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Estrogen receptor-β characterization in breast cancer: development of a reliable assay for measuring expression

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Estrogen receptor-β characterization in breast cancer: development of a reliable assay for measuring expression

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Content 1

Estrogen Receptor-b Characterization in Breast Cancer: Development of a Reliable
Assay for Measuring Expression

Ryan LaRue

Master of Science (Experimental and Molecular Pathology)

Degree Conferral Date: May, 2018

University of Southern California

Keck School of Medicine

Thesis Advisory Panel:

Chair: Dr. Michael F. Press

Dr. Michael Stallcup

Dr. Julie Lang

2

Acknowledgements

I would like to thank Dr. Michael Press for his mentorship throughout this project, and to Drs.
Stallcup and Lang for agreeing to serve on my thesis committee. I would also like to thank the
members of the Press lab for their constant support and dedication to my success with this
project. In particular, I would like to acknowledge Simon Davenport who was invaluable in the
development of my skill set in the laboratory, Angela Santiago for her help with
immunohistochemistry, Himanshu Joshi for his assistance using bioinformatics tools to collect
estrogen receptor-b mRNA data from various tissue types, and Shantae Thornton for teaching me
how to culture cells in suspension.

3
Contents

Acknowledgements…………………………………………………………….….……………2

Summary…………………………………….…………………………...……….….………….5

Chapter 1

Introduction and Background………………………………………....…………………........…..7

Steroid Hormone Receptors………………….……………….…….…………....….…….7

Estrogen Receptor-b Isoforms………………………………………………………….....9

Steroid Hormone Receptors in Breast Cancer…………………………………………...10

Contradictory Literature on Estrogen Receptor-b’s Involvement in Cancer……….……12

Tables…………………………………………………………………………………….14

Figures……………………………………………………………………………………16

Chapter 2

Methods and Results……………………..………………………………………………………17

Materials and Methods…………………………………………………………………...17

Results……………………………………………………………………………………22

Tables…………………………………………………………………………………….25

Figures……………………………………………………………………………………28

Chapter 3

Discussion and Conclusions………………………..……………………………………………42

The Search for Endogenous ER-b Amidst Contradictory Literature Sources…………...42

A Comparison of Our Data with Those from Other Studies………….……….………...44

The Foundation Our Results Create for Future Studies………………………………….47

An Analysis of our Process ……………………………………………………………...49

4
Conclusions………………………………………………………………………………52

Figures……………………………………………………………………………………54

References…………………………………………………………………………….……......57

5
Summary
Steroid hormones are lipophilic molecules synthesized in the adrenal cortex, the testes,
and the ovaries. Within target cells, steroid hormones bind to steroid hormone receptors, which
are intracellular transcription factors, to exert positive or negative effects on the expression of
target genes. For example, estrogen regulates the growth and development of breast tissues
through the action of estrogen receptor-α (ER-α) and progesterone receptor (PR).
There are multiple subgroups of patients’ breast cancers depending on their respective
phenotypes. Hormonal intervention therapies, such as Tamoxifen, have demonstrated success in
treating women with ER-α positive breast cancers. In triple-negative breast cancer (TNBC)
patients, estrogen receptor-α, progesterone receptor, and HER2 expression is low or absent (table
1-1). There are currently no “targeted therapies” for TNBC. Uncovering mechanisms that drive
this specific breast cancer subtype, and the subsequent development of targeted therapies which
inhibit them, are priorities in cancer research.
Estrogen receptor-b’s (ER-b) function is a potential factor in the progression of breast
cancer and, therefore, a potential therapeutic target for TNBC patients. For the following study,
we investigate ER-b expression in TNBC patients. The hypothesis at the onset was if ER-b
expression contributes to a proliferative phenotype, then using a validated antibody to quantify
ER-b expression levels will reliably identify breast cancer cell lines and tissues which, upon
estrogen stimulation, demonstrate an increase in proliferation. ER-b’s expression is investigated
by way of immunoblotting analysis combined with immunohistochemistry. With this in mind,
Chapter 1 is an introduction and background on ER-b’s characteristics, including its relevance to
the field of cancer research. This chapter also explores the inconsistencies in published papers of
the last two decades. Chapter 2 contains a description of results, as well as the methodology
6
used. Our data simultaneously analyzes antibody specificity among various commercially
available antibody reagents for their ability to detect a protein of ~59 kDa, the predicted
molecular weight for ER-b. The most favorable antibody, called 4D2, is used to characterize ER-
b expression in the context of breast cancer samples, both from cell lysates on an immunoblot,
and from formalin-fixed, paraffin-embedded cells. Expression by mRNA is also evaluated by
qRT-PCR in breast cancer cell lines and several lymphoma cell lines. Our conclusion is as
follows; ER-b is expressed in only one breast cancer cell line among those tested, including, but
not limited to, TNBC’s, and is thus not expected to represent a potential target for therapy due to
the infrequent expression of ER-b in breast cancer.

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Chapter 1: Introduction and Background
Steroid Hormone Receptors:
Steroid hormones are lipophilic molecules synthesized in the adrenal cortex, the testes,
and the ovaries. Steroid hormones reach their target cells via the blood and, due to their
lipophilic nature, pass through cell membranes by simple diffusion. Within target cells, steroid
hormones bind to steroid hormone receptors, which are intracellular transcription factors, to exert
positive or negative effects on the expression of target genes. For example, estrogen regulates the
growth and development of breast tissues through the action of ER-α. A component of ER-α’s
gene expression program is to initiate proliferation of breast epithelial cells during adolescence
to mediate an extensive arborizing growth of a primitive ductal system into a more mature,
developed breast in the adult.
The initial discovery of the second estrogen receptor, estrogen receptor-b (ER-b), was in
1996 (1). Its biological functions, in the context of cancer, have since been investigated, yet
remain poorly understood. ER-b is a member of the nuclear receptor superfamily (2). This
receptor has the ability to form homodimers, which then bind to estrogen response elements
(ERE’s) that consist of palindromic repeat sequences.
ER-α and ER-b are both considered type I receptors. They are similar in that they share
characteristics common to all type I nuclear receptors. Steroid hormone receptors, like ER-α,
localize to the nucleus, independent of ligand binding (3). The human gene for ER-β (ESR2) is
composed of eight exons. The peptide sequence encodes a 530-amino acid protein similar in
structure to ERα as well as other nuclear hormone receptors. Like ER-α, ER-b contains five
distinct protein domains. The A/B domain, located at the N-terminus of the protein, exhibits
8
ligand-independent activity (4). The DNA-binding domain corresponds to the C domain and is
highly conserved. This domain also functions in receptor dimerization. The hinge region is
termed the D domain and contains a nuclear localization signal (5). Lastly, the E domain
contains the ligand-binding domain.

9
Estrogen Receptor-b Isoforms:
Unlike ER-α, ER-β’s functional implications in cancer remain poorly understood. Thus
far, published data on ER-β function has been derived from studies on ER-β isoform 1, the first
isolated clone (6). Sequencing data indicates four different ER-β isoforms due to alternative
splicing in the C-terminal region, which consists of the ligand binding domain (figure 1-1). The
functional characteristics of the different ER-β isoforms are ambiguous; however, a common
theme is their differentially expressed locations in tissues and cell lines (7). Additionally,
isoform 3 contains a premature stop codon in the mRNA, leading to nonsense-mediated mRNA
decay (8).
ER-β isoform 1 is the only fully functional isoform, whereas isoform 2, 4, and 5 do not
form homodimers, and thus have no innate activities of their own (9). However, these isoforms
can heterodimerize with isoform 1 and enhance its transactivation in a ligand-dependent manner
(9). It is imperative that a complete understanding of ER-β, and its isoforms, is reached. Until
that time, ER-β’s total biological and clinical relevance to cancer remains unclear.

