A pathological high expression of Hdmx is found in a subset of human cancers, and generally correlated with the presence of wild-type p53 protein [34
]. Constitutive Hdmx overexpression contributes to the oncogenic transformation of cultured cells, thereby functionally resembling loss of p53 [34
]. These findings emphasize that high Hdmx expression in cancer appears mainly to serve to block p53 activity. Indeed, in this study, we show high Hdmx expression in a subset of cell lines and fresh-frozen tumor samples from uveal melanoma, which rarely contain p53 mutations. Increased levels of Hdm2 were also observed in some cell lines and tumor samples, although the extent of over-expression was not impressive when compared to normal uveal melanocytes. Interestingly, our experiments in uveal melanoma cell lines also suggest the existence of an additional growth promoting function of Hdmx. Reducing Hdmx levels via shRNA knockdown constructs reduced proliferation and survival of 92.1 and Mel202 cells, whereas the growth of Mel285 cells remained largely unaffected. This indicates that the proliferation in a subset of uveal melanomas depends on the high Hdmx expression. The resistance of Mel285 cells reduces the likelihood of non-specific effects caused by the Hdmx knockdown constructs in 92.1 and Mel202 cells. Unexpectedly, knocking down Hdmx still reduced growth of the 92.1 and Mel202 cells in which p53 levels were strongly reduced by shRNA. The lack of growth inhibition by Nutlin-3 in these shp53-cells confirmed the efficiency of the p53 knockdown, indicating that Hdmx promotes uveal melanoma growth through p53-dependent and -independent pathways.
Similarly, two recently described Hdmx inhibitors, although certainly less specific than knocking down Hdmx, also inhibited the growth of these uveal melanoma cells in a partly p53-independent manner. These results strongly suggest that high Hdmx levels stimulate cell proliferation and survival in a manner stretching beyond p53 inhibition.
At first sight, this finding is a little surprising, particularly with regard to the complete rescue of the embryonic lethality of Mdmx deletion by loss of p53 [57
], which would argue against the importance of p53-independent effects of Hdmx. On the other hand, the physiological role of basal Hdmx levels during development may not be identical to the pathological effects of Hdmx overexpression during tumorigenesis. In addition, it is becoming increasingly clear that Hdm2 activity, and especially pathologically high levels of Hdm2, are not restricted to p53 regulation. Because of the homology between Hdm2 and Hdmx, our search for the mechanisms underlying p53-independent activities of Hdmx was primarily based on known functions of Hdm2. Enhanced Hdm2 activity has been reported to inhibit Rb function [49
], which prompted us to investigate whether activation of Rb would be involved in the inhibition of S-phase entry upon Hdmx knockdown. However, Rb knockdown did not rescue the decrease in S-phase cells upon Hdmx knockdown, suggesting that Hdmx does not function via Rb inactivation. Interestingly, our results point to p27 protein induction as one of the factors contributing to the growth-inhibiting effects of Hdmx knockdown. Although p27 may exert some functions that are potentially oncogenic, it is generally considered to be a tumor suppressor [59
]. The main role of p27 is to regulate the G0/G1 to S transition by binding and inhibiting cyclin E/CDK2 and cyclin D/CDK4,6 complexes, and thereby reducing phosphorylation of pocket proteins. P27 itself is highly regulated at multiple levels, including transcription, translation, phosphorylation and ubiquitination [59
]. P27 protein levels are maximal during G0 and early G1, mainly due to differences in cap-independent translation [61
] and ubiquitin-dependent proteolysis [62
] in different stages of the cell cycle. However, the induction of p27 protein levels in response to Hdmx knockdown probably occurred too quickly to be a secondary event of the G1 arrest. Moreover, p27 knockdown partially prevented the G1 arrest in response to Hdmx knockdown. This indicates that Hdmx somehow limits p27 levels via an unknown mechanism and thereby stimulates cell cycle progression.
A few reports suggest that the inhibitor of apoptosis (IAP) family member Survivin is overexpressed in uveal melanoma [63
]. We found that, although endogenous levels of Survivin are slightly reduced upon Hdmx knockdown, constitutive Survivin expression did not affect the outcome of Hdmx knockdown experiments (not shown).
In conclusion, Hdmx over-expression is present in a subset of uveal melanomas, most likely to promote tumorigenesis by inhibiting p53, which is rarely mutated in this type of tumors. Interestingly, however, we show that Hdmx also has an important p53-independent role in promoting cell proliferation and survival. It will be important to analyze the relevance of this role of Hdmx in other cell types as well. Our attempts to uncover the molecular basis of a p53-independent function of Hdmx reveal a contribution for p27 in the induction of G1 arrest. Future studies are required to provide more insight into the mechanism by which Hdmx affects p27 protein levels. However, our data strongly suggest the involvement of additional, yet unknown factors, although unraveling these factors thus far proved difficult. In this respect, it may be worthwhile to investigate the involvement of proteins reported to interact with Hdm2, but not tested in this study, since they might interact with Hdmx as well. Alternatively, detailed analysis of Hdmx- containing protein complexes in uveal melanoma by mass spectrometry and subsequent functional characterization of newly found interactors might open new avenues to clarify p53-independent activities of Hdmx. Together, this will improve our understanding of Hdmx over-expressing tumors and ultimately may lead to the development of new therapeutic strategies to target such tumors.