mdm2 and its family member , mdmx , are overexpressed in numerous human cancers ( eischen and lozano , 2014 ) . both proteins are considered oncogenes due to their tumour promoting activity and ability to cooperate with other oncogenes when overexpressed ( lundgren et al . , 1997 ; jones et al . , 1998 genetic studies with knockout mice have firmly established that mdm2 and mdmx regulate the tumour suppressor p53 ( jones et al . , 1995 ; montes de oca luna et al . , 1995 ; parant et al . , 2001 ; finch et al . , 2002 ; migliorini et al . , 2002 ) . mdm2 and mdmx regulate the activity , stability , and localization of p53 ( eischen and lozano , 2014 ) . increased expression of mdm2 or mdmx dampens dna damage signalling and the p53 response ( wang et al . , 2008 ; however , a series of reports starting in the late 1990s revealed that mdm2 appeared to have effects that were independent of p53 ( melo and eischen , 2012 ) . for example , overexpression of mdm2 in the presence of wild - type p53 often led to cell death or an inability to generate clones or mice ( kubbutat et al . , 1997 ; these results suggested that mdm2 was more than just regulating p53 , as mdm2 overexpression should have led to a growth / survival advantage if it only inhibited p53 . mdm2 transgenic p53-null mice developed a different tumour spectrum compared with mice that were just p53-null . more sarcomas developed in the mdm2 transgenic p53 mice than in the p53 mice , which preferentially developed lymphomas ( jones et al . , 1998 ) . additionally , a haploinsufficiency in mdm2 , as in mdm2 heterozygous p53-null mice , resulted in a different tumour spectrum and a slightly longer tumour latency ( mcdonnell et al . , 1999 ; eischen and boyd , 2012 ) . multiple human and murine tumours have been identified that both overexpress mdm2 and have inactivated p53 ( cordon - cardo et al . , 1994 ; , 1996 ; eischen et al . , 1999 ; peng et al . , 2001 ; ramos et al . , 2001 ; , 2003 ; giglio et al . , 2005 ) . finally , principal component analysis of microarray data comparing lymphomas overexpressing mdm2 and harbouring wild - type p53 , lymphomas overexpressing mdm2 and lacking p53 , and lymphomas with both indicated separation of these lymphomas based on differential mrna expression ( unpublished data ) . together , the data indicate that mdm2 has p53-independent effects that likely contribute to tumorigenesis . the challenge was to identify the p53-independent function(s ) of mdm2 and the proteins that mediate the function(s ) . to identify proteins other than p53 that associate with mdm2 , we chose an unbiased approach by immunoprecipitating endogenous mdm2 from hela cells and performing mass spectrometry on the associated proteins . we identified all three members of the mre11/rad50/nbs1 ( mrn ) dna repair complex ( alt et al . , 2005 ) . the mrn complex is activated in response to dna breaks and facilitates dna break repair by amplifying the dna damage signal , holding the broken dna ends together , and processing the ends ( shiloh and ziv , 2013 ; rein and stracker , 2014 ) . the mrn complex also aides in the activation of the dna damage kinase atm ( rein and stracker , 2014 ) . extensive additional characterization of mdm2 association with the mrn complex indicated their interaction occurred in primary , immortalized , and malignant cells from a variety of cellular origins ( alt et al . mdm2:mrn complex association was also independent of p53 status ( wild - type , mutant , or deleted ) . mdm2 co - localized with the mrn complex to sites of dna damage ( alt et al . , 2005 ) . we determined mdm2 bound directly to nbs1 of the mrn complex and localized the binding domains of each protein to ~30 amino acids ( alt et al . , 2005 ; bouska et al . , 2008 ) . therefore , there was a direct , specific association between mdm2 and nbs1 that occurred upon dna damage and was independent of p53 . evaluation of the effects of mdm2 binding to nbs1 was revealed in assays that measured dna damage signalling and dna breaks . increased levels of mdm2 led to reduced levels of dna damage signalling and significant delays in dna break repair ( alt et al . , 2005 ; bouska et al . , 2008 ) . specifically , the levels of marks of atm - mediated dna damage signalling , such as phosphorylated h2ax ( h2ax ) and phosphorylated serine / threonine glutamine ( s / tq ) sites , were reduced shortly after dna damage induced by gamma radiation in cells overexpressing mdm2 ( bouska et al . , 2008 ) . consequently , the repair of dna breaks , as measured by comet assay , was delayed with increased levels of mdm2 . neither the e3 ubiquitin ligase domain of mdm2 nor its