MDM2/MDMX: Master negative regulators for p53 and RB

ABSTRACT

MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

Abbreviations

CDK4

cyclin-dependent protein kinase 4

E2F

E2F family of DNA binding transcription factors

MDM2

mouse double minute 2 homolog

MDMX

double minute X human homolog, also known as MDM4

p16^INK4A

inhibitor of cyclin-dependent kinase type 4

RB

retinoblastoma protein

RB1

retinoblastoma susceptibility gene

Inactivation of the retinoblastoma gene is one of the most frequent events in cancer development. The growth suppressive function of the retinoblastoma protein (RB) is inhibited by direct inactivating mutations, or more commonly by changes in expression of its upstream regulators, including overexpression of cyclin D1, activating mutations in cyclin-dependent protein kinase 4 (CDK4), and inactivating mutations in p16^INK4A (inhibitor of cyclin-dependent kinase type 4, also called INK4A).¹ In addition, RB is inactivated by the viral oncoprotein E7, which promotes RB instability, and by E1A or SV40T, which disrupt the interaction between RB and the E2F family of DNA binding transcription factors (E2F).¹

We have shown that mouse double minute 2 homolog (MDM2) directly binds to the C-pocket of RB, leading to disruption of RB–E2F interaction and accelerated RB degradation.^2,3 In our recent study, we demonstrated that double minute X human homolog, (MDMX, also known as MDM4) can also bind to and promote RB degradation in an MDM2-dependent manner, thereby promoting tumor growth.⁴

MDMX and MDM2 are highly homologous with considerable structural similarity.⁵ Both proteins contain a central acidic domain, a zinc finger domain, and a well-conserved C-terminal typical ring (RING) finger domain, yet only MDM2 possesses E3 ubiquitin ligase activity.⁵ MDM2 and MDMX are predominantly found as heterodimers, which is thought to enhance E2 protein recruitment and stabilize the ternary complex containing tumor protein p53 (best known as p53).⁶ In our study, we found that MDMX enhances the ability of MDM2 to bind to and promote RB degradation. Interestingly, although MDM2 binds RB via its central acidic domain,³ MDMX binds RB via its RING finger. Notably, a RING finger mutant MDMX (C463A) retained its ability to bind RB, but did not bind MDM2 and was not able to affect RB protein levels. Hence, MDMX may bring RB and MDM2 into close proximity by recruiting them to its C-terminal site, thus facilitating MDM2 binding to RB and inducing RB degradation. Moreover, MDM2 can promote RB degradation independently of ubiquitination by directly interacting with the C8 subunit of the 20S proteasome to deliver RB for proteolysis.² It is possible that a MDM2–MDMX heterodimer might more efficiently recruit additional players involved in RB degradation, such as the C8 proteasome subunit, in a manner analogous to E2 binding for p53 ubiquitination. These alternatives are not mutually exclusive, and the molecular mechanism may involve some combination of both possibilities.

As an important alternative pathway to inactivate p53 in addition to p53 gene mutations, amplification of MDM2 or MDMX genes is found in a variety of human tumors and cancers.⁶ Of note, the MDMX gene is amplified in more than 60% of human retinoblastomas, with an additional 10% of retinoblastomas exhibiting MDM2 gene amplification.⁷ Notably, retinoblastomas are commonly characterized by a dysfunctional RB pathway but maintain wild-type p53 expression.⁸ Hence, it is conceivable that loss or inactivation of RB leads to upregulation of E2F transcriptional activity, which in turn promotes apoptosis in a p53-dependent manner.⁹ Thus, overexpression of MDMX may be selected to inactivate p53 and inhibit p53-mediated apoptosis in the context of RB deficiency in retinoblastoma.

However, numerous cases of cancer have been reported to harbor p53 mutations and concomitant MDMX and/or MDM2 gene amplification.¹⁰ An important question then arises as to why the MDMX gene is amplified in cancers where p53 is already inactivated by mutation. Our study indicates that the oncogenic function of MDMX relies on its activity in the suppression of RB. Indeed, silencing of MDMX in p53-null H1299 cells significantly suppressed their growth as xenograft tumors in nude mice. However, concomitant silencing of both MDMX and RB dramatically reverted tumor growth to basal levels, suggesting that MDMX-mediated regulation of RB plays an important role in the pathology of cancer development. These observations suggest that in tumors with wild-type RB1 alleles, overexpression of MDM2 and/or MDMX can facilitate RB degradation and therefore promote cancer development (Fig. 1).

Figure 1.

MDM2/MDMX gene amplification in RB wild-type (WT) or RB-deficient cancers with wild-type tumor protein p53. (A) Overexpression of MDM2/MDMX in cancers with WT p53 and RB leads to inactivation of both p53 and RB pathways, resulting in cell proliferation and cancer development. (B) Overexpression of MDM2/MDMX in cancers with WT p53 but RB deficiency inhibits E2F/p53-mediated apoptosis. E2F, E2F family of DNA binding transcription factors; MDM2, mouse double minute 2 homolog; MDMX, double minute X human homolog, also known as MDM4; RB, retinoblastoma protein.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the National Key Basic Research Program (973 Program) of China (2012CB910700) and National Natural Science Foundation of China (NSFC) grant (81330054 and 31171362) to Z-XX, and NSFC grant (31350110216) to JB.