Epigenetic Control of Tamoxifen Resistant Breast Cancer Kristin Williams Arcaro Lab Thesis Resarch June 25, 2012 Kristin Williams Arcaro Lab Thesis Resarch June 25, 2012

Parent cell line Estrogen Receptor Positive Bind estrogen Non-invasive Rounded morphology Express luminal cytokeratins Majority of cells in G0/G1 phase MCF-7TMX2-28 Single clone from MCF-7 cells cultured in Tamoxifen for 6 months Estrogen Receptor Negative (both protein and mRNA) Triple-negative (ER, PR, HER2) Migratory & Invasive Similar cellular morphology to MCF-7 Express basal and luminal cytokeratins Display altered cell cycle from MCF-7 with the majority of the cells in S & G2/M phase TMX2-11 Single clone from MCF-7 cells cultured in Tamoxifen for 6 months Estrogen Receptor Positive Bind estrogen ~1.5- fold more than MCF-7 Non-invasive Cellular morphology similar to MCF-7 Unknown cytokeratin expression Cell cycle analysis preliminary data similar to MCF-7

✦ Objective: Further understand the role that epigenetics, specifically promoter methylation, plays in antiestrogen resistant breast cancer ✦ Goal: Determine the extent to which DNA methylation contributes to antiestrogen resistance through use of tamoxifen-resistant and -sensitive cell lines and breast tumor tissue samples

✦ Hypothesis: Methylation of genes involved in cell cycle control, DNA repair, apoptosis, and cell survival plays a role in acquired anti-estrogen resistance

Overview of Study Design Aim 1 ✦ Identify signaling pathways that are differentially methylated in tamoxifen-sensitive and tamoxifen-selected ER-positive and ER-negative cell cultures and confirm by pyrosequencing. Aim 2 ✦ Determine if combined treatment with methylase/demethylase and an antiestrogen reverses the methylation alterations and hormone-resistance of tamoxifen-selected ER-positive and ER-negative cell lines. Aim 3 ✦ Determine whether genes differentially methylated in tamoxifen-resistant cells are similarly methylated in vivo.

HumanMethylation450 Study Design ✦ What pathways involved in tamoxifen resistance are altered by promoter methylation?

Multidimensional Scaling Shows Methylation Changes (Arbitrary Units) ✦ Uses differences in raw Beta values of 1000 most variable CpG sites as a similarity measure ✦ MDS plot is a way of visualizing relationship between samples based on the similarity measure ✦ Differences in epigenomic profiles

Dendrogram Mimics Findings in MDS Plot ✦ Shows greatest similarity between MCF-7 and Estrogen treated MCF-7 ✦ TMX2-11 is more similar to MCF-7 than TMX2-28

MCF-7 vs. TMX2-11 MCF-7 AVG Beta TMX2-11AVG Beta

MCF-7 vs. TMX2-28 MCF-7 AVG Beta TMX2-28 AVG Beta

What has been done so far: ✦ In TamR lines, filtered for CpG sites that had >2 fold change, >0.2 average Beta value and detection p-value <0.01. ✦ 4,091 CpG sites TMX2-11 ✦ 33,748 CpG sites TMX2-28 ✦ Also filtered for <-2 fold change, <0.2 average Beta value and detection p-value <0.01. ✦ 2,591 CpG sites TMX2-11 ✦ 5,244 CpG sites TMX2-28 Selected for TMX lines for all filters

What needs to be done: ✦ Ensure data are filtered correctly ✦ Better method of data selection? ✦ Run genes represented by CpG sites in filtered data through pathway analysis program ✦ Estrogen responsive genes filtered out? ✦ Determine interesting/promising genes differentially methylated between TMX lines and MCF-7 ✦ Design and run pyrosequencing assays on selected genes

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MCF-7 vs. Estradiol (E 2 ) treated MCF-7 MCF-7 AVG Beta E 2 treated MCF-7 AVG Beta Cells were treated for 14 days with M Estradiol

MCF-7 TMX2-11 TMX2-28 Promoter region