e3 ubiquitin ligase activity was required for mdm2 to inhibit dna repair ( alt et al . , 2005 ) . however , point mutations in the nbs1-binding domain of mdm2 were sufficient to block the inhibition of dna break repair and the delay in dna damage signalling . additionally , dna break repair and resolution of dna damage foci were delayed with mdm2 overexpression , but this did not occur in cells containing nbs1 that could not bind to mdm2 ( bouska et al . elevated levels of mdm2 in cells lacking p53 resulted in chromatid and chromosome breaks , and this was prevented with mutations in the nbs1-binding domain of mdm2 . another consequence of delayed dna break repair was enhanced transformation . increased levels of mdm2 resulted in an increased transformation potential of p53-null fibroblasts , which required an intact nbs1-binding domain ( bouska et al . , 2008 ) . therefore , inhibition of dna break repair by mdm2 through its binding to nbs1 was identified as a specific and novel function of mdm2 that would contribute to genomic instability and tumorigenesis . mdmx shares homology with mdm2 and also regulates p53 ( shvarts et al . , 1996 ) . mdm2 and mdmx have overlapping functions in their regulation of p53 , but differences also exist ( marine et al . , 2007 ) . recently , we determined that similar to mdm2 , increased levels of mdmx inhibit dna break repair independent of p53 status . mdmx delayed the dna damage signalling response , resulting in dna breaks persisting . specifically , kinetic studies showed reduced levels of atm - mediated dna damage - induced phosphorylation events ( h2ax and phosphorylation of s / tq sites ) ( carrillo et al . , 2015a ) . notably , elevated levels of mdmx in mdm2/p53-null cells or mdmx lacking its ring domain in cells containing mdm2 resulted in a delay in dna break repair ( carrillo et al . these results indicate that the effects of mdmx on dna repair do not require mdm2 and are independent of its association with mdm2 . moreover , these data indicate that a conserved function of both mdm2 and mdmx is to inhibit dna break repair . mice lacking mdmx and p53 have an accelerated rate of tumorigenesis ( matijasevic et al . , 2008b ) , whereas deletion of mdm2 does not alter the rate of tumour development in a p53-null background ( jones et al . , 1996 ) . it was determined that mdmxp53 fibroblasts had multi - polar mitotic spindles and lost chromosomes , which was not evident in fibroblasts that lacked only p53 ( matijasevic et al . it will be important in the future to determine whether the genome instability caused by loss of mdmx is mediated by alterations in the regulation of the mrn complex or through a different mechanism . nevertheless , together , the data highlight a p53-independent role for mdmx in dna break repair and genome instability . evidence indicates that at least part of the explanation for this is through their interactions with the mrn complex , but many experiments never tested the effects of mdm2 or mdmx overexpression in the absence of p53 . mdm2 transgenic mice overexpressing mdm2 in breast epithelial cells show the development of polyploid breast epithelial cells irrespective of p53 status ( lundgren et al . , 1997 ) . increased levels of mdm2 lead to increased dna breaks , fusions , other structural chromosomal abnormalities , and aneuploidy in mdm2 transgenic mice with mdm2 driven by its native promoter ( ~34-fold increased expression ) and in fibroblasts with ectopic overexpression ( > 5-fold ) ( alt et al . , 2005 ; bouska et al . , 2008 ; wang et al . , 2008 ; lushnikova et al . , 2011 ) . the structural and numerical chromosome abnormalities increased with age in the mdm2 transgenic mice and appeared prior to the development of tumours ( lushnikova et al . , 2011 ) . in non - transformed cell lines , elevated mdm2 resulted in genome instability from centrosome amplification and aneuploidy ( carroll et al . , 1999 ) . decreased levels of mdm2 led to increased genome stability with mdm2 heterozygous fibroblasts containing fewer breaks and fusions than mdm2 wild - type fibroblasts ( wang et al . , 2006 ) . overexpression of mdm2 with point mutations in its nbs1-binding domain was not able to induce chromosome instability , indicating mdm2:nb1 interactions are required ( bouska et al . , 2008 ) . additionally , nutlin3a ( nutlin ) , a compound that blocks mdm2:p53 interactions also stabilizes mdm2 , leading to increased protein levels of mdm2 regardless of p53 status ( vassilev et al . , 2004 ; li et al . , 2012 ; carrillo et al . , 2015b ) . exposure of multiple cell types lacking p53 to nutlin results in increased dna breaks and