Epigenetic Control of Tamoxifen Resistant

Breast Cancer

Epigenetic Control of Tamoxifen Resistant

Breast Cancer

Kristin WilliamsArcaro Lab

Thesis ResarchJune 25, 2012

Kristin WilliamsArcaro Lab

Thesis ResarchJune 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-7 TMX2-28• 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)

(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

MC

F-7 A

VG

Beta

TMX2-11AVG Beta

MCF-7 vs. TMX2-28

MC

F-7 A

VG

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 TMX2-28Selected 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

END

MCF-7 vs. Estradiol (E2) treated MCF-7

MC

F-7 A

VG

Beta

E2 treated MCF-7 AVG Beta

Cells were treated for 14 days with 10-10 M Estradiol

MCF

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X2-2

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