10
Steroid Hormone Receptors in Breast Cancer:
In the emerging field of personalized medicine, translational oncology has become a
subspecialty with a focus on developing diagnostic and therapeutic tools for patients with life-
threatening cancers. One such example is the FDA-approved, clinically effective drug
trastuzumab, which is a humanized monoclonal antibody, for HER2/neu oncogene amplification,
which occurs in 20-25% of human breast cancers. Other targeted therapies for breast cancer
patients include tamoxifen, aromatase inhibitors, and olaparib.
There are several subgroups of breast cancers in patients depending on their respective
phenotypes. Targeted therapies, such as tamoxifen, have demonstrated success in treating women
with ER-α positive breast cancers. For three decades, estrogen receptor-α (ER-α) has been an
important prognostic and predictive marker in the treatment of women with endocrine sensitive
breast cancer. Interfering with its function dramatically reduces proliferation in tumor cells.
Olaparib is a targeted therapy for breast cancer patients with BRCA1 and BRCA2
mutations. These genes normally maintain the integrity of the genome by mediating a DNA
repair process, known as homologous recombination. Olaparib induces a persistent DNA lesion
because there is a loss of function mutation in BRCA proteins responsible for DNA repair
through homologous recombination; tumor cells are thus unable to effectively repair these DNA
lesions and die, while non-tumor cells are unaffected (10).
The identification of genetic alterations, respective to a patient’s genotype, which are
candidates for targeted therapeutic intervention, will lead to innovative advances in personalized
medicine. In triple-negative breast cancer patients, estrogen receptor-α, progesterone receptor,
and HER2 expression is low or absent (table 1-1). Therefore, uncovering mechanisms that drive
this specific phenotype, and the subsequent development of targeted therapies which inhibit
11
them, are priorities in cancer research. In triple-negative breast cancer patients, estrogen
receptor-α, progesterone receptor, and HER2 expression is low or absent (table 1-1). Uncovering
mechanisms that drive this specific phenotype, and the subsequent development of targeted
therapies which inhibit them, are priorities in breast cancer research.
Following the discovery of a second estrogen receptor, estrogen receptor-β (11),
researchers began to investigate the possible role of this protein in mediating breast cancer
development and response to therapy. Patients with triple negative breast cancers are widely
disadvantaged with regard to administering effective therapies. As a potential therapeutic target
for triple negative breast cancer patients, elucidation of ER-β’s role is an important factor in the
progression of breast cancer research.

12
Contradictory Literature on Estrogen Receptor-β’s Involvement in Cancer:
Conflicting reports regarding estrogen receptor β’s (ER-β) expression, or lack thereof,
have led researchers to believe it plays an anti-proliferative, but also oncogenic role (11, 12, 13,
14, 15). Inconsistencies in the reported expression of ER-β in breast and prostate cancers as
determined by methods such as immunohistochemistry (IHC) have contributed to this confusion.
Published data on prostate cancer suggest that ER-β is highly expressed in benign epithelial cells,
with expression decreasing with cancer development, inversely correlating with increasing
Gleason grade (11, 16, 17, 18, 19, 20). However, there are also data which support the
hypothesis that high ER-β expression correlates with poor clinical prognosis (18, 21). And
finally, in breast cancer, high ER-β expression has been described as both a poor (22) and
favorable (23, 24, 25, 26, 27, 28, 29) prognostic marker, as well as researchers finding no
correlation between pathological outcome and ER-β expression (30).
The reason for these conflicting conclusions is largely the result of poorly validated
antibodies. Variability in the specificity of ER-β targeted antibodies directly contribute to the
uncertainties surrounding tissue expression and functional relevance to cancer. Previous ER-β
antibody validation studies have been published (31, 32, 33, 34, 35), but many of them are
limited in that they rely on two assumptions. First, that when evaluating an antibody by Western
blot in a cell line model, the protein of interest is expressed; and second, when testing an
antibody's specificity by IHC in tissue, the tissue expression of the protein has been well
characterized. With respect to ER-β, these assumptions are a weakness often overlooked, as its
expression in commonly used cell line models and tissues is not universally agreed upon (11, 16,
17, 18, 19, 20, 22, 24, 25, 28, 30, 33, 36, 37, 38, 39, 40, 41, 42).
A number of published studies utilize Thermo Scientific’s PPG 5/10 monoclonal
13
antibody to identify ER-β in cell lines (7, 14, 16, 25, 40). There are also various literature sources
indicating its frequent expression in uterine, prostate, and testis tissues (14, 16, 17, 21, 35, 43).
However, recent evidence suggests that some of these studies are compromised by off-target
binding to a different protein of larger molecular weight than ER-β (44). There is, therefore, a
need to address these technicalities in evaluating ER-β’s characterization and functional role in
cancer.
To address these assumptions, we sought to test and validate four commonly used,
commercially available ER-β antibodies in a systematic manner. We performed a number of
assays for antibody validation, and then successfully applied a validated antibody to 19 cell line
models of breast cancer to assess them for ER-β expression.

14
Tables:
Table 1-1: Immunohistochemistry staining for ER-α, PR, and HER2 status in breast cancer cell
lines
Cell line ER-α PR HER2
HCC70 - -
EFM19 + +
JIM-T1 - - ++**
MDA-MB-436 - -
SK-N-SH N/A* N/A* N/A*
MDA-MB-134 + -
MDA-MB-157 - -
MDA-MB-468 - -
SK-BR-3 - - ++
MDA-MB-453 - -
UACC812 + - ++
HCC1419 - - ++
15
HCC2218 - -
HCC1187 - -
HCC38 - -
BT474 + + ++
MCF-7 + +
T47D + +
MDA-MB-231 - -

*SK-N-SH denotes a neuroblastoma cell line
**‘++’ indicates amplification of HER2/neu copy number

16
Figures:
Figure 1-1: Protein sequence analysis and molecular modeling of ER-β isoforms

Figure 1-1: (A) Protein sequence alignment of the C-terminal regions of ER-β1, -β2, -β4, and -β5
by using the Clustalw alignment program. The ligand binding domain of ER-β1 is boxed. The
protein sequence forming helix 11 in each isoform is shown in red, whereas the protein sequence
participating in helix 12 is in green. (B) Molecular models of ER-β isoforms. The common helix
11 region of each isoform is labeled in pink, whereas the isoform-specific region of helix 11 is
highlighted in dark red. The orientation of helix 12 (green) in ER-β2 is different from that of ER-
β1, which has “tight” configuration in the ER-β1 binding pocket (orange oval). (C and D)
Molecular models of ER-β1 (C) and ER-β2 (D) show the coactivator binding pocket created by
electrostatic potential of the amino acid residues in helices 3–5 and 12. The size of the coactivator
binding pocket in ER-β2, which is indicated by a yellow arrow, was determined, by using PyMol
software, to be smaller than that of ER-β1 (9).

