Abstract 3701: Transcriptomic consequences of MDMX (MDM4) oncoprotein expression knockdown in MDMX-amplified breast cancer cells

Abstract

Abstract The tumour suppressor p53 is activated by cellular stress to induce cell cycle arrest and/or apoptosis. Although the TP53 gene is frequently mutated in cancer, approximately half of human cancers express wild type and functional p53. However, p53 activity is often suppressed by its negative regulators, MDM2 and MDMX. Small molecule antagonists have been developed to inhibit p53-MDM2 binding to release the growth inhibitory and/or pro-apoptotic functions of p53. Previous studies have indicated that MDMX amplification and/or increased protein expression may be associated with resistance to MDM2-p53 binding antagonists. However, specific inhibitors for MDMX have yet to be developed and an MDM2/MDMX-p53 co-inhibitor (RO-5963) does not show better efficacy against high MDMX expressing cells than single target MDM2 inhibitors. To better understand the cellular function and therapeutic potential of targeting MDMX, we have investigated the downstream transcriptomic consequences of knocking down MDMX expression. Knockdown of MDMX protein expression, demonstrated by Western blot analysis, was achieved in MRK-nu-1 MDMX amplified and TP53 wild-type breast carcinoma cells using both lentiviral shRNA and siRNA. MRK-nu-1 cell growth suppression was observed after knockdown of MDMX. Affymetrix Human Transcriptome Array 2.0 was used to detect differences in the expression of full genes and alternatively spliced forms between negative control and MDMX knockdown groups. Transcriptome Analysis Console 3.0 software was used to analyse the microarray data to identify significant changes in gene expression, alternative splicing and associated functional pathways. Alterations in mRNA expression for a selected panel of genes was confirmed by qRT-PCR. A greater degree of MDMX expression knockdown was achieved with the siRNA (>95%) than the lentiviral shRNA (>50%) This was reflected by increased suppression of growth and larger changes in gene expression patterns with the siRNA knockdown. Although a similar set of genes was affected by both the siRNA and shRNA knockdown, the magnitude of changes was greater following the more pronounced knockdown with siRNA. The expression of a number of p53 transcriptional target genes was found to be altered, including CDKN1A, MDM2, CCNG2, RRM2B, BTG2, ZMAT3 and FAS consistent with a role for MDMX in suppression of p53 function in these MDMX amplified cells. Citation Format: Yi-hsuan Ho, Claire Hutton, Herbie Newell, John Lunec. Transcriptomic consequences of MDMX (MDM4) oncoprotein expression knockdown in MDMX-amplified breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3701.