activation of the dna damage response ( verma et al . nutlin delays dna break repair , which can be prevented with loss of mdm2 ( carrillo et al . , 2015b ) . therefore , mdm2 mediates nutlin - induced inhibition of dna break repair in cells lacking p53 . furthermore , elevated mdmx levels in fibroblasts result in increased genome instability with increased dna breaks , fusions , and aneuploidy ( carrillo et al . , 2015a ) . these genomic alterations also occurred in cells lacking p53 , indicating that the inhibition of p53 by mdm2 or mdmx was not responsible for these effects ( alt et al . therefore , mdm2 and mdmx contribute to genome instability when overexpressed , which is mediated , in part , through their interaction with nbs1 . the question arises as to whether the delay of dna break repair by mdm2 and mdmx is aberrant and just something that cancer cells select for , or whether this is a normal function of these proteins . experiments utilizing fibroblasts with mutant nbs1 have indicated that this is a normal function of mdm2 . specifically , primary murine fibroblasts lacking functional nbs1 were reconstituted with nbs1 containing point mutations in its mdm2-binding domain that inhibit endogenous mdm2 from binding , but not impact on its association with mre11 . in these cells , dna repair occurred at a faster rate than cells reconstituted with wild - type nbs1 ( bouska et al . , 2008 ) . these data indicate physiological levels of mdm2 that exist in non - transformed cells regulate mrn - mediated dna break repair . cancer cells that have increased levels of mdm2 and have inactivated p53 appear to have selected for this function of mdm2 . although similar studies are needed for mdmx , it is likely that analogous results will be obtained . why would normal cells have evolved this mechanism ? alterations in the rate or ability to repair dna breaks can cause dna aberrations and chromosome instability ( stracker et al . , 2013 ; mladenov et al . , 2016 ) . therefore , since mdm2 and mdmx are able to modulate the speed of dna repair , this may ensure that dna is repaired properly or alternatively , that it is not repaired in a timely manner , depending on its levels and likely other signals in the cell . cancer cells that have selected for a higher level of mdm2 have either adjusted to the delays in dna break repair or tolerated an increased amount of dna breaks , as these can lead to translocations and other chromosome abnormalities that may confer survival and/or growth advantages to the malignant cell . upon dna damage or a variety of cellular stresses , mdm2/mdmx : p53 interactions are inhibited and p53 is free to transcribe genes needed for cell cycle inhibition and/or apoptosis ( eischen and lozano , 2014 ) . therefore , if a cell has p53 , mdm2/mdmx regulate it , but mdm2/mdmx also simultaneously regulate dna break repair through the mrn complex ( figure 1 ) . the two mdm2/mdmx functions are not mutually exclusive of each other , as dna breaks lead to a dna damage signal that activates p53 . moreover , when mdm2/mdmx inhibit dna break repair , which leads to a delay in the resolution of the breaks , dna damage signals persist ( bouska et al . , 2008 ; therefore , in cells containing p53 , increased levels of mdm2/mdmx inhibit mrn - mediated dna break repair and suppress p53 activation , limiting the dna damage response , which can lead to dna breaks persisting . dna breaks can induce apoptosis , but they also facilitate chromosomal abnormalities , such as translocations , fusions , ringed chromosomes , and other structural changes ( morgan et al . , 1998 ; bunting and nussenzweig , 2013 ) . dna damage activates the mrn complex to induce dna break repair and p53-mediated transcription that facilitates dna repair through multiple mechanisms . compounds that inhibit mdm2/mdmx : p53 interactions and/or increase mdm2 levels , resulting in increased inhibition of the mrn complex , impact dna break repair and genome instability . dna damage activates the mrn complex to induce dna break repair and p53-mediated transcription that facilitates dna repair through multiple mechanisms . compounds that inhibit mdm2/mdmx : p53 interactions and/or increase mdm2 levels , resulting in increased inhibition of the mrn complex , impact dna break repair and genome instability . these compounds combined with dna damaging agents can lead to increased cancer cell apoptosis . currently , it is not entirely clear how a cell makes the decision to die instead of undergoing cell cycle arrest upon cellular stresses that result in damage ( carvajal and manfredi , 2013 ) . a large amount of cellular damage is more likely