Estrogen and its receptors play an important role in breast carcinogenesis. In humans, there are two subtypes of estrogen receptors (ER), ER and ER, both of which are expressed in normal breast and breast cancer. Breast tumorigenesis has been reported to be associated with an increase in ER and a decrease in ER (1). Thus, it is hypothesized that ER plays a role as a tumor suppressor in breast cancer. The mechanism involved in ER downregulation is poorly understood, however epigenetic changes such as DNA methylation may be involved in gene regulation (2). We (3) demonstrated that loss of ER expression in prostate cancer was associated with hypermethylation of the ER ON promoter. The goal of this study was to determine if increased CpG island (CGI) methylation in the ER ON promoter correlated with loss of ER expression in breast cancers. Sections of breast cancer and adjacent normal tissue were stained for ER expression. Representative breast cancer and adjacent normal foci were microdissected using laser capture microscopy. Bisulfite genomic sequencing was then used to examine the methylation status of all 41 CGIs in the ER ON promoter. Immunocytochemistry data revealed 1) normal epithelial cells expressed high levels of nuclear and cytosolic ER, 2) well differentiated tumors expressed weak nuclear but significant cytosolic staining, and 3) poorly differentiated tumors had either no or very weak nuclear staining and some cytosolic staining. Bisulfite genomic analyses revealed that well differentiated breast tumors had a lower degree of ER ON promoter CGI methylation in both cancerous and normal cells, whereas, poorly differentiated tumors had a higher degree of cytosine methylation in both cancerous and normal cells. However, an apparent mono-allelic methylation pattern was observed in the normal epithelial cells, whereas bi-allelic methylation seemed to occur in the cancerous cells. These results provide support that hypermethylation of the ER ON promoter may contribute to loss of ER expression in breast tumorigenesis. Also, it suggests that the ER ON promoter in the normal epithelial cells in poorly differentiated tumors may have already undergone aberrant methylation when compared to well differentiated tumors. The difference in the methylation status of the ER- ON promoter in normal epithelial cells found between the poorly differentiated and well differentiated breast tumors suggest that each may evolve through a separate pathway.

References:

1.Balfe et al Eur J Surg Oncol. 30:1043-1050 (2004).  

2.Zhao et al Oncogene. 22:7600-7606 (2003).  

3.Zhu X et al, Am J Pathology 164: 2003-2012, 2004

Abstract

• Breast cancer is the second leading cause of cancer death in women in the USA

• Estrogen and its receptors play important roles in the genesis and malignant progression of breast cancer

• Estrogen receptors (ER):– Are members of the nuclear hormone receptor superfamily

and act as ligand-activated nuclear transcription factors– The first ER was cloned in 1986 and named ER– The second ER, named ER, was identified in 1996

• Estrogen Receptor- (ER): – Various isoforms of ER have been described which are

alternatively spliced variants (ER1-5)– Another level of complexity provided by diversity in the 5’

untranslated region-2 alternatively spliced exons-ON and OK

• Breast Cancer:– There appears to be a relative increase in ER and a decrease

in ER expression in breast cancer compared with normal breast

– It is hypothesized that ER might play a role as a tumor suppressor in breast carcinogenesis

– Epigenetic modifications such as DNA methylation could be involved in ER downregulation

• DNA methylation:– Mammalian cells can epigenetically modify their genome via

covalent addition of a methyl group to the 5’-position of the cytosine ring

– About 3-5% of cytosines in the human genome are methylated– 70-80% of the 5-methylcytosines are located in CpG islands

found in the 5’ promoter region and first exon– Methylation of CpG islands is frequently associated with gene

silencing

Introduction

Immunohistochemistry to identify ER status in normal breast and breast cancer

Laser Capture Microscopy of representative areas

DNA ExtractionBisulfite Sequencing

Protocol For Determining Methylation Status of ER Promoter/Exon ON

Methylated CpG IslandMethylated CpG Island Unmethylated CpG IslandUnmethylated CpG Island

