Sunday, July 29, 2012

MDM4 and Melanoma: More Pathways

Recent efforts in managing melanoma have focused upon BRAF and a mutation of a V600 BRAF form. By targeting this mutation and the pathway related thereto one can find ways to block the mutated pathway and this in principle block the continuing metastasis. This works for a while and then the cells find ways around this. There are undoubtedly many other changes in cellular pathways that result in uncontrolled proliferation and failure of apoptosis. Namely the cells continue to grow and fail to die off.

Focus on other pathway defects is continuing and there has been recent focus on MDM4, which is a control element of p53, the product of TP53 which is a key control element of proliferation and apoptosis. In a recent paper by Gembarska et al the authors state the following[1]:

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma—a highly chemotherapy-resistant disease—TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (~65%) of stage I–IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras.

MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

Now MDM4, also called Mdm4 p53 binding protein homolog, is located at 1q32. It acts in a somewhat complex manner to control p53 functions. From NCI we have the following description of the gene and its product[2]:

This gene encodes a nuclear protein that contains a p53 binding domain at the N-terminus and a RING finger domain at the C-terminus, and shows structural similarity to p53-binding protein MDM2. Both proteins bind the p53 tumor suppressor protein and inhibit its activity, and have been shown to be overexpressed in a variety of human cancers. However, unlike MDM2 which degrades p53, this protein inhibits p53 by binding its transcriptional activation domain. This protein also interacts with MDM2 protein via the RING finger domain, and inhibits the latter's degradation. So this protein can reverse MDM2-targeted degradation of p53, while maintaining suppression of p53 transactivation and apoptotic functions.

The sources for information on p53 pathway and its relation to MDM4 are extensive[3]. Specific details of the p53 pathway are shown in the NCI data[4] bases for pathways. However, we shall present a simplified description based upon KEEG pathway data. This we do below (We combine from the KEGG genome database[5]).

Note in the above we have a complex control path between MDM2 and MDM4 with p53. The p53 is activated when DNA damage is perceived or from other factors. p53 then activates a collection of pathways which in turn block the cell cycle or initiate apoptosis. If p53 does not function then we have an uncontrolled cell. The control of p53 can be blocked by MDM4 blockage as shown above. That is the principle which the authors have presented.

Prior work by Macchiarulo et al stated the following regarding this combination[6]:

Alterations of p53 signalling pathway is the most frequent event in human cancers. About 50% of these, albeit showing wild-type p53, have flaws in the control mechanisms of p53 levels and activity. MDM2 and MDMX (MDM4) are the main negative regulators of p53.

The relevance of MDM2 on the regulation of p53 levels and activity has fostered the development of strategies aimed at restoring p53 functions by blocking the physical interaction between MDM2 and p53. As a consequence, a number of different small molecules and peptidomimetics have been disclosed in the last decade as inhibitors of MDM2/p53 interaction.

Recent studies, however, have thrust MDMX into the limelight as an additional chemotherapeutic target, suggesting the presence of a more complex relationship between MDM2, MDMX and p53. In this review article, we report key aspects of MDMX-mediated regulation of p53, recent advances in the structural characterization of the protein, and the progress made so far in the medicinal chemistry of MDMX ligands.

Note that MDMX is now called MDM4, to avoid confusion. The Macchiarulo paper was published a year ago (2011) and it presented the connection of MDM4 and loss of p53 control in a broader context of cancer development and spread. The Gembarska paper on the other hand has focused on melanoma. Earlier work was performed in a Doctoral Thesis in 2007 in Rotterdam, by Meulmeester who states[7]:

The p53 tumor suppressor gene encodes a sequence-specific transcription factor whose activity is either disabled or attenuated in the vast majority of human cancers. Its inactivation occurs in about 50% of human tumors through mutations affecting the p53 locus directly.

p53 transcriptionally activates a vast, constantly growing number of target genes, resulting in various biological outcomes such as cell-cycle arrest and apoptosis. Several types of stress, such as oncogene activation, hypoxia and DNA damage, result in an increase in p53 levels and the subsequent activation of p53 target genes (Vogelstein et al., 2000). One of the best-characterized target genes of p53 is the mdm2 gene, which contains two promoters.

The first promoter (P1) drives mdm2 expression constitutively (Jones et al., 1996), while p53 binds two adjacent p53-responsive elements within the second promoter (P2), thereby promoting transcription of the mdm2 gene. Under normal circumstances, p53 is tightly regulated through the interaction with its negative regulator Mdm2, which counteracts p53 function in a number of ways.

 The autoregulatory negative feedback loop, whereby p53 induces Mdm2 expression resulting in the repression of p53 function, most probably serves as an important mechanism to restrain p53 activity in normal cells. Therefore, uncontrolled, high expression of Mdm2 may result in improper inactivation of p53 function. It has been shown that in 5-10% of all human tumors Mdm2 is overexpressed, due to gene amplification, transcriptional- or posttranscriptional mechanisms. In most of these cases the p53 gene is wild type, presumably because Mdm2 overexpression alleviates the selective pressure for direct mutational inactivation of the p53 gene.

