P4-01-09: Reduced CYBA Expression Leads to the Development of Fulvestrant Resistance in a Breast Cancer Cell Line.
Authors: Skerry BJO , Ramsahoye BH 
Published: June 2012

Background

Acquired resistance to endocrine therapy is an important problem in the management of advanced breast cancer. In this study we used a cell line model to investigate the evolution of fulvestrant resistance. We examined gene expression and promoter methylation in estrogen-dependent MCF7 cells and compared two derived cell lines, estrogen-hypersensitive LCC1 and fulvestrant-resistant LCC9, using array technology. Thus we determined the epigenetic changes associated with the evolution of estrogen independence and fulvestrant resistance, which revealed a role for CYBA/p22phox in the acquisition of resistance.

Material and Methods: Estrogen-dependent gene expression was measured in MCF7, LCC1 and LCC9 cells using Illumina HT-12 microarrays. In parallel, differential methylation between MCF7, LCC1 and LCC9 cells was examined using Illumina Infinium methylation 27 arrays.

Results: Despite marked physiological differences between MCF7, LCC1 and LCC9 cells there were few genes which were both differentially methylated and differentially expressed. However, CYBA was underexpressed in LCC1 (p&lt;10−5) and LCC9 (p&lt;10−4) and had a methylated promoter in LCC1 (p&lt;10−5) and LCC9 (p&lt;10−4), with reduced expression correlated to increased estrogen independence. This gene codes for the protein p22phox, a critical component of membrane NADPH oxidase. We verified expression by qRT-PCR and Western blot, and methylation by CpG island bisulphite sequencing, demonstrating significant and biologically relevant changes. Fulvestrant resistant LCC9 cells exhibited a 20-fold reduction in CYBA expression compared to MCF7, corresponding to undetectable levels of p22phox. Due to its role in the generation of reactive oxygen species (ROS), we investigated the cellular response to oxidant damage, finding that LCC9 cells showed reduced sensitivity to oxidant damage when compared to MCF7 (p&lt;10−7), in keeping with impaired NADPH oxidase function. We then treated MCF7 cells with a ROS scavenger, N-acetyl cysteine, which reduced their sensitivity to fulvestrant (p&lt;10-13). Using siRNA, we knocked down CYBA expression, which conferred slight fulvestrant resistance on MCF7 cells, mitigating fulvestrant-induced growth arrest (p&lt;10−7).

Discussion: The striking differences in methylation in the CYBA promoter and gene expression between these cell lines lead us to conclude that the repression of this gene represents an important step in the development of fulvestrant resistance. As these cells develop estrogen independence and fulvestrant resistance, CYBA/p22phox levels drop, making the cells more resistant to oxidant damage caused by estrogen deprivation and, ultimately, providing a selective advantage against fulvestrant-induced growth arrest and cell death. CYBA/p22phox may represent a novel druggable target in breast cancer, either in the prevention of the acquisition of fulvestrant resistance, or the further treatment of resistant tumours.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-09.