We and others have shown that the expression of tumor-derived mutant p53 in cells leads to the up-regulation of expression of a set of genes, some of which are involved in oncogenesis; this is accompanied with GOF phenotypes. However, the mechanism of up-regulation of gene expression and the relationship to the GOF phenotype are only starting to be defined. Earlier, we had suggested that the GOF activities observed in H1299 cells after the expression of mutant p53 can be explained in part by mutant p53’s ability to enhance the expression of NF-κB2.9
Axl has been identified as another player, which has previously been implicated in oncogenesis35
and is significantly up-regulated by tumor-derived p53 mutants ( and ) in lung cancer cell lines and human lung tumors. One interesting and important aspect of Axl being up-regulated by p53 mutants is the fact that p53 mutants do not apparently require an intact transactivation domain for this up-regulation (). Thus, this mutant p53 target seems to be a good candidate for a gene that has been up-regulated by mutant p53 perhaps through interaction with p63/p73.52
We note that there is a second transactivation subdomain that may contribute to transactivaton, or alternatively, mutant p53 may be utilizing another transcriptional activator subdomain as a surrogate for transactivation. Using human lung cancer cell lines and lung tumor samples expressing mutant p53, we demonstrate that the expression of GOF mutant p53 induces Axl expression (). We found that knocking down Axl levels by RNAi in H1048 cells led to a decrease in cell motility and growth rate () while mutant p53 levels remained constant. This suggests that GOF mutant p53 may induce part of its GOF activity via the induction of Axl.
We have examined the promoter sequences of Axl and identified putative transcription factor binding sites (, top) and have undertaken an in vivo transient transcriptional analysis to determine the mechanism of activation of the promoter by GOF mutant p53. Analysis showed that mutant p53 indeed transactivated the Axl promoter in H1299 cells (). Interestingly, our promoter deletion analysis indicated that the p53/p63 binding site present is not needed for in vivo transactivation by mutant p53 in H1299 cells (). This shows that GOF p53 mutants transactivate the Axl promoter without the necessity of p63 binding to the promoter, suggesting that mutant p53 is not activating the Axl promoter by p63 interaction.
ChIP assay analysis demonstrated that GOF mutant p53 induces histone acetylation at the Axl promoter (), suggesting that mutant p53 causes chromatin modifications on the promoter, indicative of increased transcriptional activity. Transcription factor ChIP analyses indicated that mutant p53 induced interactions between p300, CREB, and E2F1 and the Axl promoter (), suggesting their involvement, in general, in the transactivation of Axl by mutant p53. The observed increased histone acetylation may lead to increased interactions of the transcription factors p300, CREB, and E2F1 with their consensus binding sites on the Axl promoter, which may lead to increased Axl promoter activity. Since CBP/p300 has histone acetylase activity associated with it, these data suggest that mutant p53 may be enhancing histone acetylation through the use of CBP/p300. Interestingly, interactions of CBP/p300 and E2F1 and CREB53
may further nucleate p300 on the Axl promoter and lead to further acetylation of histones, increasing its promoter activity.
Figure 8. ChIP assay shows the mutant p53–mediated increase in transcription factor binding on the Axl promoter. ChIP analysis was performed on H1299 cells stably transfected with a vector (HC5) or mutant p53-R273H (R273H) as described for to test (more ...)
ChIP assays using p53 antibodies demonstrate unequivocally that mutant p53 in H1299 cells expressing the p53-R273H protein is nucleated on the Axl regulatory sequences (). We have demonstrated that there is more mutant p53–mediated activation of the Axl promoter further away from the transcription start site, indicating that mutant p53 may bind to an enhancer-like region to regulate transcription. Our transient transcriptional analysis ( and ) demonstrates that Axl promoter activation by mutant p53 does not require the p53/p63 binding site. When we examined the in vivo interaction of p63 with the Axl promoter by ChIP assays, we could detect a marginal increase in binding over the background in the chromatin setting in the presence of mutant p53 (data not shown). This may suggest that mutant p53 helps to open up the Axl promoter region, enhancing the interaction of transcription factors with the promoter. Thus, one has to speculate the involvement of other transcription factors in this transactivation unless it is assumed (an unlikely event) that mutant p53 itself interacts with the DNA. Further in vivo and in vitro work is needed to clarify the mechanism of transactivation by mutant p53.
At present, we envision 3 ways that mutant p53 may induce the up-regulation of target gene expression (). 1) Mutant p53 may induce histone acetylase(s) and/or inhibit histone deacetylase(s) and thereby cause the acetylation of histones () and chromatin reorganization on the promoters of mutant p53 target genes; this would lead to activation of the promoter and transactivation. 2) Mutant p53 may induce interactions of one or more transcription factor(s) with the promoter, leading to transactivation of the target gene (). 3) Mutant p53 itself is nucleated on the target promoter directly or indirectly () through another transcription factor and then modulates the recruitment of other factors on the promoter with eventual promoter activity enhancement. A combination of these possible mechanisms may also be at work.
Figure 9. Model of up-regulation of gene expression by mutant p53. Three different molecular mechanisms of how mutant p53 may activate transcription from its target promoter are depicted. (A) Mutant p53 may induce histone acetylase(s) and/or inhibit histone deacetylase(s) (more ...)