Abstract B05: WHSC1L1 and estrogen-independent activation of estrogen receptor-alpha (ERα) in 8p11 amplicon-bearing cell lines

The 8p11-p12 genomic region is amplified in 15% of breast cancers and 21% of lung squamous cell carcinomas (LSCC) and is associated with poorer prognosis. This genomic region harbors several oncogenes, three of which are epigenetic modifiers of chromatin (WHSC1L1, KAT6A, ASH2L). WHSC1L1 is a histone methyltransferase (HMT) that is expressed in 2 isoforms. The long isoform (WH-long) encompasses the entire coding region and is associated with dimethylation of lysine 36 on histone 3 (H3K36me2) to facilitate transcriptional elongation. The short isoform (WH-short) is produced by alternative splicing at exon 10, resulting in a truncated protein that retains the ability to bind lysine residues but lacks the catalytic SET domain required for methylation. Our studies have shown that the short isoform is consistently more highly expressed than the long isoform, both in breast and lung cancer lines, consistent with data in the Cancer Genome Atlas (TCGA). Upon investigation of the role of WHSC1L1 in regulation of gene transcription, shRNA knockdown of WH-short resulted in reduced expression of several important genes, including CD44, HER4, and ESR1, which encodes estrogen receptor alpha (ERα). SUM-44 cells are an 8p11 amplicon-bearing breast cancer cell line which overexpresses WHSC1L1 and ERα. Knockdown of WHSC1L1 in this cell line resulted in decreased expression of ERα. ERα ChIP-seq in SUM-44 cells, which are ER+ but do not require estrogen for growth, revealed that ERα, which is not mutated or deleted in this cell line, retains the ability to bind to chromatin and participate in gene transcription in the absence of estrogen. Estrogen response elements (EREs) and FOXA1 sites were identified as the most common binding motifs for ERα, and RT-PCR following siRNA knockdown of ESR1 or FOXA1 demonstrated decreased transcription of a panel of genes identified as bound by ERα. Knockdown of WHSC1L1 abolished the ability of ERα to bind chromatin in the absence of estrogen. Despite estrogen-independence, shRNA knockdown of ESR1 in this cell line demonstrated that these cells are indeed dependent on ERα for growth and survival. Similarly, treatment with Fulvestrant, a selective estrogen receptor degrader (SERD), resulted in downregulation of ERα protein and inhibition of growth in SUM-44 cells. Together, these results indicate that overexpression of WHSC1L1 is associated with ERα overexpression, leading to several downstream changes in gene expression dependent upon ERα and FOXA1 but independent of estrogen signaling. Ongoing studies in the lab are focused on identifying the alterations in gene expression directly due to WHSC1L1 and its potential epigenetic actions on transcriptional regulation as opposed to those due to the transcription factor ERα as an indirect result of WHSC1L1 overexpression. These mechanistic studies are essential to understanding the role of the oncogenes on the 8p11 amplicon in ER+ breast cancer and how the presence of this genomic alteration can be exploited to identify patients that would benefit from additional therapies, such as the SERDs currently under development. Citation Format: Jamie N. Mills, Jonathan Irish, Brittany Turner-Ivey, Stephen P. Ethier. WHSC1L1 and estrogen-independent activation of estrogen receptor-alpha (ERα) in 8p11 amplicon-bearing cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B05.