to lead to apoptosis , but how much damage is too much and what type of damage defaults to apoptosis remain unresolved . the possibility of mdm2/mdmx inhibiting dna break repair evolving to facilitate p53-induced apoptosis of normal cells that have sustained too much or unrepairable damage is intriguing . it is known that following p53 activation , a transcriptional program is induced , which includes mdm2 being transcriptionally upregulated by p53 , leading to increased levels of mdm2 protein ( barak et al . , 1993 ) . with elevated mdm2 levels , dna double - strand break repair that is occurring should be delayed ( alt et al . , 2005 ; bouska et al . , 2008 ; carrillo et al . , 2015a ) . with breaks persisting and not getting repaired in a timely manner , apoptosis could ensue . therefore , if p53 is attempting to induce apoptosis , an mdm2 and/or mdmx - mediated delay in dna break repair may allow breaks to be maintained to facilitate apoptosis . however , evaluation of apoptosis in cultured primary fibroblasts overexpressing mdm2 has not shown an overall increase in the levels of cell death in the population ( alt et al . , 2005 ) , but a small number of individual cells could be dying due to this mechanism . additional research will be needed to further investigate the possibility of mdm2/mdmx inhibiting dna break repair to facilitate apoptosis . the identification of mdm2 and mdmx overexpression leading to inhibition of dna break repair has opened up new therapeutic avenues for cancer therapy . recently , therapeutic approaches have tested inhibiting dna damage pathways / dna repair in combination with dna damaging agents to induce cancer cell death ( o'connor , 2015 ) . this approach was slow in coming , possibly because there was fear that making a cancer cell more genomically unstable would result in a more difficult to treat cancer . additionally , it is possible that knowledge gained from tumours , which contained wild - type p53 but went on to lose functional p53 , resulting in the tumour becoming more genomically unstable , aggressive , and resistant to specific therapies , also led some to reject the idea of making genomes more unstable . however , data are emerging that such an approach may be beneficial and could include mdm2 modulation . approaches to combine mdm2 modulation with dna damaging agents in malignant cells that lack functional p53 have begun to be investigated . specifically , prostate cancer cells with mutant or deleted p53 showed increased sensitivity to radiation in the presence of nutlin ( supiot et al . , 2008 ) . in cutaneous t cell lymphoma and sarcoma that did or did not have functional p53 , there was increased cell death with combined treatment with nutlin and doxorubicin or cisplatin ( ohnstad et al . 2012 ) . increased dna replication stress induced by gemcitabine , together with the mdm2 inhibitor mi-63 , led to increased lymphoma cell death ( jones et al . , 2011 ) . utilizing p53 deleted or mutated ovarian cancer cells , we showed that addition of nutlin led to increased levels of mdm2 protein and a delay in dna break repair . a combination treatment with the dna damaging agent cisplatin or etoposide together with nutlin significantly increased ovarian cancer cell apoptosis . notably , doses of cisplatin or etoposide that had little effect alone were able to kill ovarian cancer cells in the presence of nutlin ( carrillo et al . , 2015b ) . similarly , treatment of breast cancer cells that harbour mutant p53 with nutlin and carboplatin synergistically induced cell death . in addition , combined treatment of mice with nutlin and carboplatin reduced breast cancer growth and metastasis in xenograft studies ( tonsing - carter et al . , 2015 ) . together , these studies are proof of principle that combining dna damage with inhibition of dna break repair through mdm2 stabilization should have therapeutic potential in treating human cancers . future studies should focus on further characterizing the effect of increased mdm2 or mdmx levels on dna damage for therapeutic purposes .
abstractmdm2 and mdmx are critical regulators of the p53 tumour suppressor and are overexpressed in many human malignancies . 
 however , in recent years , their impact on genome instability was shown to be at least , in part , independent of p53 . 
 both mdm2 and mdmx inhibit dna break repair through their association with the mre11/rad50/nbs1 dna repair complex . 
 recent evidence indicates that harnessing mdm2 and/or mdmx - mediated inhibition of dna break repair in cancer cells could provide a therapeutic opportunity , particularly for those malignancies that have lost functional p53 .