Reproduced from Proceedings of the National Academy of Sciences under the terms of the
Creative Commons Non-Commercial Attribution License.

d

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Chapter 2: Methods and Results
Materials and Methods:
Cell Lines and Culture Conditions
Cell lines were seeded and cultured to confluency in accordance with accepted methods
from the ATCC (45). The following breast cancer cell lines were cultured in RPMI 1640 with
10% heat-inactivated FBS (Fisher-Scientific), 2 mM L-glutamine, 50 U/ml penicillin, and 50
mg/ml streptomycin: HCC70, EFM19, JIM-T1, MDA-MB-436, MDA-MB-134, MDA-MB-157,
MDA-MB-468, SK-BR-3, MDA-MB-453, UACC812, HCC1419, HCC2218, HCC1187,
HCC38, BT474, T47D, MDA-MB-231. The following lymphoid cell lines were cultured in
RPMI 1640 with 10% heat-inactivated FBS (Fisher-Scientific), 2 mM L-glutamine, 50 U/ml
penicillin, and 50 mg/ml streptomycin: DB, KARPAS 422, KU812, and SU-DHL-5. The breast
cancer cell line MCF-7, neuroblastoma cell line SK-N-SH, and lung cancer cell line 273T were
cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated FBS
(Fisher-Scientific), 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin. All cells
were incubated at 37°C with 5% CO2 and cultured to 80-90% confluency. All cell lines were
obtained from ATCC (American Type Culture Collection, Manassas, VA).
Lysate Preparation and Extraction
Cells were harvested for nuclear extract using the Ne-Per nuclear extraction kit (Thermo
Scientific Pierce, Rockford, IL USA) according to the manufacturer's instructions. Extracted
protein was quantified using BCA assay analysis (Thermo Scientific Pierce, Rockford, IL USA)
according to the manufacturer’s instructions.
18
Western Blotting
Nuclear extracts were prepared with 4X protein sample loading buffer (LICOR
Biosciences, USA), 10X NuPage sample reducing agent (Thermo Scientific Pierce, Rockford, IL
USA) and water, and 20 µg protein per lane loaded into 10% polyacrylamide gels (BioRad,
Hercules, CA USA). Commercially available ER-β transient overexpression lysate (LY400556)
(Origene, Rockville, MD USA) from HEK293 cells and empty vector transfected control cell
lysate from HEK293 cells were also added to indicated lanes at 20 µg protein per lane. Gels were
run with running buffer for 30 minutes at 60 V followed by 30 minutes at 120 V. Western
transfer was performed using the sandwich transfer cassette system (BioRad, Hercules, CA
USA) according to the manufacturer's instructions. Odyssey blocking buffer (LICOR
Biosciences, USA) was added to membranes for one hour at room temperature. Primary
antibodies (Table 2-1) were added, as well as a Β-actin loading control antibody (TA811000)
(Origene, Rockville, MD USA) 1.0 mg/mL, and incubated overnight at 4°C: anti-ER-β PPG 5/10
(MAI-81281) (Thermo Scientific Pierce, Rockford, IL USA) 1.0 mg/mL, anti-ER-β 4D2 (MA1-
23219) (Thermo Scientific Pierce, Rockford, IL USA) 1.0 mg/mL, anti-ER-β MC10 (14-9336-
80) (Thermo Scientific Pierce, Rockford, IL USA) 0.5 mg/mL, anti-ER-β EPR3777 (Ab3576)
(Abcam, Cambridge, UK) 1.0 mg/mL, anti-ER-β 68-4 (05-824) (Merck Millipore, Watford, UK),
1.0 mg/mL. The membranes were washed three times with PBS/0.1% tween and incubated with
secondary antibodies for one hour at room temperature: Goat anti-mouse (green) 2.0 mg/mL with
Goat anti-rabbit (red) 2.0 mg/mL or Goat anti-rabbit (green) 2.0 mg/mL with Goat anti-mouse
(red) 2.0 mg/mL according to the species of the anti-ER-β antibody. Membranes were imaged
using the LICOR Odyssey fluorescent imaging system (LICOR Biosciences, USA).
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Immunohistochemistry
Immunohistochemistry was performed on the following formalin-fixed breast cancer cell
lines embedded in paraffin blocks: HCC70, EFM19, JIM-T1, MDA-MB-436, MDA-MB-134,
MDA-MB-157, MDA-MB-468, SK-BR-3, MDA-MB-453, UACC812, HCC1419, HCC2218,
HCC1187, HCC38, BT474, T47D, MDA-MB-231, MCF-7, and the neuroblastoma cell line, SK-
N-SH. Cell lines were cultured in accordance with accepted methods from the ATCC website
(ATCC Animal Cell Culture Guide, 2017) until confluency was reached. Cells were detached
from the surface of the flask using 0.05% trypsin and centrifuged for 5 minutes at 500 rcf. Cells
were resuspended in PBS and centrifuged for 5 minutes at 500 rcf. Cells were incubated on a
rotator in 1mL of 10% neutral- buffered formalin for 20 minutes. Cells were then centrifuged for
5 minutes at 12,000 rpm. Cells were embedded in paraffin and 4um sections were affixed to
glass slides. Immunohistochemistry was performed on slides using the anti-ER-β 4D2 (MA1-
23219) (Thermo Scientific Pierce, Rockford, IL USA) 1.0 mg/mL or MC10 (Wu et al., 2012)
1:1000 antibodies.
Transient Transfection
MDA-MB-231 cells were transiently transfected with the ESR2 expression vector
(SC119216) (Origene, Rockville, MD USA) using Lipofectamine 3000 (Thermo Scientific
Pierce, Rockford, IL USA) according to manufacturer protocol (46). Cells were incubated for 48
hours post-transfection.
qRT-PCR
In order to evaluate expression with an antibody-independent method, qRT-PCR was
20
used to analyze ESR2 expression at the mRNA level. The breast cancer cell lines MDA-MB-453,
MDA-MB-231, transiently transfected MDA-MB-231 with an expression vector encoding for
the ESR2 gene, SK-BR-3, and the lymphoid cell lines DB, KARPAS 422, KU812, and SU-DHL-
5 cells were harvested for collection of mRNA using the RNEasy Mini Kit (Qiagen, California
USA). On-column DNase digestion was performed to remove contaminating genomic DNA.
RNA was quantified with the NanoDrop 8000 (Thermo Scientific, Delaware USA). Samples
containing 250 ng random primers, 1 mg RNA, 1 ml 10 mM dNTP mix and water to a total
volume of 13 ml were heated to 65º C for 5 minutes, followed by 1 minute incubation on ice. To
each sample 4 ml 5X First-strand buffer, 1 ml 0.1 M DTT, 1 ml RNaseOUT and 1 ml
SuperScript III reverse transcriptase (RT) (Thermo-fisher Scientific, Leicestershire, UK) were
added and incubated at 25º C for 5 minutes then 50º C for 60 minutes followed by heating at 70º
C for 15 minutes. Custom primers for wild-type ER-β were ordered from Thermo-Fisher
Scientific based on published primer sequence data validated for use in qRT-PCR (44). Alternate
ER-β primers and actin primers were obtained from Origene. Each qRT-PCR reaction contained
7.5 ml Power SYBR Green PCR Master Mix (Applied Biosystems, California USA), 0.5 ml of
10 mM primer mix, 2 ml of a 1:5 dilution of cDNA and nuclease-free water to a final volume of
15 ml. Reactions were performed with the Stratagene Mx3005P RealTime machine in triplicate.
Hot-start Taq polymerase was heat-activated at 95º C for 10 minutes followed by 40 cycles of 15
s at 95º C and 30 s at 60º C. Fluorescence was read in each cycle and a melting curve constructed
as the temperature was increased from 65º C to 95º C with continuous fluorescence readings.
Actin was used as a control gene to normalize between the samples and relative expression
determined using the delta-delta Ct method (47). Statistical significance for cell line and
transfection data was determined by 2-way ANOVA with multiple comparisons and the
21
Student’s two-tailed t-test, respectively.
Bioinformatics Analysis
Microarray analysis for mRNA expression of the ESR2 gene was identified among
differential tissue types using the cBio cancer genomics portal, an open-access resource for
interactive exploration of multidimensional cancer genomics data sets, which provides access to
data from more than 5,000 tumor samples from 20 cancer studies (48, 49).

