Abstract 5148: Overexpression of histone methyltransferase WHSC1L1 regulates expression of ER-alpha in SUM44 breast cancer cells
Authors: Jonathan Curtis Irish, Alexandria Rutkovsky, Stephen P. Ethier
Journal: Cancer Research (2014)

Abstract The WHSC1L1 gene is amplified and overexpressed in ∼12% of breast cancers, and is thought to be a driving oncogene when amplified and overexpressed, however the mechanism of transformation is not known. SUM44 cells harbor an amplification of WHSC1L1 as part of the 8p11-p12 amplicon, and express high levels of both the long and short isoforms of the gene. The short isoform, containing only a PWWP domain and no catalytic SET domain, is the major species present in SUM44 cells. To create a model to study the effect of WHSC1L1 on gene regulation, SUM44 cells were lentivirally transduced with either a control shRNA for LacZ (shLacZ), an shRNA specific for both isoforms of WHSC1L1 (shWHtotal), or a short isoform-specific shRNA (shWHshort). Cell growth was significantly reduced in shWHtotal cells compared to shLacZ, however, the greatest decrease in growth was observed in cells transduced with the short isoform-specific shRNA. To identify genes transcriptionally regulated by WHSC1L1, we performed genome-wide expression profiling of SUM44 breast cancer cells transduced with either shWHtotal or shLacZ. A total of 5185 genes were found to be differentially expressed in shWHtotal SUM44 cells compared to shLacZ control. Among the 5185 differentially expressed genes, we found ESR1 to be significantly downregulated (-7.3-fold, p &amp;lt; 0.0001) in the shWHtotal cells compared to shLacZ controls. In addition MYCN, which is amplified in SUM-44 cells was reduced by 7.43-fold, (p &amp;lt; 0.004), ERBB3 was reduced by 2.5-fold, (p &amp;lt; 0.0009), and ERBB4, which is highly expressed in SUM-44 cells was reduced by 2.12-fold, (p &amp;lt; 0.00001). Immunoblotting confirmed downregulation of estrogen receptor alpha (ERα) in the shWHtotal compared to shLacZ control. Interestingly, knockdown of WHshort resulted in an even greater decrease of ERα than the shWHtotal knockdown. To determine whether downregulation of ERα affects sensitivity to Tamoxifen, we measured the Tamoxifen LD50 in SUM44 shLacZ and SUM44 shWHtotal cells, and found a 10-fold decrease in Tamoxifen LD50 compared to shLacZ control (3.8nM vs. 35nM). Tamoxifen affected SUM-44 cells in a dose-dependent manner, however 500nM Tamoxifen resulted in complete growth arrest in SUM44 shWHtotal cells, while the cells infected with the shLacZ virus cells grew at the same dose, albeit slowly. Our data indicate that WHSC1L1 regulates ERα expression in SUM44 cells, and WHSC1L1 knockdown increased the cells' sensitivity to Tamoxifen. The decrease in both cell growth and ERα expression in the SUM44 WHshort-specific knockdown cells suggests that the catalytically inactive short isoform of WHSC1L1 may be the isoform driving the oncogenic activity of WHSC1L1 amplification, perhaps through a dominant-negative interaction with the long isoform of WHSC1L1. Data showing increases in global H3K36 methylation levels following knock down of WHSC1L1 are consistent with this hypothesis. Citation Format: Jonathan Curtis Irish, Alexandria Rutkovsky, Stephen P. Ethier. Overexpression of histone methyltransferase WHSC1L1 regulates expression of ER-alpha in SUM44 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5148. doi:10.1158/1538-7445.AM2014-5148