GC-selective DNA-binding antibiotic, Mithramycin A, reveals multiple points of control in the regulation of Hdm2 protein synthesis

Phillips, Anna; Darley, Matthew; Blaydes, Jeremy P.

doi:10.1038/sj.onc.1209451

Cited by 10 publications

(12 citation statements)

References 53 publications

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“…The analyses done on bladder cancer cells also served to confirm the association of SNP309 with the tumor transcript profiles shown above. Further analyses will underline functional interaction of the different components of the p53 pathway (39).…”

Section: Resultsmentioning

confidence: 99%

A Polymorphism in HDM2 (SNP309) Associates with Early Onset in Superficial Tumors, TP53 Mutations, and Poor Outcome in Invasive Bladder Cancer

Sänchez‐Carbayo¹,

Socci

Kirchoff

et al. 2007

Clinical Cancer Research

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Purpose: The HDM2 gene represents one of the central nodes in the p53 pathway. A recent study reported the association of a single nucleotide polymorphism (SNP309) in the HDM2 promoter region with accelerated tumor formation in both hereditary and sporadic cancers. In this study, we aim to evaluate the SNP309 in bladder cancer and to link it to TP53 status. Experimental Design: SNP309 genotyping and TP53 mutation status were done on 141 bladder tumors and 8 bladder cancer cell lines using a RFLP strategy andTP53 genotyping arrays, respectively. Transcript profiling of a subset of cases (n = 41) was done using oligonucleotide arrays to identify genes differentially expressed regarding their SNP309 status. Results: Of 141 bladder tumors analyzed, 36.9% displayed the SNP309 wild-type (WT; T/T) genotype, whereas 11.3% were homozygous (G/G) and 51.8% were heterozygous (T/G) cases. Patients with superficial disease and the G/G genotype had an earlier age on onset than those with the T/G or T/T genotypes (P = 0.029). Tumors with SNP309 WT genotype significantly displayed TP53 mutations when compared with tumors harboring G/G or T/G genotypes (P < 0.05). SNP309 WT cases had a poorer overall survival than cases with G/G and T/G genotypes (P < 0.05).TP53 mutation status provided enhanced prognostic value (P < 0.001).Transcript profiling identified TP53 targets among those differentially expressed between tumors displaying G/G orT/G SNP309 versus WTcases. Conclusions: SNP309 is a frequent event in bladder cancer, related to earlier onset of superficial disease and TP53 mutation status. SNP309 genotypes were found to be associated with clinical outcome.The HDM2 gene is a transcriptional target of p53. It encodes a negative feedback regulator (p90-Hdm2) that binds to p53 and acts as an ubiquitin-ligase, targeting p53 to proteasomal degradation (1). The association of a single nucleotide polymorphism (SNP309) in the HDM2 promoter region with accelerated tumor formation in both hereditary and sporadic cancers has been studied by several groups (2 -24). As Hdm2 expression levels seem to be critical to a well-regulated p53 response, naturally occurring sequence variations in the HDM2 promoter may result in altered expression of the Hdm2, affecting p53 tumor suppression activity (2). In addition, the association of SNP309 to an early age of onset in cancer predisposition syndromes has also been reported (2, 3), although it has also been reported lack of evidence that the HDM2 SNP309 accelerates tumor development in carriers of known pathogenic germ-line mutations, such of BRCA1 (4). Several case-control studies of squamous head and neck tumors, breast, ovarian, lung, and colon carcinomas did not found either such correlation (6 -10, 12, 16).Bladder tumors are known to commonly harbor TP53 mutations and less frequently HDM2 amplifications (25 -31). Although the abrogation of normal p53 function may be one of the permissive events promoting proto-oncogene amplification, predisposition to gene amplification of HDM2 in uroth...

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Section: Resultsmentioning

confidence: 99%

A Polymorphism in HDM2 (SNP309) Associates with Early Onset in Superficial Tumors, TP53 Mutations, and Poor Outcome in Invasive Bladder Cancer

Sänchez‐Carbayo¹,

Socci

Kirchoff

et al. 2007

Clinical Cancer Research

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“…The P1 promoter is located upstream of exon 1 and is active at basal constitutive levels in most cells (Mendrysa and Perry, 2000). Although motifs of the P1 promoter important for its activity have been defined, its control is still not understood (Chang et al, 2004;Phillips et al, 2006). The second promoter (P2) is located in intron 1, it has two p53-responsive elements (Zauberman et al, 1995) and p53 is believed to initiate MDM2 transcription from this promoter, thus forming an auto-regulatory feedback loop.…”