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Results:
Western Blotting
To validate previous claims made in the literature, western blots were performed testing
various commercially available antibodies with cell lines that have been reported to express ER-
β (figures 2-1, 2-2, 2-3, and 2-4). The results showed some antibodies as less specific, showing
non-specific binding to proteins of varying molecular weight, with only one shown to be highly
specific for the positive control engineered lysate. The antibody that had the most success
identifying ER-β is the 4D2 antibody from Thermo-Fisher. This mouse monoclonal antibody
confirmed suspicions that cell lines previously reported as positive, such as the 273T lung cancer
cell line (50), for ER-β expression are, in fact, negative, since only the transfected HEK293 line
with the ER-β expression vector resulted in a positive band (figure 2-2). This demonstrated
specificity for the protein, and the reportedly ER-β positive breast cancer cell line MCF-7 (36,
51), appears negative for ER-β by western blot as well.
When used in western blotting, the 4D2 antibody identifies a protein in the positive
control overexpression lysate, as well as a protein of the predicted molecular weight for ER-β
(~59 kDa) in the MDA-MB-453 cell line (figure 2-5). This antibody demonstrates a highly
specific antigenic targeting of a protein whose molecular weight coincides with the predicted
molecular weight of ER-β. In addition, these results implicated this protein’s lack of expression
in samples, excluding MDA-MB-453 and the overexpression lysate, as compared to MC10’s
results which demonstrates positivity.
Because 4D2 was successful in identifying an endogenous protein corresponding to ER-
β’s predicted molecular weight (figures 2-5 and 2-13), MC10 is likely detecting a different
protein in samples, such as SK-BR-3 and MDA-MB-436 (figures 2-6 and 2-12). MC10 yielded
23
consistently positive results, for a protein of slightly larger molecular weight than ER-β’s
predicted ~59 kDa (figures 2-6, 2-7, and 2-8). MC10 western blot results displayed non-specific
binding to proteins of a larger molecular weight (~65 kDa), more visibly apparent by higher
fluorescent intensity (figure 2-8). In contrast, using 4D2 generates a band of the appropriate size
for both the engineered lysate and MDA-MB-453 (figure 2-5). MC10 even detects this larger
protein in the negative control cell line, MDA-MB-231; this is more easily seen in the
black/white image. Collectively, these data suggest MC10’s efficacy in recognizing ER-β may be
hindered by off-target binding of other proteins.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on samples using the 4D2 antibody as well
as the MC10 antibody. Further confirmation of 4D2’s specificity to ER-β was demonstrated by
IHC data which showed differential staining between MDA-MB-453 cells and other cell lines
like SK-BR-3 and MDA-MB-436 (figure 2-13). In addition to detecting a larger protein by
western blot, IHC data for MC10 show a prominent amount of cytoplasmic staining in various
cell lines for a nuclear hormone receptor (figure 2-12).
qRT-PCR
In order to validate ER-β expression, or lack thereof, qRT-PCR was performed as an
antibody-independent method of analyzing mRNA expression for the ESR2 gene corresponding
to ER-β’s peptide sequence. The qRT-PCR data indicated significantly high mRNA levels in the
MDA-MB-453 cell line, while other cell lines, like SK-BR-3 and MDA-MB-231, yielded low
amounts of mRNA corresponding to the ESR2 gene (figure 2-14). When using template primers
from Origene, high mRNA levels for ER-b were detected in the lymphoid cell lines DB,
24
KARPAS 422, KU812, and SU-DHL-5. When custom primers used by Jason Carroll (44) were
tested in these same cell lines, low mRNA levels were seen. Lastly, the transient transfection was
successful due to mRNA detection increasing ~600 fold in transfected MDA-MB-231 cells.
Bioinformatics Analysis
Publically available expression array data was analyzed which employed the use of
probes to detect mRNA corresponding with the ESR2 gene in cancer cell lines across 24 different
tissues, all of which were organized in a single database (48, 49). The lymphoid cell lines DB,
KARPAS 422, KU812, and SU-DHL-5 demonstrated high levels of ER-b mRNA relative to
other tissue types.

25
Tables:
Table 2-1: List and properties of various antibodies screened for ER-β specificity
Antibody Immunogen Host
Species
Class Binding
Region
Application
PPG 5/10 Synthetic peptide
C terminus of wt
ER-β
Mouse Monoclonal C terminus IF, IHC, WB
4D2 Amino acids 1-153
of human ER-β
expressed in E.
coli
Mouse Monoclonal N terminus IHC, WB
EPR3777
Synthetic peptide
corresponding to
the N terminus of
ER-β
Rabbit Monoclonal N terminus IP, WB
68-4 KLH-conjugated
linear peptide
corresponding to
amino acids 108-
127 from the N-
terminal half of
ER-β
Rabbit Monoclonal N terminus IP, WB

26
Antibodies
Table 2-2: Western blot and immunohistochemistry results for both 4D2 and MC10 antibodies

4D2/MC10 4D2/MC10
Overexpression lysate +/+ +/+
Transfected negative control -/- -/-
HCC70* -/+ -/+
EFM19 -/+ -/+
JIM-T1 -/+ -/+
MDA-MB-436* -/+ -/+
SK-N-SH -/+ -/+
MDA-MB-134 -/+ -/+
MDA-MB-157* -/+ -/+
MDA-MB-468 -/+ -/+
SK-BR-3 -/+ -/+
MDA-MB-453* +/+ +/+
UACC812 -/+ -/+
Samples IHC WB
27
HCC1419 -/+ -/+
HCC2218* -/+ -/+
HCC1187* -/+ -/+
HCC38* -/+ -/+
BT474 -/+ -/+
MCF-7 -/+ -/+
T47D -/- -/-
MDA-MB-231* -/- -/-

* Indicates triple-negative breast cancer cell line

28
Figures:
Figure 2-1: Western blot using PPG 5/10 primary antibody against ER-β

anti-ER-β clone PPG5/10, 1:1000
M 3 1 2 4 M 3 1 2 4
1: ER-β overexpression lysate
2: transfection control
3: MCF-7
4: 273T
75 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
Figure 2-1: Western blot using PPG 5/10 primary antibody against ER-β. Lanes were
loaded with the following cell lysates, respectively: an ER-β transient overexpression
positive control from HEK293 cells, an empty vector transfected negative control from
HEK293 cells, MCF-7, and 273T. Using this antibody, protein was detected in both the
positive and negative control at a higher molecular weight than what is predicted for ER-β
(~59 kDa) (Left side: color, right side: black/white).

29
Figure 2-2: Western blot using 4D2 primary antibody against ER-β

M 3 1 2 4 M 3 1 2 4
1: ER-β overexpression lysate
2: transfection control
3: MCF-7
4: 273T
75 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
anti-ER-β clone 4D2, 1:1000
Figure 2-2: Western blot using 4D2 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an ER-β transient overexpression positive
control from HEK293 cells, an empty vector transfected negative control from HEK293
cells, MCF-7, and 273T. Using this antibody, protein was detected in only the positive
control lane at the appropriate molecular weight for ER-β’s predicted ~59 kDa with
minimal non-specific binding (Left side: color, right side: black/white).

30
Figure 2-3: Western blot using EPR3777 primary antibody against ER-β

anti-ER-β clone EPR3777, 1:1000
M 3 1 2 4 M 3 1 2 4
1: ER-β overexpression lysate
2: transfection control
3: MCF-7
4: 273T
250 kDa
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
Figure 2-3: Western blot using EPR3777 primary antibody against ER-β. Lanes were
loaded with the following cell lysates, respectively: an ER-β transient overexpression
positive control from HEK293 cells, an empty vector transfected negative control from
HEK293 cells, MCF-7, and 273T. Using this antibody, protein corresponding to ER-β’s
predicted molecular weight (~59 kDa) was detected in both the positive and negative
control, although at a much fainter level in the negative control, as well as MCF-7 (Left
side: color, right side: black/white).

