In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 29 ( 2011-07-19)
Abstract:
Our results support a model in which the tumor suppressor role of Prep1 is associated with the maintenance of genomic stability ( Fig. P1 ). Reduced expression of Prep1 (as in the majority of human cancers) is advantageous in the multistep process of tumorigenesis, because it accelerates the rate of accumulation of cancer-favoring mutations. It is noteworthy that, despite the occurrence of irreversible genetic events in Prep1 i/i MEFs, the cells still require reduction of Prep1 to manifest a fully transformed potential. Prep1 expression reverts some of the properties of these transformed cells, suggesting that a strategy based on the restoration of Prep1 function might have therapeutic effects in the fraction of human tumors ( 1 ) expressing very low levels of Prep1 . To determine the cellular processes that are implicated in the tumor suppressor function of Prep1 , we analyzed the behavior of cells in which Prep1 had been knocked down. We examined hypomorphic Prep1 i/i fetal liver cells and mouse embryonic fibroblasts (MEFs) using a biochemical technique for detecting DNA breaks (Comet assay) and immunological assays for markers of the DNA damage signaling cascade. We found that Prep1 -deficient cells exhibit increased basal DNA damage and a normal cellular response to DNA damage after γ-irradiation compared with WT cells. DNA content and cytogenetic analyses in MEFs also revealed a tendency for chromosomal aberrations, including alterations in chromosome copy number, in Prep1 i/i MEFs. To determine whether the observed DNA damage is an early or late consequence of the absence of Prep1 , we down-regulated Prep1 in normal human fibroblasts and used markers of DNA damage signaling to check for both DNA damage and the cellular response to damage. Down-regulation of Prep1 is rapidly followed by DNA damage. Furthermore, Prep1 knockdown is followed by an increase of chromosome-related modifications, such as a change in the compactness of heterochromatin, a higher-order chromosomal structure, which was indicated by a rapid increase in the trimethylation of a specific histone protein that helps compose chromatin. Additional molecular analysis confirmed the overall increase of this modification on Prep1 down-regulation in both mouse and human cells. However, chromatin modifications appear as a consequence rather than as a cause of DNA damage. Because genomic instability is associated with cell immortalization and cancer, we studied the immortalization kinetics of two WT and two Prep1 i/i littermate MEF cell lines in culture. Prep1 -deficient cells but not WT cells markedly increased their proliferative rates at later stages of cell culture. The causal link between genomic instability and cellular behavior is supported by a spontaneous genetic deletion in a genetic locus dubbed the Ink4a-Arf locus in one of the Prep1 i/i MEF lines analyzed. We also analyzed transformation, the cellular process that is known to trigger cancer, in MEFs by overexpressing the oncogene Ras and performing in vitro and in vivo transformation assays. Prep1 i/i MEFs were much more efficiently transformed by Ras compared with WT cells. Remarkably, expression of Prep1 partially rescued the transformation phenotype of Prep1 i/i MEFs. Finally, we studied whether a stable knockdown of Prep1 can affect oncogene-mediated senescence in human cells. In fact, in the absence of other lesions, introduction of an oncogene blocks cell proliferation-inducing senescence. This phenomenon is mediated by the increased expression of one specific tumor suppressor gene, p19-Arf. We showed that Prep1 down-regulation favors the bypass of oncogene-induced senescence and partially compromises Arf induction. Prep1 is essential during embryonic development. Mouse embryos lacking Prep1 die before gastrulation, a critical early step in embryonic development, because epiblast cells, which help determine the formation of the embryo, undergo apoptosis ( 2 ). Genetic data suggest that the Prep1 phenotype is related to DNA damage. However, embryos carrying a different Prep1 mutation—called a hypomorphic Prep1 mutation—that does not eliminate the gene but leads to very low (2%) levels of Prep1 mRNA—show incomplete development of organs, and in 75% of cases, they die at embryonic day 17.5 ( 3 ). The idea that Prep1 is a tumor suppressor stems from the observation that the fraction of homozygous mice bearing this Prep1 mutation and survive embryonic lethality often develops tumors. In addition, Prep1 insufficiency drastically accelerates the development of a type of lymphoma. Furthermore, a survey of more than 1,000 human cancers shows a dramatic reduction of Prep1 expression in a large proportion (70%) of the patients ( 1 ). The down-regulation of tumor suppressor genes, which help keep cancer in check, has been reported in a range of cancer types. Our recent studies revealed that the human homolog of one such mouse tumor suppressor protein (Prep1, which acts as a gene switch bearing a canonical structure called a homeodomain) is absent or down-regulated in 70% of human cancers ( 1 ). Extending those earlier findings, here, we report that Prep1 acts as a tumor suppressor most likely by helping cells maintain genomic stability. Although many tumor suppressor genes are known, the large proportion of human tumors in which the Prep1 gene is absent or down-regulated prompts a deep investigation of its function.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1105216108
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2011
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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