Abstract
Isoprenyl cysteine carboxyl methyltransferase (ICMT) plays a key role in post-translational regulation of prenylated proteins. On the basis of previous results, we hypothesized that the p53 pathway and ICMT expression may be linked in cancer cells. Here, we studied whether WT p53 and cancer-associated p53 point mutants regulate ICMT levels and whether ICMT overexpression affects tumor progression. Studying the effect of p53 variants on ICMT mRNA and protein levels in cancer cells, we found that WT p53 and p53 mutants differentially affect ICMT expression, indicating that p53 status influences ICMT levels in tumors. To investigate the underlying mechanisms, we constructed ICMT-luciferase reporters and found that WT p53 represses ICMT transcription. In contrast, p53 mutants showed a positive effect on ICMT expression. Promoter truncation analyses pinpointed the repressive effect of WT p53 to the -209 and -14 region on the ICMT promoter, and ChIP assays indicated that WT p53 is recruited to this region. Instead, a different promoter region was identified as responsible for the mutant p53 effect. Studying the effect of ICMT overexpression on tumor-associated phenotypes in vitro and in vivo, and analyzing breast and lung cancer databases, we identified a correlation between p53 status and ICMT expression in breast and lung cancers. Moreover, we observed that ICMT overexpression is correlated with negative clinical outcomes. Our work unveils a link between postprenylation protein processing and the p53 pathway, indicating that the functional interplay between WT and mutant p53 alters ICMT levels, thereby affecting tumor aggressiveness.
Highlights
Isoprenyl cysteine carboxyl methyltransferase (ICMT) plays a key role in post-translational regulation of prenylated proteins
Previous results from microarray analysis on MDA-MB-231 breast cancer cells [25], which endogenously express p53R280K and lack a functional WT the p53 gene (TP53) allele, indicated to us that ICMT expression was reduced upon mutant p53 knockdown, suggesting a positive regulation
We found that knockdown of endogenous mutant p53 reduced ICMT mRNA levels (Fig. 1a)
Summary
The presence of p53 point mutants was proposed to affect gene expression in tumor cells through different mechanisms. Previous results from microarray analysis on MDA-MB-231 breast cancer cells [25], which endogenously express p53R280K and lack a functional WT TP53 allele, indicated to us that ICMT expression was reduced upon mutant p53 knockdown, suggesting a positive regulation To test this hypothesis, we first confirmed the effect of endogenous p53R280K knockdown on ICMT mRNA steady-state levels in independent experiments. A similar effect on ICMT mRNA and protein levels was observed when we knocked down endogenous p53R273H in breast adenocarcinoma MDA-MB468 and colorectal adenocarcinoma HT29 cells (Fig. 1, b and c) These results are in agreement with the hypothesis that mutant p53 promotes ICMT expression. By performing ChIP assays in H1299 cells transfected with p53R280K, we were able to amplify a fragment between positions Ϫ2178 and Ϫ2071 on the ICMT promoter (ICMT up) upon immunoprecipitation with an anti-p53 antibody (Fig. 4c). Our results show that p53R280K is recruited on the ICMT promoter on a different region than WT p53, indicating that each protein regulates promoter activity by different mechanisms
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