31
Figure 2-4: Western blot using 68-4 primary antibody against ER-β

anti-ER-β clone 68-4, 1:1000
M 3 1 2 4 M 3 1 2 4
1: ER-β overexpression lysate
2: transfection control
3: MCF-7
4: 273T
250 kDa
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
Figure 2-4: Western blot using 68-4 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an ER-β transient overexpression positive
control from HEK293 cells, an empty vector transfected negative control from HEK293
cells, MCF-7, and 273T. Using this antibody, results were unclear, with prominent non-
specific binding to proteins of varying molecular weights occurring in all samples tested
(Left side: color, right side: black/white).

32
Figure 2-5: Western blot using 4D2 primary antibody against ER-β in breast cancer cell lines,
including MDA-MB-231 transiently transfected with an expression vector which encodes for the
ER-β peptide sequence

80 kDa
20 kDa
15 kDa
M 2 1 3 4
anti-ER-β clone 4D2, 1:1000
1: Blank
2: MDA-MB-231
3: MDA-MB-231, expression vector
4: MDA-MB-453
5: SK-BR-3
6: MDA-MB-436
7: JIM-T1
8: HCC1419
9: Blank
5 6 7 8 9
60 kDa
42 kDa
Green: ER-β,approximately 59 kDa
M: Marker
Red: β-Actin, 42 kDa
Figure 2-5: Western blot using 4D2 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: blank (no protein lysate loaded), MDA-MB-
231, MDA-MB-231 transiently transfected with an ER-β expression vector, MDA-MB-
453, SK-BR-3, MDA-MB-436, JIM-T1, HCC1419, blank (no protein lysate loaded). Using
this antibody, protein corresponding to ER-β’s predicted molecular weight (~59 kDa) was
detected in MDA-MB-453 cells, with no non-specific interactions elsewhere.

33
Figure 2-6: Western blot using MC10 primary antibody against ER-β in breast cancer cell lines,
testis tissue, and prostate tissue

1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: SK-BR-3
7: HCC1419
8: MDA-MB-436
9: JIMT1
10: T47D
11: MCF-7
12: HCC1187
13: Blank
14: Blank
ER-β: approximately 59 kDa
M: Marker
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
M 3 1 2 4 5
anti-ER-β clone MC10, 1:1000
6 7 8 9 10 11 12 13 14
1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: SK-BR-3
7: HCC1419
8: MDA-MB-436
9: JIMT1
10: T47D
11: MCF-7
12: HCC1187
13: Blank
14: Blank
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
M 3 1 2 4 5
anti-ER-β clone MC10, 1:1000
6 7 8 9 10 11 12 13 14
ER-β: approximately 59 kDa
M: Marker
Figure 2-6: Western blot using MC10 primary antibody against ER-β. Lanes were
loaded with the following cell lysates, respectively: an ER-β transient overexpression
positive control from HEK293 cells, an empty vector transfected negative control from
HEK293 cells, testis tissue, prostate tissue, HCC70, SK-BR-3, HCC1419, MDA-MB-
436, JIM-T1, T47D, MCF-7, HCC1187, blank (no protein lysate loaded), and blank (no
protein lysate loaded). Using this antibody, protein of a slightly larger molecular weight
than ER-β was detected in each breast cancer cell line tested, except for T47D (Top:
color, bottom: black/white).

34
Figure 2-7: Western blot using MC10 primary antibody against ER-β in additional breast cancer
cell lines, testis tissue, and prostate tissue

150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
M 2 1 3 4
anti-ER-β clone MC10, 1:1000
1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
5 6 7 8 9
ER-β: approximately 59 kDa
M: Marker
M 2 1 3 4
anti-ER-β clone MC10, 1:1000
5 6 7 8 9
1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
Figure 2-7: Western blot using MC10 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an ER-β transient overexpression positive
control from HEK293 cells, an empty vector transfected negative control from HEK293
cells, testis tissue, prostate tissue, HCC70, MDA-MB-468, SK-N-SH, MCF-7, and MDA-
MB-231. Using this antibody, protein of a slightly larger molecular weight than ER-β was
detected in each breast cancer cell line tested (Top: color, bottom: black/white).

35
Figure 2-8: Western blot using MC10 primary antibody against ER-β in additional breast cancer
cell lines, testis tissue, and prostate tissue (overexposed fluorescence intensity)

M 2 1 3 4
anti-ER-β clone MC10, 1:1000
5 6 7 8 9
1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
M 2 1 3 4
anti-ER-β clone MC10, 1:1000
5 6 7 8 9
1: ER-β overexpression lysate
2: transfection control
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
ER-β: approximately 59 kDa
M: Marker
Figure 2-8: Western blot using MC10 primary antibody against ER-β (overexposed
fluorescence intensity). Lanes were loaded with the following cell lysates, respectively:
an ER-β transient overexpression positive control from HEK293 cells, an empty vector
transfected negative control from HEK293 cells, testis tissue, prostate tissue, HCC70,
MDA-MB-468, SK-N-SH, MCF-7, and MDA-MB-231. Using this antibody, protein of a
slightly larger molecular weight than ER-β was detected in each breast cancer cell line
tested (Top: color, bottom: black/white).

36
Figure 2-9: Western blot using 4D2 primary antibody against ER-β in breast cancer cell lines,
testis tissue, and prostate tissue

M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
1: transfection control
2: ER-β overexpression lysate
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
6 7 8 9
ER-β: approximately 59 kDa
M: Marker
M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
1: transfection control
2: ER-β overexpression lysate
3: testis tissue lysate
4: prostate tissue lysate
5: HCC70
6: MDA-MB-468
7: SK-N-SH
8: MCF-7
9: MDA-MB-231
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
6 7 8 9
ER-β: approximately 59 kDa
M: Marker
Figure 2-9: Western blot using 4D2 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an empty vector transfected negative control
from HEK293 cells, an ER-β transient overexpression positive control from HEK293 cells,
testis tissue, prostate tissue, HCC70, MDA-MB-468, SK-N-SH, MCF-7, and MDA-MB-
231. Using this antibody, protein was only detected in the positive control, while no other
proteins corresponding to ER-β’s predicted molecular weight (~59 kDa) were detected in
each breast cancer cell line tested (Top: color, bottom: black/white).

37
Figure 2-10: Western blot using 4D2 primary antibody against ER-β in additional breast cancer
cell lines

M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
1: ER-β overexpression lysate
2: transfection control
3: UACC812
4: MDA-MB-134
5: SK-BR-3
6: EFM19
7: HCC38
8: HCC1419
9: BT474
75 kDa
25 kDa
6 7 8 9
ER-β: approximately 59 kDa
M: Marker
M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
1: ER-β overexpression lysate
2: transfection control
3: UACC812
4: MDA-MB-134
5: SK-BR-3
6: EFM19
7: HCC38
8: HCC1419
9: BT474
75 kDa
25 kDa
6 7 8 9
ER-β: approximately 59 kDa
M: Marker
Figure 2-10: Western blot using 4D2 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an ER-β transient overexpression positive
control from HEK293 cells, an empty vector transfected negative control from HEK293
cells, UACC812, MDA-MB-134, SK-BR-3, EFM19, HCC38, HCC1419, and BT474. Using
this antibody, protein was only detected in the positive control, while no other proteins
corresponding to ER-β’s predicted molecular weight (~59 kDa) were detected in each breast
cancer cell line tested (Top: color, bottom: black/white).

