Mechanisms of action of PPARy ligands and cellular role of PPARy in childhood neuroblastoma

Abstract

The level of PPARγ activation in neuroblastoma cells correlated with their biological response to the natural PPARγ ligand 15-deoxyΔ^12,14 prostaglandin J₂ (15dPGJ₂).  Higher levels of PPRE-mediated transcription were associated with more pronounced growth inhibition of neuroblastoma cells by 15dPGJ₂. The transcriptional activity of PPARγ in neuroblastoma cells may be regulated by members of the retinoblastoma family, since their ectopic expression in ND-7 neuroblastoma cells in vitro repressed induction of PRE-mediated transcription by 15dPGJ₂ by a mechanism that was dependent on histone deacetylase recruitment. Furthermore, the growth inhibitory effect of 15d PGJ₂ on neuroblastoma cells was enhanced by co-treatment with the HDAC inhibitor Trichostatin A, which suggests that using PPARγ ligands in combination with agents that enhance their ability to stimulate PPARγ transactivation might improve their efficacy in neuroblastoma treatment. In IMR-32 neuroblastoma cells, 15dPGJ₂ was dependent on its receptor to mediate its anti-proliferative effects, since stable expression of a PPARγ dominant negative receptor blocked  growth inhibition by 15dPGJ₂. Conversely, growth arrest induced by another PPARγ ligand ciglitazone was not affected by expression of the receptor mutant, suggesting that PPARγ ligands can also regulate neuroblastoma growth by PPARγ-independent pathways.  In primary neuroblastoma cells PPARγ protein levels correlate with the maturational status of the neuroblast, with high expression of PPARγ observed in cells with a more differentiated phenotype although the function of the receptor remains elusive.  To address the cellular role of PPARγ in neuroblastoma, the factors which control its expression were examined.  A transiently transfected PPARγ1 promoter reporter was differentially activated  in neuroblastoma cell lines with the level of induction mirroring endogenous expression of PPARγ mRNA in these cell types.  Analysis of the PPARγ1 promoter sequence revealed a number of putative binding sites that could mediate regulation by Myc family transcription factors. Co-transfection of the PPARγ1 promoter reporter with c-Myc or N-Myc expression plasmids attenuated its activity in vitro.  c-Myc inhibition of the PPARγ1 promoter in neuroblastoma cells occurred by a Miz-1 and HDAC independent mechanism.  The site of c-Myc repression on the PPARγ1 promoter was mapped to a region close to the start site of transcription.  This region also mediated transactivation by the Sp1 transcription factor. The finding that PARγ1 promoter activity was negatively influenced by Myc oncogenes involved in the pathogenesis of many human cancers lends support to the hypothesis that PPARγ functions as a tumour suppressor.

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