents.     

Mouse models dissect the role of p53 in cancer and development

Mice lacking one or two copies of the p53 gene have provided invaluable insight into the process of tumorigenesis. The importance of apoptosis in suppression of tumorigenesis in vivo became evident from analysis of these mice. Moreover, the timing and kinds of tumors that develop in these mice are altered by the presence of additional inherited mutations, by strain differences, and by food intake. Developmental abnormalities are also visible in mice with loss of p53 and with overexpression of p53 suggesting that p53 levels are critical for normal cellular processes. While mice do not necessarily recapitulate all the tumor types found in inherited cancers, they offer the unique opportunity to decipher the critical pathways in tumorigenesis. These findings can then be applied to humans.     

Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice.

Germline mutations in the tumor suppressor gene BRCA1 predispose women to breast cancer, however somatic mutations in the gene are rarely detected in sporadic cancers. To understand this phenomenon, we examined mouse models carrying conditional disruption of Brca1 in mammary epithelium in either p53 wild type (wt) or heterozygous backgrounds. Although a p53(+/-) mutation significantly accelerated tumorigenesis, both strains developed mammary tumors in a stochastic fashion, suggesting that multiple factors, in addition to p53 mutations, may be involved in Brca1 related tumorigenesis. A unique feature of Brca1 mammary tumors is their highly diverse histopathology accompanied by severe chromosome abnormalities. The tumors also display extensive genetic/molecular alterations, including overexpression of ErbB2, c-Myc, p27 and Cyclin D1 in the majority of tumors, while they were virtually ERalpha and p16 negative. Translocations involving p53 were also identified which lead to abnormal RNA and protein products. In addition, we generated cell lines from mammary tumors and found that the cells retained many of the genetic changes found in the primary tumors, suggesting that these genes may be players in Brca1-associated tumorigenesis. Despite their distinct morphology, all cultured tumor cells were Tamoxifen resistant but highly sensitive to Doxorubicin or gamma-irradiation, suggesting that these methods would be effective in treatment of this disease.     

Role of p53 mutations in endocrine tumorigenesis: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, p53 mutations in human endocrine tumors were analyzed by using polymerase chain reaction-single strand conformation polymorphism. Exons 5 through 10 of the p53 gene were studied in genomic DNAs from 134 primary endocrine tumors and 6 human endocrine cancer-derived cell lines. Mutations were detected and identified in 4 endocrine tumors, including one parathyroid adenoma and three thyroid carcinoma cell lines. The sites of these mutations were in exons 5 (codon 151 and 152) and 7 (codon 248 and 255). In all of three tumor cell lines, but not in a parathyroid adenoma, the normal allele encoding the p53 gene was lost. However, p53 mutations were not found in any other endocrine tumors or cell lines. Based upon these results, we concluded that the p53 gene may play a role in the tumorigenesis of a limited number of parathyroid adenoma and thyroid cancers, and that the p53 mutation with an allelic loss of the p53 gene is an important factor in malignant tumorigenesis of the thyroid gland.