Alternate Splicing of the p53 Inhibitor HDMX Offers a Superior Prognostic Biomarker than p53 Mutation in Human Cancer

Lenos, Kristiaan; Grawenda, Anna M.; Lodder, Kirsten; Kuijjer, Marieke L.; Teunisse, Amina F.A.S.; Repapi, Emmanouela; Grochola, Lukasz F.; Bartel, Frank; Hogendoorn, Pancras C.W.; Wuerl, Peter; Täubert, Helge; Cleton‐Jansen, Anne‐Marie; Bond, Gareth L.; Jochemsen, Aart G.

doi:10.1158/0008-5472.can-12-0215

Cited by 53 publications

(64 citation statements)

References 38 publications

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“…Notably, the frequency of p53-inactivating missense mutations (28) was lower in samples predicted to express MDM4 (4.6%) than in the nonexpresser samples (9.3%). This is consistent with previous observations that a low PSI index is associated with inactivating p53 mutations or MDM2 overexpression (29). However, this also indicates the presence in some cancers of p53-inactivating mutations in MDM4-expressing samples, an observation that is consistent with the p53-independent oncogenic functions of MDM4.…”

Section: Introductionsupporting

confidence: 92%

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

Dewaele

Tabaglio

Willekens

et al. 2015

Journal of Clinical Investigation

147

178

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Section: Introductionsupporting

confidence: 92%

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

Dewaele

Tabaglio

Willekens

et al. 2015

Journal of Clinical Investigation

147

178

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“…Although it has been suggested that mdmx-s RNA may act as a means of reducing full-length MDMX levels rather than being translated into protein, 20 we found that the mdmx-s transcript in our WD/DD LPS samples was translated into protein, albeit to varying extents.…”

Section: Mdmx Protein Expressioncontrasting

confidence: 84%

“…In this way, the ratio between the mdmx-s splice variant and the full-length mdmx becomes the means by which mdmx-s mRNA expression reduces that of the full-length MDMX protein, 19 and it has recently been reported that the reduced MDMX protein expression mediated by mdmx/mdmx-s is associated with a worse prognosis in patients with osteosarcoma. 20 The role of MDMX and MDMX-S in WD/DD LPS has not yet been defined. It has been reported that MDMX protein expression is very low in WD/DD LPS cell lines, 21 and MDMX gene amplification has been described in association with MDM2 co-amplification in one WD/DD LPS surgical specimen.…”

mentioning

confidence: 99%

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

Bozzi

Conca

Laurini

et al. 2013

Laboratory Investigation

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The molecular marker of well-differentiated/de-differentiated liposarcomas is MDM2 gene amplification coupled with protein overexpression and wild-type TP53. MDMX is a recently identified MDM2 homolog and its presence in this tumor is unexplored. Our aim was to investigate the role of full-length MDM2 and MDMX proteins and their isoforms in surgical specimens of well-differentiated/de-differentiated liposarcomas in view of Nutlin-3A (a MDM2 inhibitor) treatment. Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens was examined by means of: (1) fluorescence in situ hybridization to determine MDM2 and MDMX gene copy numbers; (2) RT-PCR and densitometry to analyze alternative splicing forms of mdm2 and mdmx; (3) immunoblotting and immunohistochemistry to assess the corresponding translated proteins; and (4) in vitro and in silico assays to determine their affinity for Nutlin-3A. All these cases showed MDM2 gene amplification with an MDMX disomic pattern. In all cases, the full-length mdm2 transcript was associated with the mdm2-b transcript, with ratios ranging from 0.07 to 5.6, and both were translated into protein; mdmx and mdmx-s were co-transcripted, with ratios ranging from 0.1 to 5.6. MDMX-S was frequently more upregulated than MDMX at both transcriptional and protein level. Each case showed different amounts of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and the corresponding proteins. In vitro assays showed that Nutlin-3A was ineffective against MDM2-B and was unable to disrupt the MDMX/TP53 and MSMX-S/TP53 complexes. Molecular simulations confirmed these in vitro findings by showing that MDM2 has high Nutlin-3A affinity, followed by MDMX-S, MDMX, and MDM2-B. Nutlin-3A is predicted to be a good therapeutic option for well-differentiated/de-differentiated liposarcomas. However, our findings predict heterogeneous responses depending on the relative expression of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and proteins. Well-differentiated/de-differentiated (WD/DD) liposarcomas (LPS) account for 40% of all liposarcomas. With a few exceptions, 1,2 the DD component is defined as a usually abrupt and generally a phenotypic time-dependent transition to a non-lipogenic sarcoma.WD/DD LPS characteristically involve the retroperitoneum and are characterized by a high rate of local recurrences. The inability of even aggressive surgery to obtain negative margins leads to high local failure rates, worsening the long-term prognosis of WD/DD patients and favors the use of multiple treatment modalities albeit with limited benefit. 3,4 The molecular hallmark of WD/DD LPS is MDM2 gene amplification coupled with protein overexpression and wild-type TP53, 5-7 which provides the rationale supporting the therapeutic potential of the MDM2 antagonist Nutlin-3A. MDM2 is an E3 ubiquitin ligase that, in addition to targeting TP53 for proteasomal degradation, blocks TP53