38
Figure 2-11: Western blot using 4D2 primary antibody against ER-β in additional breast cancer
cell lines

M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
1: ER-β overexpression lysate
2: transfection control
3: HCC1187
4: ZR-75-1
5: JIM-T1
6: HCC2218
7: MDA-MB-436
8: T47D
9: MDA-MB-157
75 kDa
25 kDa
6 7 8 9
ER-β: approximately 59 kDa
M: Marker
M 3 1 2 4 5
anti-ER-β clone 4D2, 1:1000
75 kDa
25 kDa
6 7 8 9
1: ER-β overexpression lysate
2: transfection control
3: HCC1187
4: ZR-75-1
5: JIM-T1
6: HCC2218
7: MDA-MB-436
8: T47D
9: MDA-MB-157
ER-β: approximately 59 kDa
M: Marker
Figure 2-11: Western blot using 4D2 primary antibody against ER-β. Lanes were loaded
with the following cell lysates, respectively: an ER-β transient overexpression positive
control from HEK293 cells, an empty vector transfected negative control from HEK293
cells, HCC1187, ZR-75-1, JIM-T1, HCC2218, MDA-MB-436, T47D, and MDA-MB-
157. Using this antibody, protein was only detected in the positive control, while no other
proteins corresponding to ER-β’s predicted molecular weight (~59 kDa) were detected in
each breast cancer cell line tested (Top: color, bottom: black/white).

39
Figure 2-12: Immunohistochemical stains of various cell lines using MC10 antibody at 1:100
concentration

Negative
control:
MDA-
MB-231
MDA-
MB-436
SK-BR-3
Positive
control:
MDA-
MB-453
4X 40X 60X
Figure 2-12: Immunohistochemistry using MC10 primary antibody against ER-β in the
following paraffin-embedded cell lines: MDA-MB-453, MDA-MB-231, SK-BR-3, MDA-
MB-436. In each cell line tested, there is a varying degree of cytoplasmic staining present
for this antigen, despite being a nuclear hormone receptor.

40
Figure 2-13: Immunohistochemical stains of various cell lines using 4D2 antibody at 1:100
concentration

Negative
control:
MDA-
MB-231
MDA-
MB-436
SK-BR-3
Positive
control:
MDA-
MB-453
4X 40X 60X
Figure 2-13: Immunohistochemistry using 4D2 primary antibody against ER-β in the
following paraffin-embedded cell lines: MDA-MB-453, MDA-MB-231, SK-BR-3, MDA-
MB-436. Positive staining resembling nuclear morphology is observed for MDA-MB-453
cells, while negative staining is observed for MDA-MB-231, SK-BR-3, and MDA-MB-
436 cells.

41
Figure 2-14: qRT-PCR data detailing mRNA relative expression among cell lines

MDA-MB-231
MDA-MB-453
SK-BR-3
DB
KARPAS 422
KU812
SU-DHL-5
0
20
40
60
80
Cell Type
ER-β mRNA Fold Change
(Normalized to Actin)
ER-β qRT-PCR
ER-β
Custom
***
***
***
***
***
***
Figure 2-14: qRT-PCR using custom primers for wild-type ER-β, as well as primers for
wild-type ER-β and actin primers from Origene. All three primer sets were used with
extracted mRNA in the following cell lines: MDA-MB-231, MDA-MB-453, SK-BR-3,
DB, KARPAS 422, KU812, SU-DHL-5, and MDA-MB-231 transiently transfected with
an ER-β expression vector. High levels of ER-β mRNA are seen in MDA-MB-453, DB,
KARPAS 422, KU812, and SU-DHL-5 cells. Low amounts of ER-β mRNA are expressed
in MDA-MB-231 and SK-BR-3 cells. In transfected MDA-MB-231 cells, ~600 fold ER-β
mRNA was detected compared to non-transfected MDA-MB-231 cells.

‘***’ indicates statistical significance compared to the minimum mRNA
expression value with a p<0.001

MDA-MB-231 MDA-MB-231, Transfected
0
200
400
600
800
ER-β mRNA Fold Change
(Normalized to Actin)
MDA-MB-231 ER-β Transiently Transfected Expression Vector
***
42
Chapter 3: Discussion and Conclusions
The Search for Endogenous ER-β Amidst Contradictory Literature Sources:
A multi-faceted approach to characterize cell lines using both antibody-dependent
(western blotting and immunohistochemistry) and antibody-independent (qRT-PCR) approaches
has shown that multiple cell lines do not express detectable ER-b, despite numerous publications
making conclusions about ER-β biology using the same cell line models (13, 14, 17, 36, 37, 52,
53, 54, 55, 56, 57, 58, 59, 60).
The search for a positively expressed endogenous source of ER-β was misleading at
times because many publications implicate ER-β positivity in breast cancer lines, like MCF-7
(36, 51). However, bands on western blots were present at inappropriate molecular weights as
well as other non-specific bands to the ER-β protein (figures 2-1, 2-2, 2-3, and 2-4). Once this
antibody screening process was completed, a paper was published which verified our findings
with regard to PPG 5/10 preferentially binding a protein of a higher molecular weight than ER-β
(44). We then began to investigate a particular antibody because of their RIME data which
demonstrated its effectiveness in recognizing ER-β. The antibody, called MC10, was shown to
detect ER-β with the highest frequency compared to other antibodies tested (44).
MC10 was a top candidate to identify endogenous ER-β because of Jason Carroll’s work.
However, retrospective analysis of immunohistochemistry and western blotting results using
4D2 demonstrated abundant negativity (figures 2-5, 2-9, 2-10, 2-11 and 2-13). We decided to
pursue this further, with the idea that MC10 might have been identifying another protein of a
larger molecular weight (~65 kDa) (figures 2-6, 2-7, 2-8, and 2-12). The idea that MC10 is not
detecting ER-β in these cell lines, and is recognizing a different protein of similar molecular
43
weight, is reinforced by data using Thermo Scientific’s 4D2 antibody.
For a long period of time, the search for an endogenous source had been an obstacle in
validating the 4D2 antibody due to the nature of publications falsely suggesting its positivity in
various tissues ranging from breast to lung; however, 4D2’s ability to identify ER-β in MDA-
MB-453 effectively demonstrates that 4D2 can indeed detect endogenous ER-β in cell lines. And
the negative results it yields in breast cancer cell lines do, in fact, correlate with mRNA data
indicating little to no expression of the ESR2 gene mRNA in a majority of breast cancer cell line
tested (figure 3-3). The results obtained using the 4D2 antibody correlate with these microarray
data for the following cell lines: HCC70, EFM19, JIM-T1, MDA-MB-436, SK-N-SH, MDA-
MB-134, MDA-MB-157, MDA-MB-468, SK-BR-3, UACC812, HCC1419, HCC2218,
HCC1187, HCC38, BT474, MCF-7, T47D, MDA-MB-231, and 273T. Taken together, these
data are evidence supporting 4D2’s competency over MC10, with higher specificity for the ER-β
antigen.
Our data clearly demonstrate that while MC10 may recognize ER-β with the highest
affinity, it detects another protein, or multiple different proteins, of slightly larger molecular
weight (~65 kDa) (figures 2-6, 2-7, and 2-8). Prominent cytoplasmic staining in cell lines
provides additional support for this claim as the antigenic target is a nuclear hormone receptor
(figure 2-12). Importantly, these results are not due to background generated by improper
immunohistochemistry technique. This is because the same results were generated by the
developers of this antibody at the Mayo Clinic in Minnesota (35). The developers of the antibody
also detected antigen in the cytoplasm as shown by their sub-cellular localization data.

