Biochem Biophys Res Commun. 2016 Sep 16;478(2):976-81.
Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.
Dong Min Kim1, Seung-Hyun Choi, Young Il Yeom, Sang-Hyun Min2*, and Il-Chul Kim3*
1Center for Applied Life Science, Hanbat National University, Taejon 305-719, Korea
2New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea
3Department of Biology, Chonnam National University, Gwangju 500-757, Korea
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MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected 1,089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.
Inactivating p53 by mutation or by interaction with oncogene products of DNA tumor viruses can lead to cancer. p53 mutations are found in almost 50% of all human cancers, whereas p53 normally inhibits tumor formation in many tissues. Interestingly, leukemia represents a class of cancers that does not contain a significant number of p53 mutations. Although there are few direct p53 mutations in leukemia, the p53 pathway has been suggested to be defective due to alterations in either upstream or downstream regulators. Leukemia provides an example for such a phenomenon, whereby MDM2, the critical negative regulator of p53, is often overexpressed. p53 is a very unstable protein in unstressed cells, with a half-life of 5–30 min, and exists at very low levels in normal unstressed cells due to continuous degradation largely mediated by MDM2.
MDM2 also has p53-independent functions in cell cycle control, differentiation, cell fate determination, DNA repair, basal transcription, and other processes . For example, expression of MDM2 during development is tissue-specific and is independent of p53 in different organs . High MDM2 expression levels are observed in human tumor cell lines with little or no functional p53 . MDM2 expression increases following fibroblast growth factor-2 cell treatment  and in cells harboring a chimeric macrophage colony-stimulating factor/platelet-derived growth factor receptor . Furthermore, the MDM2 appears to contribute to the transformed phenotype in the absence of wild-type p53 . Nevertheless, the number of studies is still limited, and the evidence in some cases does not unequivocally show that the functions are p53 independent.
Overexpression of human MDM2 induces chromosome instability and polyploidy independent of p53 status , whereas reducing MDM2 expression levels in ARF-deficient cells enhances resistance to Ras-induced transformation . Although overexpression of Mdm2 can arrest some cells in G1, it drives cell-cycle progression in many cell types . Specifically, MDM2 activates E2F-1 transcription and cell-cycle progression in tumor cells .
Figure 1. MDM2-p53 pathway alteration in cancer cells. The expression of MDM2 and p53 is balanced in normal cells, however, MDM2 is often overexpressed in cancer cells regardless of p53 mutations and the balance is disrupted in cancer cells. This imbalance can be explained by the high level of expression of NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, YWHAB, MAGEA12, SERPINA3, LOC51035, PRCC, Enigma, and PRL-1 in combination with lower expression of KLF6, Sirt3, and ING1. Those activations of multiple cancer signals can induce transcriptional activation of Mdm2 in p53 independent manner resulting in Rb degradation, E2F1 transactivation, p21 degradation, and hnRNP degradation that enhance cell proliferation. Also, Mdm2 accumulation can lead to FOXO3a degradation and the enhanced translation of XIAP that suppress apoptosis as well as E-cadherin degradation that promote EMT and metastasis in cancer cell. Moreover, Mdm2 accumulation can induce genomic instability by inhibition of DSB repair through interaction with Nbs1.
In this study, we performed genome-scale functional screening of the human genes modulating MDM2 expression and composed a screening library including 368 genes after searching the co-expression profiles of genes showing positive or negative patterns with MDM2 expression from a DNA microarray database. Using MDM2 promoter-reporter systems, we screened nine clones that enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells, suggesting that these clones act in a p53 independent manner. Also we discriminated the clones which can mediate the MAPK signal from others.
MDM2 has three different promoter regions and second promoter P2 shows the strongest expression trends according to p53 transcription factor (Fig.2). We also screened the P2 mutant promoters without E2F, MYC and AP-1. Most clones did not show different reporter activity without E2F and MYC (data not shown). Because AP-1 activity affected the transcription of MDM2 from P2 promoter, we focused this signal in p53 independent expression of MDM2.
One of the p53-independent oncogenic roles of MDM2 is its association with the Mre11/Rad50/Nbs1 DNA repair complex by specifically binding to Nbs1 . Expression of MDM2 is at levels two- to four-fold higher than endogenous levels leads to centrosome amplification and chromosome instability . Overproduction of MDM2 leads to DNA duplication independent of p53 . In addition, MDM2 rescues transforming growth factor-β-induced growth arrest in a p53-independent manner . Overexpression of MDM2 activates E2F and inhibits the Rb growth suppressing function . These results suggest that p53-independent upregulation of MDM2 by increasing the levels of selected clones may lead to oncogenesis, and that MDM2-modulating genes are novel potential targets for cancer treatment.
Figure 2. The nucleotide sequence of promoter region in human MDM2. Bald shows the alternative first exon and the underlined sequences represent the binding motives of transcription factors. The position in the sequence is determined by at the position of exon start site as +1. The binding motives was expected by TransExplorer algorithm in Korean Research Institute of Bioscience and Biotechnology.
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (2013-R1A1A1007596 and 2015M3A9C7030181). Also, this study was financially supported by Chonnam National University (Grant number: 2012-0828).
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