Abstract 3359: TET1 mediated hypomethylation activates oncogenic signaling pathways in triple negative breast cancer
Authors: Good Charly Ryan , Kelly Andrew , Madzo Jozef , Jelinek Jaroslav , Issa Jean-Pierre 
Published: July 2017

Triple negative breast cancer (TNBC) is a subtype of breast cancer that occurs in 15-20% of patients, and is defined by tumors that do not overexpress the estrogen, progesterone and HER2 receptors. This aggressive subtype has a significantly worse overall survival compared to non-TNBC and importantly, these patients lack options for targeted therapy. To identify novel subtype-specific therapeutic targets, we must first understand the biological underpinnings of the disease. DNA methylation is a hallmark of cancer, as it can regulate gene expression of both tumor suppressor genes and oncogenes. We have found that TN tumors have widespread genome-wide hypomethylation when compared to other breast cancer subtypes and normal breast controls. TET1 is a DNA demethylase that converts 5-methyl cytosine into 5-hydroxymethyl cytosine, which can be further oxidized into un-methylated cytosine. To determine if TET1 could possibly play a role in TNBC hypomethylation, we analyzed genome-wide DNA methylation, DNA mutation and RNA-seq datasets from the TCGA breast cancer cohort. We identified a subset of TN patients that upregulate TET1 and display DNA hypomethylation. To identify the hypomethylated TET1 target genes, we computed Spearman correlations between TET1 expression and methylation % for 450,000 sites (450K array) across 67 TNBC patients. Filtering for sites with r&lt;-0.3 revealed 42,559 sites negatively correlated with TET1 expression. Cluster analysis of the sites that lose methylation compared to normal breast (12,807 CpG sites) revealed two distinct clusters. Cluster 1 (42% of TNBC cases) were TET1 high and hypomethylated, while cluster 2 (58%) looked more like normal breast controls. Gene set enrichment analysis of the hypomethylated genes revealed Hippo Signaling, Pathways in Cancer and PI3K-Akt Signaling as significantly enriched, with p&lt;0.001. In addition, only 4% of patients in cluster 1 have mutations/genomic alterations in the PI3K pathway, compared to 29% of cluster 2 patients, p=0.01. Most strikingly, analysis of phosphorylated EIF4EBP1 RPPA proteomic data revealed TET1 high patients have increased PI3K pathway activity, even though they lack mutations in the pathway, p=0.02. We hypothesize that the PI3K hyper-activation, in part, can be explained by TET1 upregulation and target gene demethylation. In TET1 knock out MDA-MB-231 cells, we observed a reduction in phosphorylated 4E-BP1, suggesting loss of PI3K activity is concomitant with loss of TET1 as well as decreased cellular proliferation (p&lt;0.01). Furthermore, breast cancer cell lines with high TET1 (but not low TET1) are sensitive to drugs targeting the PI3K/ERK pathway (XMD8-85, AZ628, TGX221). In addition to explaining and predicting PI3K inhibitor sensitivity in breast cancer, our studies may establish TET1 as a TNBC specific oncogene that could serve as a novel druggable target for therapeutic intervention.

Citation Format: Charly Ryan Good, Andrew Kelly, Jozef Madzo, Jaroslav Jelinek, Jean-Pierre Issa. TET1 mediated hypomethylation activates oncogenic signaling pathways in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3359. doi:10.1158/1538-7445.AM2017-3359