Hidehiko Kawai

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. Typically, HNSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. However, molecular mechanisms associated with the invasion and metastasis of HNSCC remain poorly understood. Here, we identified periostin as an invasion-promoting factor in HNSCC by comparing the gene expression profiles between parent HNSCC cells and a highly invasive clone. Indeed, periostin overexpression promoted invasion and anchorage-independent growth both in vitro and in vivo in HNSCC cells. Moreover, periostin-overexpressing cells spontaneously metastasized to cervical lymph nodes and to the lung through their aggressive invasiveness in an orthotopic mouse model of HNSCC. Interestingly, periostin was highly expressed in HNSCCs in comparison with normal tissues, and the level of periostin expression was well correlated with the invasiveness of HNSCC cases. In summary, these findings suggest that periostin plays an important role in the invasion and anchorage-independent growth of HNSCC.

There are currently two distinct models proposed to explain why both MDM2 and MDMX are required in p53 control, with a key difference centered on whether these two p53 inhibitors work together or independently. To test these two competing models, we generated knockin mice expressing a point mutation MDMX mutant (C462A) that is defective in MDM2 binding. This approach allowed a targeted disassociation of the MDM2/MDMX heterocomplex without affecting the ability of MDMX to bind to p53, and while leaving the MDM2 protein itself completely untouched. Significantly, Mdmx(C462A/C462A) homozygous mice died at approximately day 9.5 of embryonic development, as the result of a combination of apoptosis and decreased cell proliferation, as shown by TUNEL and BrdU incorporation assays, respectively. Interestingly, even though the MDMX mutant protein abundance was found slightly elevated in the Mdmx(C462A/C462A) homozygous embryos, both the abundance and activity of p53 were markedly increased. A p53-dependent death was demonstrated by the finding that concomitant deletion of p53 completely rescued the embryonic lethality in Mdmx(C462A/C462A) homozygous mice. Our data demonstrate that MDM2 and MDMX function as an integral complex in p53 control, providing insights into the nonredundant nature of the function of MDM2 and MDMX.

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.