Abstract 212: Reexpression of hSNF5 regulates the transcriptional activity of the NOXA promoter through p53 independent mechanisms in malignant rhabdoid tumors
Authors: Kuwahara Yasumichi , Durand Joel , Weissman Bernard E. 
Published: July 2012

Malignant rhabdoid tumor (MRT), a highly aggressive cancer of young children, displays inactivation of the hSNF5 gene in primary tumors and cell lines. We have previously reported that hSNF5 can modify p53 target genes such as p21, PUMA and NOXA. However their expression varies among MRT cell lines. It suggested that hSNF5 generally operates through a p53-independent mechanism to regulate these genes.

NOXA is one of p53 target genes associated with apoptosis pathway, and many transcriptional regulators have been described. However the relationship between NOXA and SWI/SNF complex has not been reported. Because reexpressed hSNF5 induced NOXA mRNA in 2 MRT cell lines, in this study, we addressed the mechanism of NOXA promoter activation by hSNF5.

First, we assessed NOXA mRNA induction in the five MRT cell lines (TTC642, TTC549, TM87-16, TTC709 and G401) after reexpression of hSNF5 using adenoviral vectors expressing either hSNF5 and GFP (Ad-hSNF5) or GFP (Ad-GFP). The levels of NOXA mRNA in MRT cell lines are lower than MCF7 (NOXA-positive control), especially, G401 and TTC709 that express no NOXA mRNA. The levels of NOXA mRNA increased significantly at 24 hours, followed by a marked increase at 48 hours after infection in these all MRT cell lines. These findings suggested that hSNF5 induced NOXA transcriptional activity. In MRT cells, loss of hSNF5 may cause the repression of NOXA expression.

Next, we used chromatin immunoprecipitation (ChIP) analysis of the NOXA promoter to determine where hSNF5 appeared at the NOXA promoter and whether its recruitment altered binding of other transcription factors or the chromatin landscape in TTC642 cell line and TTC549 cell line. Our results indicated that reexpressed hSNF5 binds within 1 kb of transcript start site (TSS), with maximal enrichment at the TSS in both cell lines. RNA polymerase II (RNAPII) assembly, along with H3K4 tri-methylation, likely resembles the hSNF5 binding pattern in both cell lines. However, BRG-1 binding increased throughout the NOXA locus with no apparent peak. Furthermore, we did detect p53 at the promoter even after hSNF5 reexpression in either cell line. Therefore, hSNF5 may initiate increased expression of the NOXA promoter by itself. While loss of p53 expression by p53 knock-down significantly inhibited NOXA transcriptional activity induced by hSNF5 in TTC549 cells at 24 hours, it did not alter its activity by hSNF5 after 24 hours in the TTC642 cells. These findings suggested that NOXA transcriptional activity induced by hSNF5 is not directly correlated with p53 activity.

Our results demonstrate that hSNF5 regulates NOXA transcriptional activity in MRT cell lines by a p53-independent mechanism. Furthermore, our results suggest that NOXA pathway modification by hSNF5 loss might be related with the therapy resistant character of MRT patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 212. doi:10.1158/1538-7445.AM2011-212