GSK-3β phosphorylates numerous substrates that govern cell survival. It
phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent
ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit
programmed cell death, depending on the nature of the pro-apoptotic stimulus. We
previously showed that the multikinase inhibitor sorafenib activated GSK-3β and
that this activation attenuated the cytotoxic effects of the drug in various
BRAF-mutant melanoma cell lines. In this report, we describe the results of
studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity
of sorafenib combined with the HDM2 antagonist MI-319. MI-319 alone increased p53 levels and p53-dependent gene expression in
melanoma cells but did not induce programmed cell death. Its cytotoxicity,
however, was augmented in some melanoma cell lines by the addition of sorafenib.
In responsive cell lines, the MI-319/sorafenib combination induced the
disappearance of p53 from the nucleus, the down modulation of Bcl-2 and
Bcl-xL, the translocation of p53 to the mitochondria and
that of AIF to the nuclei. These events were all GSK-3β-dependent in that they
were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive
melanoma cell lines by the introduction of a constitutively active form of the
kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the
sorafenib/MI-319 combination were the exact reverse of its effects on the
activities of sorafenib alone, which induced the down modulation of Bcl-2 and
Bcl-xL and the nuclear translocation of AIF only in cells
in which GSK-3β activity was either down modulated or constitutively low. In A375
xenografts, the antitumor effects of sorafenib and MI-319 were additive and
associated with the down modulation of Bcl-2 and Bcl-xL,
the nuclear translocation of AIF, and increased suppression of tumor
angiogenesis. Our data demonstrate a complex partnership between GSK-3β and HDM2 in the
regulation of p53 function in the nucleus and mitochondria. The data suggest that
the ability of sorafenib to activate GSK-3β and alter the intracellular
distribution of p53 may be exploitable as an adjunct to agents that prevent the
HDM2-dependent degradation of p53 in the treatment of melanoma.