As regards to the pathway discussion we presented above Meulmeester remarks:

The complex web of ATM-mediated activation of the p53 pathway. ATM mediates direct and indirect phosphorylation of p53, while 14-3-3 binding to p53 is augmented by ATM-mediated de-phosphorylation of p53. Phosphorylation of Strap by ATM results in the recruitment of Strap/p300 complexes towards p53 that elevates its acetylation.

A safeguard mechanism exists to ensure proper p53 activation by inhibiting its inhibitors Mdm2 and Mdmx. Phosphorylation of Mdmx/Mdm2 attenuates their interaction with the ubiquitin protease HAUSP, resulting in the instability of Mdmx and Mdm2. Thus ATM activates p53 via a sophisticated mechanism, while it ensures proper activation by inhibition of its negative regulators .

Again note that MDMX is now MDM4. He does raise the issue of a complex feedback loop which may have some internal instabilities. Namely the loop between MDM2, MDM4, and p53 may have unstable points under certain conditions. Some have approached this via rate reaction equations but as we have discussed elsewhere the rate reaction equations require large concentrations. In a cell we have a few protein molecules with limited binding sites. This specific low density case has particular concerns of instabilities. Thus there may not just be a mutation of MDM4 but also some instabilities in the internal dynamics.

In a paper by Mancini et al (2009), the authors state[8]:

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function.

Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46P) and (iii) promotes binding between p53Ser46P and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription.

Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46P to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Thus they make the connection of control of MDM4 products as key to the ultimate functionality of p53. They also have examined the effects of cisplatin in the case of ovarian cancers. They did not examine ways to block MDM4 and its protein. The principle here is that the pathway control element such as p53 can be dysregulated by another gene MDM4. The question of course is what has happened to MDM4. We address that later.

In a discussion of the paper by Gembarska et al, Azvolinsky then states[9]:

While the TP53 gene, which encodes the tumor protein 53, is found mutated in the vast majority of tumors, TP53 is intact in more than 95% of melanomas. The p53 tumor suppressor pathway is important for most cancers, preventing neoplastic growth, and inactivation of the pathway is a common driver mutation for cancer.

Exploring alternative mechanisms of dysregulation of the p53 tumor suppressor pathway, Jean-Christophe Marine, the Center for Human Genetics in Leuven, Belgium, and colleagues show that the pathway is indeed altered in as many as 65% of human melanomas. Rather than a mutation in TP53, the researchers find that melanomas have upregulated Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53. According to the authors, the results identify “MDM4 as a key determinant of impaired p53 function in human melanoma.”

They continue:

“Melanomas do not harbor MDM4 mutations per se,” explained Jean-Christophe Marine. “They select for mechanisms that cause MDM4 protein levels to go up.” Marine and colleagues are currently investigating the mechanisms of MDM4 upregulation.

Approximately 50% of melanoma cases harbor a mutation in the BRAF gene, part of the mitogen-activating protein kinase (MAPK) pathway that results in constitutive activation of the MAPK pathway. A specific inhibitor of BRAF, vemurafenib, was approved last year for metastatic melanoma patients with the BRAF mutation and two other targeted inhibitors of the MAPK pathway, dabrafenib and trametinib, have recently completed phase III trials. While treatment with a BRAF inhibitor results in rapid tumor shrinkage and symptom relief, resistance is still a major issue and new agents and combinations are needed for sustaining prolonged responses.

“The whole field is looking for drugs that can prevent relapse,” said Marine. “Awakening the p53 pathway in melanoma, by targeting MDM4, could be one way to achieve this—a possibility that has so far been completely overlooked.” The p53 pathway has been majorly overlooked in melanoma because the mechanism by which p53 is inactivated in this tumor were unknown until this study.

The issue is both what happened to MDM4 and is MDM4 just another patch for some melanomas. Let us discuss this issue at some length.


p53 is a powerful gene which regulates the cell from the cell cycle point of view through apoptosis. It is an essential gene and has been found as a mutated version in many cancers. However in melanoma it appears that loss of p53 expression resulting from a mutation is not observed frequently. Thus, although p53 does not appear to do what it should do, p53 looks just fine when examined by its lonesome. Thus the authors had identified the controller of p53, namely MDM4 as a changed element and as a cause of the potential loss of cell cycle control and of apoptosis.

1. What causes the mutation of MDM4? What is it mutated to?

2. What melanoma cells contain the mutated or dysfunctional MDM4 gene?

3. Could this be a more complex issue when one looks at the complex set of pathways?

4. Are we trying to target aberrant MDM4 products and thus eliminate blockage on p53 functions. What of the instabilities in the control loop we have already discussed. Will this function in low density environments. Also what are we targeting, sites on MDM4 products for blockage.

5. The stem cell issue always raises its head. Frankly this may very well be a stem cell only problem as compared to a BRAF issue. If the stem cell has unstable MDM4 characteristics than we would anticipate longer term survival, perhaps.