CHCH33

CG

CHCH33

CG

CHCH33

CG

CHCH33

CG CG CG CG CG

CHCH33

CG

CHCH33

CG

CHCH33

CG

CHCH33

CG

CG CG CG CG TG TG TG TG

UG UG UG UG

Step 1: Step 1: Bisulfite Bisulfite treatmenttreatment

Step 1: Step 1: Bisulfite Bisulfite treatmenttreatment

Step 2: Step 2: PCRPCR

Step 2: Step 2: PCRPCR

Step 3: Step 3: SequencingSequencing

Step 3: Step 3: SequencingSequencing

Protocol For Bisulfite Sequencing

2. BPH

3. HG-PIN

1. Normal

A B

10. Bone Met

10 20 30 40

exon 0Npromoter

cancer5. GR3

cancer6. GR4

cancer7. GR5

8. LymphNode Met

2. BPH (Positive) 150

6. GR4 cancer (Negative) 175

9. LymphNode Met

7. GR5 cancer (Negative) 200 8. Lymph node Met (Mixed) 375

9. Lymph node Met (Positive) 375 10. Bone Met (Positive) 300

4. HG-PIN (Mixed) 400

5. GR3 cancer (Positive) 425

3. HG-PIN (Positive) 375

1. Normal (Positive) 350

4. HG-PIN

(Positive)

(Positive)

(Positive)

(Mixed)

(Mixed)

(Positive)

(Positive)

(Negative)

(Negative)

(Positive)

2. BPH

3. HG-PIN

1. Normal

A B

10. Bone Met

10 20 30 40

exon 0Npromoter

cancer5. GR3

cancer6. GR4

cancer7. GR5

8. LymphNode Met

2. BPH (Positive) 150

6. GR4 cancer (Negative) 175

9. LymphNode Met

7. GR5 cancer (Negative) 200 8. Lymph node Met (Mixed) 375

9. Lymph node Met (Positive) 375 10. Bone Met (Positive) 300

4. HG-PIN (Mixed) 400

5. GR3 cancer (Positive) 425

3. HG-PIN (Positive) 375

1. Normal (Positive) 350

4. HG-PIN

(Positive)

(Positive)

(Positive)

(Mixed)

(Mixed)

(Positive)

(Positive)

(Negative)

(Negative)

(Positive)

(Zhu et al, Am J of Path, 2004)

Methylation Status of ER Promoter/Exon ON in Prostate Cancer

Adjacent normal-10X

Well differentiated Breast Ca-40X

Poorly differentiated Breast Ca-100X

Adjacent normal-40X

Normal Breast (breast reduction)-10X

Normal Breast (breast reduction)-40X

Immunohistochemistry of ERin Breast Tissue

•ER +++

exon ONpromoter

20 30 4010

Methylation Status of ER Promoter/Exon ON in Normal Breast Tissue

Bre

ast T

issu

e fr

om R

educ

tion

Mam

mop

last

y

Cas

e #1

Cas

e #2

Cas

e #3

Cas

e #4

Cas

e #5

•ER +++

•ER +++

•ER +++

•ER +++

IHC

Methylation Status of ER Promoter/Exon ON in Breast Cancer and Adjacent Normal

• ER +++ to ++++

• ER +++

• ER , PR

exon ONpromoter

20 30 40

• ER +++ to ++++

• ER 95% • ER , PR

10

• ER ++ to +++• ER , PR

• ER +++

• ER 75% +++, 25% -

• ER , PR

• ER +++

• ERb +++• ER +++, PR

• ERb +++

Poo

rly D

iffer

entia

ted

Wel

l Dif

fere

nti

ated

Cas

e #1

Cas

e #2

Cas

e #3

Cas

e #4

Cas

e #5

N

C

N

N

N

N

C

C

C

C

IHC

• Normal breast tissue has a different cytosine methylation pattern of promoter/exon ON compared with normal prostate tissue

• The methylation pattern suggests that there may be mono-allelic methylation in normal breast tissue

• Well differentiated tumors have a lower degree of cytosine methylation of the promoter/exon ON in, which correlates with a higher ER expression

• Poorly differentiated tumors have a higher degree of cytosine methylation of the promoter/exon ON, which correlates with lower ER expression

• The methylation pattern suggests that there may be mono-allelic methylation in adjacent normal breast tissue, whereas bi-allelic methylation seems to occur in the cancerous cells

Summary

Normal Breast Tissue

Luminal cells

Myoepithelial cells

Stromal cells

Breast Cancer

Well Differentiated Breast CancerPoorly Differentiated Breast Cancer

Adjacent Normal Breast Adjacent Normal Breast

Breast Cancer