44
A Comparison of Our Data with Those from Other Studies:
There have been conflicting reports on estrogen receptor-β expression among the
scientific community in recent years. It has been an elusive antigen as antibody reagents used to
identify its expression in various cell lines and tissues have been shown to be non-specific for the
protein product (44). One such example is the widely-used antibody from Bio-Rad, named PPG-
5/10, a monoclonal mouse antibody. Jason Carroll’s group at the University of Cambridge used a
novel proteomic-based pull down method called Rapid Immunopreciptation Mass spectrometry
of Endogenous protein (RIME). RIME uses an antibody-based purification followed by mass
spectrometry (MS) to identify enriched peptides. They conducted RIME in transfected ER-β
positive MDA-MB-231 and ER-β negative, wild-type MDA-MB-231 cells via doxycycline-
inducible vector using 8 different antibodies. In order to illustrate respective antibody specificity,
they ranked all the proteins purified by immunoprecipitation and identified by mass
spectrometry. This system assigned each antibody a ranking which correlated with its specificity
for ER-β against other proteins. If, for example, an antibody ranks ER-β as having the greatest
number of unique peptides relative to all other proteins, ER-β is ranked first.
They used RIME to determine the PPG 5/10 antibody’s ability to detect ER-β in relation
to other proteins. This technique utilizes immunoprecipitation and an algorithm designed to
analyze the frequency of the antibody in question binding to the various proteins in a given
lysate. Jason Carroll used a doxycycline-inducible vector encoding ER-β’s nucleotide sequence
which was transfected into MDA-MB-231 cells to accurately determine this information from
various antibodies. The system ranked PPG 5/10’s ability to detect ER-β as number three, behind
two other proteins, which would explain the antibody identifying a different protein of larger
molecular weight in our samples, as well as Jason Carroll’s. Our data supports Jason Carroll’s
45
report that PPG 5/10 recognizes a different protein with higher specificity than ER-β with a
larger molecular weight (figure 2-1). His group was also able to verify a different antibody as
being more competent in identifying ER-β with higher specificity, called MC10 from Mayo
Clinic (35, 44).
Our data with regard to MC10’s efficacy in detecting the ER-β protein verifies Jason
Carroll claims; it is able to detect the ER-β protein in the overexpressed lysate (figures 2-6, 2-7,
and 2-8). Jason Carroll’s RIME data ranks MC10 as identifying ER-β with the highest frequency
compared to any other protein in the lysate. However, MC10 binds a protein of larger molecular
weight than ER-β (~65 kDa) in nearly every breast cancer cell line tested, except for T47D,
where no band is detected (figure 2-6). Jason Carroll’s group never expressed ER-β as the only
protein MC10 would bind to, and, as it turns out, it detects a protein of a larger molecular weight
in nearly every breast cancer cell line tested (figures 2-6, 2-7, and 2-8).
MC10 even detects this larger protein in the negative control cell line, MDA-MB-231, by
western blot and immunohistochemistry. On a western blot, this result is more easily visualized
in the black/white image (figure 2-7). This is a cell line validated as negative by results presented
here using 4D2 (figure 2-5), qRT-PCR data (figure 2-14), and even Jason Carroll’s qRT-PCR
analysis of mRNA expression of the ESR2 gene for this cell line (44). However, Jason Carroll’s
data using MC10 on a western blot only depicts results in color, when a faint band is indeed
present at that same molecular weight of ~65 kDa when the same black and white image is
visualized.
A critical component of an antibody reagent is its specificity for the protein in question, not
only its ability to detect it or not. MC10, by Mass Spectrometry and RIME analysis, has been
demonstrated to recognize ER-β (44). But, importantly, it has not been shown to not cross-react
46
with other proteins, and in the case of breast cancer cell lines, one with a molecular weight
slightly larger than ER-β’s.
Jason Carroll’s published work provided sufficient evidence that MCF-7 does not express
ER-β (44). His group also demonstrated that previous data which implicated ER-β as positive in
various cell lines and tissues were likely false-positive results. Since then, it has been challenging
to identify a reputably positive sample for endogenous expression of ER-β. What Jason Carroll’s
group did was essentially disprove previous reports, without providing data on an endogenous
ER-β expressing source.

47
The Foundation Our Results Create:
As a validated assay for ER-β detection, the data presented here are an important advance
in addressing technical issues, which require attention for future studies. Those of which aim to
assess ER-β expression in different tissues, as well as general ER-β biological action, will benefit
from the assay developed in this study. In characterizing breast cancer cell lines with this assay,
the identification of a positive result by western blot in MDA-MB-453, a triple negative breast
cancer line, is a first step in designating ER-β as a potential therapeutic target in triple negative
breast cancer patients.
From the data presented, ER-β is low or absent in 18 breast cancer cell lines, and its
expression is undetectable by western blot in 7 triple negative breast cancer cell lines. The results
presented here are sufficient in the development of a standardized method of assessing for high
levels of ER-β expression in future assays from a given tissue type.
Breast cancer patients with ER-α, PR, or HER2 expression are candidates for various
types of clinical intervention, such as anti-estrogen therapies or antibodies against HER2. For
TNBC patients, these types of therapies cannot be effectively administered. In triple negative
breast cancers, 7 results generated with this validated reagent were negative, while MDA-MB-
453 gave a positive result (table 2-2). According to the data presented, ER-β may be a possible
target for TNBC patients with a similar genotype to that of MDA-MB-453. Indeed, Tamoxifen
may be effective in these patients as it is a hormonal intervention therapy which is prescribed to
ER-α positive patients. As a steroid hormone receptor which binds estrogen, similar to that of
ER-α, inhibiting the action of estrogens may demonstrate clinical benefit in these patients (61). If
a TNBC patient expresses ER-β like MDA-MB-453, a triple negative cell line, ER-β could be
playing a significant role in cell growth and proliferation, among other oncogenic characteristics.
48
Further work is required to identify predictive markers, in line with an MDA-MB-453
resembling genotype, which will confer sensitivity to anti-estrogen therapies like Tamoxifen in
such patients.
Fortunately, some of the confusion surrounding ER-β’s detection has been resolved. It
thus follows that the most pressing data generated is of those from triple negative lines to
implicate a possible therapy for patients with triple negative breast cancer phenotypes. Since the
discovery of ER-β, its involvement in the origin, treatment, and progression of breast cancer has
been highly elusive. Because of this, its clinical utility lacks effective standardization. This
directly contributes to the highly variable results in identifying this protein of interest across
different tissues. Another crucial factor that has led to these inconsistencies is the use of
disreputable, poorly characterized, antibodies in measuring ER-β expression.