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“…Therefore, both studies reveal that MDMX predominantly adopts an inactive conformation for p53 binding. Our results also shed light on a puzzling observation that full-length MDMX is a poor inhibitor of p53 in reporter gene assays, whereas the MDMX-S alternative splice product without the central and C-terminal domains is a strong inhibitor of p53 and its expression in cancer correlates with poor prognosis (22).…”

Section: Discussionmentioning

confidence: 60%

Autoinhibition of MDMX by intramolecular p53 mimicry

Chen¹,

Borcherds

Wu³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.T he p53 tumor suppressor is activated by numerous cellular and environmental signals and induces the expression of genes that regulate metabolism, cell growth, cell cycle, apoptosis, and senescence (1). MDM2 and MDMX are key regulators that control the cellular level and transcriptional activity of p53 through direct binding. Mouse knockout experiments showed that both MDM2 and MDMX are essential for controlling p53 activity during embryogenesis. Somatic knockout experiments showed that MDM2 is indispensable for regulating p53 in adult tissues, whereas MDMX deletion does not lead to cell death (2). MDM2 is a well-established p53 transcriptional target that forms a negative feedback loop by binding to the N-terminal transcriptional activation domain of p53 and, subsequently, ubiquitinating the C-terminal regulatory domain, which leads to degradation of p53 by the proteasome. p53 binding sites are also found in intron 1 of human MDMX, and p53 activation leads to moderate induction of MDMX transcription (3). Therefore, MDMX is a p53 target gene that may also provide dynamic feedback in response to p53 activation.MDMX alone does not have E3 ligase activity, but it is important for regulating p53 transcriptional function. MDMX expression and phosphorylation by the ATM/Chk2 pathway is important for the p53-mediated DNA damage response in mice (4, 5). MDMX levels are controlled by MDM2-mediated ubiquitination in a stress-dependent fashion (6, 7). Significant degradation of MDMX occurs after DNA damage through phosphorylation at several C-terminal sites (S342 and S367 by Chk2, S403 by ATM) (8). Furthermore, ribosomal stress promotes MDMX degradation through L11-MDM2 interaction (9), and oncogenic stress promotes MDMX degradation through ARF expression (10). Therefore, key signaling mechanisms that block p53 degradation simultaneously enhance MDMX degradation by MDM2. These observations underscore the co...

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Alternate Splicing of the p53 Inhibitor HDMX Offers a Superior Prognostic Biomarker than p53 Mutation in Human Cancer

Cited by 53 publications

References 38 publications

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

Autoinhibition of MDMX by intramolecular p53 mimicry

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