Mutant p53 cooperates with ETS2 to promote etoposide resistance

Abstract

Mutant p53 (mtp53) promotes chemotherapy resistance through multiple mechanisms, including disabling proapoptotic proteins and regulating gene expression. Comparison of genome wide analysis of mtp53 binding revealed that the ETS-binding site motif (EBS) is prevalent within predicted mtp53-binding sites. We demonstrate that mtp53 regulates gene expression through EBS in promoters and that ETS2 mediates the interaction with this motif. Importantly, we identified TDP2, a 59-tyrosyl DNA phosphodiesterase involved in the repair of DNA damage caused by etoposide, as a transcriptional target of mtp53. We demonstrate that suppression of TDP2 sensitizes mtp53-expressing cells to etoposide and that mtp53 and TDP2 are frequently overexpressed in human lung cancer; thus, our analysis identifies a potentially ''druggable'' component of mtp53's gain-of-function activity.[Keywords: TDP2; cancer; p53] Supplemental material is available for this article. One of the definitive characteristics of the mutant p53 (mtp53) protein is that it can alter the cellular phenotype, resulting in the acquisition of gain-of-function activities such as abnormal cell growth, suppression of apoptosis, chemotherapy resistance, increased angiogenesis, and metastasis ( For example, mtp53 can interact with its family members, p63 and p73, and disable their ability to induce apoptosis (Di Como et al. 1999;Marin et al. 2000;Strano et al. 2000Strano et al. , 2002Gaiddon et al. 2001;Bergamaschi et al. 2003;Irwin et al. 2003;Lang et al. 2004). mtp53 can also interact with other transcription factors (such as NF-Y, E2F1, VDR, and p63) and thereby can be recruited to target genes that have consensus binding sites for these transcription factors (Di Agostino et al. 2006;Adorno et al. 2009;Fontemaggi et al. 2009;Stambolsky et al. 2010). Notably, some of these interactions help explain how the mtp53 protein can deregulate gene expression and promote abnormal cell growth, angiogenesis, and metastasis (Di Agostino et al. 2006;Adorno et al. 2009;Fontemaggi et al. 2009;Muller et al. 2009Muller et al. , 2011. However, thus far, none of these transcription factors have been shown to play a fundamental role in regulating the expression of genes that can confer chemotherapy resistance by modulating the response to DNA damage. The main goal of this study was to identify a transcriptional regulatory mechanism through which mtp53 can promote chemotherapy resistance. Results Identification of mtp53 target genesTo identify transcriptional targets of mtp53, we employed two different approaches: chromatin immunoprecipitation (ChIP)-on-chip and ChIP combined with deep sequencing (ChIP-seq). The ChIP-on-chip was performed with Nimblegen arrays that have oligonucleotide probes for all of the promoters in the human genome (Nimblegen Promoter Arrays). The ChIP-seq analysis was performed using the Illumina platform. We conducted these analyses in the Li-Fraumeni cell line MDAH087, which expresses only the R248W mtp53 protein (Bischoff et al. 1990). The ChIP-on-chip analysis identif...

Genetic and molecular alterations in olfactory neuroblastoma: implications for pathogenesis, prognosis and treatment

“…Cytotoxicity of cisplatin in pancreatic cancer cell lines was positively correlated with MAP4K2 expression, whereas NLRC4 deficiency may lead to radio-resistance of the respective tumor [69, 70]. Similarly p53 dysfunction results in resistance to radiotherapy and chemotherapy, including drugs widely used in ONB like etoposide [71]. Interestingly, only TP53 mutations were found by both research groups performing genome sequencing of metastatic ONBs.…”

Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity

“…Notably, a number of promoters that were reported to be regulated by bound mutp53 in other studies also overlap with CpG islands, e.g., MYC, 41 ID2, 42 ID4, 16 TWIST1, 9 EGR1, 22 MAP2K3, 43 target genes of the transcription factors VDR (CYP24A1) 14 and NF-Y (CCNA1, CCNB2, CDK1, CDC25C), 13 genes containing sterol regulatory elements in their promoters (CYP51A1, FDPS, FDFT1, HMGCR, HMGCS1, MVK, SQLE) 17 and the TDP2 gene. 18 G/C-rich DNA stretches are of particular interest regarding gene regulation. They are not only subjected to epigenetic control via cytosine methylation, but under favorable conditions (e.g., superhelical tension and stabilization by bound proteins), are also prone to form left-handed (Z), triplex (H), cruciform and G-quadruplex structures, which have been implied in transcriptional regulation.…”

“…This scenario is illustrated, e.g., by the cooperation of mutp53 with NF-Y in positive regulation of CCAT-box containing cell cycle genes upon DNA damage, 13 the functional interaction of mutp53 with the vitamin D receptor (VDR) on its target genes after exposure of cells to vitamin D, 14 the SMAD-mediated antagonism between mutp53 and p63 activity in regulation of metastasis-related, TGF-β-induced genes, 15 the co-stimulation of the pro-angiogenic ID4 gene via mutp53-E2F1 protein complexes, 16 the binding of mutp53 to promoters of mevalonate pathway genes at least partly by interaction with SREBP transcription factors 17 and the promotion of etoposide resistance via ETS2-dependent co-regulation of the DNA repair related TDP2 gene. 18 A second scenario is based on the capacity of mutp53 to interact with structured DNA, like stem-loop structures 19 and non-B DNA structures formed by trinucleotide [(CAG)·(CTG)] n repeats. 20 Along with the binding of mutp53 to intronic and intergenic sequences enriched in repetitive DNA and prone to form non-B DNA structures, these data led to a model that attributes mutp53 GOF to its ability to modulate transcription on a global level via binding to structured DNA.…”

Characterization of cancer-associated missense mutations in MDM2

“…29 Human MDM2 cDNA (GE Dharmacon) was amplified by PCR and subcloned into pCMV-Myc (Clontech), which was subsequently used as a template to generate MDM2 point mutants. All mutagenesis was accomplished using the Q5® SiteDirected Mutagenesis Kit from New England BioLabs and appropriate mutagenesis primers were generated using the NEBaseChanger TM tool.…”

“…28 More than 50% of human tumors have a mutated form of p53 (mtp53) that is more stable and has gain-of-function activities such as promoting cell growth, chemotherapy resistance, angiogenesis, and metastasis. 29,30 An alternative mechanism by which p53 is thought to be inactivated in human cancers is through increased expression of MDM2. To date, there are 92 mutations in the MDM2 gene reported in the Catalog of Somatic Mutations in Cancer (COSMIC) website.…”