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p53-independent mechanisms regulate the P2-MDM2 promoter in adult astrocytic tumours

et al. 2008

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The MDM2 gene is amplified and/or overexpressed in about 10% of glioblastomas and constitutes one of a number of ways the p53 pathway is disrupted in these tumours. MDM2 encodes a nuclear phosphoprotein that regulates several cell proteins by binding and/ or ubiquitinating them, with p53 being a well-established partner. MDM2 has two promoters, P1 and P2 that give rise to transcripts with distinct 5 0 untranslated regions. Transcription from P2 is believed to be controlled by p53 and a single-nucleotide polymorphism (SNP309, T4G) in P2 is reported to be associated with increased risk for, and early development of, malignancies. The use of P1 and P2 has not been investigated in gliomas. We used RT -PCR to study P1-and P2-MDM2 transcript expression in astrocytic tumours, xenografts and cell lines with known MDM2, TP53 and p14 ARF gene status. Both promoters were used in all genetic backgrounds including the use of the P2 promoter in TP53 null cells, indicating a p53-independent induction of transcription. Transcripts from the P1 promoter formed a greater proportion of the total MDM2 transcripts in tumours with MDM2 amplification, despite these tumours having two wild-type TP53 alleles. Examination of SNP309 in glioblastoma patients showed a borderline association with survival but no apparent correlation with age at diagnosis nor with TP53 and p14 ARF status of their tumours. Our findings also indicate that elevated MDM2 mRNA levels in tumours with MDM2 amplification are preferentially driven by the P1 promoter and that the P2 promoter is not only regulated by p53 but also by other transcription factor(s).

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“…HDM2 has two promoters, which generate transcripts with alternate 5 0 untranslated regions (UTRs) (exon 1 or 2) but a common coding region and 3 0 UTR (exons 3-12). The P1 promoter is constitutively expressed (Phillips et al, 2006a), whereas the P2 promoter is highly inducible by p53 (Zauberman et al, 1995). Growth factor signalling through RAS-RAF-MEK-ERK can also regulate HDM2 protein synthesis, with readily identifiable consequences for cancer cell survival (Shaulian et al, 1997;Ries et al, 2000).…”

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confidence: 99%

“…Compared to pMV7-transfected controls, GAPDH levels in pMV7-4E-transfected cells were 118.8 ± 6.57, 80.8 ± 6.88 and 76.0 ± 7.28% in total, nuclear and cytoplasmic fractions respectively. Cellular fractionation was validated using semiquantitative reverse transcription-PCR for snRNA U6, scRNA Y4 and GAPDH as described previously (Phelps et al, 2005;Phillips et al, 2006a). Two PCRs for each fraction are shown, with a threefold difference in the amount of input cDNA to confirm PCRs have not plateaued.…”

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confidence: 99%

“…At 48 h post-transfection, western blotting was performed using the following antibodies: HDM2 (monoclonal antibody 2A9; Chen et al, 1993), Cyclin D1 (Calbiochem, La Jolla, CA, USA), eIF4E (BD Biosciences, Oxford, UK), rabbit anti-b-actin antibody (Sigma Aldrich Co, Poole, UK). (b) MCF-7 cells in 100 mm plates were transfected with 18 mg pMV7 or pMV7-4E 48 h before cellular fractionation by hypotonic lysis (Phillips et al, 2006a). Following RNA extraction using RNABee (Biogenesis Inc., Poole, UK), cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) and Taqman quantitative (q)PCR analysis was performed using Taqman Universal PCR Mastermix (Applied Biosystems, Warrington, UK).…”

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MNK1 and EIF4E are downstream effectors of MEKs in the regulation of the nuclear export of HDM2 mRNA

Phillips

Blaydes

2007

Oncogene

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Regulation of the synthesis, function and degradation of HDM2 (Mdm2 in mouse) plays a key role in controlling the abundance and activity of the transcription factor p53, with consequent implications for the proliferation and survival of normal and cancer cells. We have previously identified the regulation of export of HDM2 mRNA from the nucleus as a novel point of control of HDM2 synthesis. This process is dependent on the activity of the growth factor-regulated MAP-kinase kinases (MEKs). Here, we provide evidence that the eIF4E kinase MNK1 is a key downstream effector of MEKs in this regulatory pathway. We show that HDM2 mRNA export in breast cancer cells is promoted by overexpressed eIF4E in a MEK-and MNK1-dependent manner, and inhibition of MNK1 suppresses endogenous HDM2 mRNA export pathways. This MNK1-and eIF4E-dependent HDM2 regulation occurs through sequences in the 3 0 untranslated region of HDM2 mRNA, and consequently HDM2 mRNA transcripts from both the constitutive P1 and inducible P2 promoters are regulated by this pathway. eIF4E is a known oncogene that is overexpressed in human tumours, including the majority of breast cancers. This pathway, therefore, may play an important role in the dysregulation of HDM2 oncoprotein expression that occurs in many human tumours.

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GC-selective DNA-binding antibiotic, Mithramycin A, reveals multiple points of control in the regulation of Hdm2 protein synthesis

Cited by 10 publications

References 53 publications

A Polymorphism in HDM2 (SNP309) Associates with Early Onset in Superficial Tumors, TP53 Mutations, and Poor Outcome in Invasive Bladder Cancer

A Polymorphism in HDM2 (SNP309) Associates with Early Onset in Superficial Tumors, TP53 Mutations, and Poor Outcome in Invasive Bladder Cancer

p53-independent mechanisms regulate the P2-MDM2 promoter in adult astrocytic tumours

MNK1 and EIF4E are downstream effectors of MEKs in the regulation of the nuclear export of HDM2 mRNA

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