49
An Analysis of our Process:
One goal of our research was to identify a monoclonal antibody that recognizes ER-β
with high specificity. In antibody screening/selection, western immunoblots were performed to
assess specificity for ER-β with an appropriate molecular weight (~59 kDa) to identify the
commercially available reagent with high affinity for ER-β, and low affinity for other proteins.
These assays were performed comparing 4 commercially available anti-ER-β antibodies: Thermo
Scientific’s clone PPG 5/10, Abcam’s clone EPR3777, EMD Millipore’s clone 68-4, and
Thermo Scientific’s clone 4D2. Accordingly, 4 immunoblots were performed with the same
samples, but different antibodies. The samples tested were a positive control (an estrogen
receptor-β overexpression lysate from Origene engineered from HEK293T cells via
transfection), a negative control (transfection control from Origene), a commonly used breast
cancer cell line (MCF-7), and a lung cancer cell line (273T) previously shown to express ER-β
(50)
Another goal was to accurately characterize ER-β in breast cancer cell lines and tissues
by using a specific anti-ER-β antibody to perform western immunoblot analysis and
immunohistochemical assays. To confirm the validity of studies indicating frequent expression in
uterine and testis tissues, we used tissue lysates in western blots (figures 2-6, 2-7, 2-8, and 2-11)
to test for any positive results. According to our western blot results using the 4D2 antibody,
there was no detection of protein in these tissues with the appropriate molecular weight, despite
being reported as positive (35, 43, 44). Once obtained, these data provided additional insight on
the ability of these various reagents to detect, or with equal importance, not detect, ER-β’s
presence in control samples, as well as samples to test for endogenous activity.
50
Here, we describe a standardized method of approach with a reliable antibody to measure
ER-β expression. We hypothesized that the majority of controversial data with regard to ER-β’s
role in breast cancer progression and therapeutic responses was mainly due to the scarcity of
commercial antibodies with high specificity for this receptor. We ultimately intended to
characterize expression and investigate the role of estrogen receptor-β (ER-β) in breast cancer.
Due to the confusion in the ER-b field and concern stemming from variable and likely non-
specific reagents, we sought to extensively validate antibodies that did not rely upon previous
data regarding ER-β expression in cell lines. To generate a positive control, we used cell line
systems with a transfected plasmid containing a CMV promoter to express the ER-b protein,
which allowed me to test the 4D2 antibody in ER-b negative and matched ER-b positive
conditions. The increase of ER-b mRNA in MDA-MB-231 transfected and non-transfected cells
was confirmed by qRT-PCR (figure 2-14). Western blots of ER-b positive MDA-MB-231 and
ER-b negative MDA-MB-231 cell lysates with the 4D2 anti-ER-b antibody were performed as
well, however results were negative, likely because cells required longer incubation time post-
transfection.
In order to reliably detect ER-β, it was important to establish positive and negative
controls of our own, despite the lack of reputable sources that indicate what those could be. In
addition, extensive validation of an endogenous source was required, notably, the cell line MDA-
MB-453. This cell line is ER-β positive by western blot and immunohistochemistry using 4D2
(figures 2-5 and 2-13). In order to further validate these results, we employed qRT-PCR, an
antibody-independent method of verification of ER-β mRNA presence (figure 2-14).
Importantly, in screening various antibodies from commercial vendors, the PPG 5/10
antibody was found to recognize a protein band of 77 kDa when the actual molecular weight of
51
the protein is approximately 59 kDa. This suggests the detection of a non-specific protein
different than ER-β. Similarly, the MC10 antibody identifies ER-β in the overexpression lysate
and not the negative control, confirming its ability to detect ER-β in line with Jason Carroll’s
Mass Spec + RIME analysis (figure 3-2). However, this does not correlate with its specificity to
ER-β in other cell lines. The protein frequently recognized by MC10 is of larger molecular
weight (~65 kDa) than ER-β’s predicted molecular weight of ~59 kDa.

52
Conclusions:
Our results have demonstrated marked variation in the ability of commonly used,
commercially available ER-β antibodies to accurately detect ER-β by western blotting and
immunohistochemistry. Most notably, MC10, an antibody shown to target ER-β with the highest
frequency in a protein lysate by Mass Spectrometry and RIME analysis (44) did not detect ER-β
in breast cancer cell lines by any methodological approach used in this study. It did, however,
consistently yield bands on western blots of a larger size (~65 kDa) than ER-β (~59 kDa) in all
tested conditions (figures 2-6, 2-7, and 2-8). We have confirmed that this protein band does not
represent ER-β in each cell line tested through the use of another antibody, 4D2. We successfully
demonstrated endogenous ER-β positivity in MDA-MB-453 using this antibody (figure 2-5),
validated by qRT-PCR (figure 2-14), an antibody-independent method. Negative results
generated using 4D2 with cell lines like MDA-MB-231 and SK-BR-3 were validated by qRT-
PCR as well. Notably, because 4D2 detects a band of appropriate molecular weight in an
endogenous source, and not in any other cell line tested, MC10 is likely detecting a different
protein in these cell lines. Otherwise, a band would be present for each respective cell line
recognized by MC10 as positive in western blots using 4D2.
Our findings with regard to ER-β expression emphasize the attention that must be paid to
proper validation of antibodies for individual experimental approaches, rather than false
assumptions stemming from previous literature. Inadequate reagents which lack validation led
researchers to publish contradictory data on ER-β’s involvement in tissues, and more
specifically, cancer. Investigators have shown ER-β to be anti-proliferative, but also tumorigenic
in function (11, 12, 13, 14, 15). Yet, many studies utilize reagents, such as Thermo Scientific’s
PPG 5/10 monoclonal antibody, to identify ER-β in cell lines and tissues. It is noteworthy that
53
reagents like this are prone to off-target specificity for different proteins of differing molecular
weights than ER-β (44). These are some of the technical issues that require attention in order to
fully understand ER-β in the context of cancer.
This reality highlights the need to develop a more reliable and consistent strategy to
measure ER-β expression. By doing so, it will enable researchers to accurately depict the role of
this protein in breast cancer pathogenesis. In working toward this goal, we have engaged a
process using a controlled cell model system and approach to verify that certain commercially
available ER-β antibodies are non-specific for antigenic detection. Therefore, erroneous
conclusions are made after analyzing the data for a large number of past publications on ER-β’s
role in various cancers. In order to address this issue, we successfully characterized an
unpublished, highly specific, monoclonal anti-ER-β antibody (4D2).

54
Figures:
Figure 3-1: Validation of ER-b antibodies using doxycycline-inducible MDA-MB-231 cells

Figure 3-1: (A) MDA-MB-231- ER-b cells were treated with doxycycline to induce ER-b
expression. Untreated cells provided an ER-b-negative control. Messenger RNA was extracted for
qRT-PCR and protein for Western blotting. ER-b positive MDA-MB-231and ER-b negative MDA-
MB-231 cells were crosslinked and immunoprecipitated with antibody for RIME. (B) qRT-PCR
confirmed 100-fold induction of ER-b mRNA in ER-b positive MDA-MB-231 cells versus
untreated MDA-MB-231 cells. Data are mean ± S.D. of technical triplicate experiments. (C)
Western blots of ER-b positive MDA-MB-231 and ER-b negative MDA-MB-231 cells with the 8
antibodies undergoing assessment. The MC10, CWK-F12, Abcam 288[14C8] and sc8974
antibodies detected bands of 59 kDa, with differential signal in the ER-b positive versus ER-b
negative conditions, indicating specificity to ER-b. GeneTex 70182 detected ER-b, although there
was non-specific signal at 65 kDa. Millipore 06-629 appears to detect ER-b, although there is also a
59 kDa band in the ER-b negative condition. Review of the RIME data suggests this may be cross-
reactivity with LACTB. NCL-ER-Β, the most commonly used ER-b antibody, gives bands of the
correct size for ER-b, but there is no difference between ER-b positive and ER-b negative
conditions, confirming that this antibody is not specific to ER-b (44).
Reproduced from Molecular and Cellular Endocrinology under the terms of the Creative Commons
Non-Commercial Attribution License.

55
Figure 3-2: Rapid Immunoprecipitation Mass Spectrometry of Endogenous Protein (RIME)
demonstrates specificity and peptide coverage of ER-b antibodies

Figure 3-2: Eight ER-b antibodies were assessed by RIME in ER-b positive and ER-b negative
MDA-MB-231 cells. Coverage of the protein relates to green areas on the peptide maps, indicating
peptides identified by MS with false discovery rate of 1% (mean of 2 biological replicates). (A)
E2F1 antibody was applied to ER-b positive and ER-b negative MDA-MB-231 conditions as a
positive control, as E2F1 is a ubiquitously expressed protein. (B) ER-b antibody tests: ‘ER-b
ranking’ indicates where ER-b features in a list of proteins purified by the antibody, ranked by
number of unique peptides identified in MS, giving an indication of antibody specificity. NCL-ER-
Β failed to identify ER-b. (C) Negative controls: All of the ER-b antibodies were tested in ER-b
negative MDA-MB-231-ER cells, to confirm absence of ER-b expression in the non-induced
condition. Mouse IgG antibodies were used to identify non-specific peptides pulled down by the IP.
None of the IgG antibodies purified ER-b (44).

Reproduced from Molecular and Cellular Endocrinology under the terms of the Creative Commons
Non-Commercial Attribution License.

d

56
Figure 3-3: Relative mRNA expression levels for the ESR2 gene among differential tissue types

Figure 3-3: Microarray analysis depicting relative mRNA expression levels for the ESR2 gene
among different tissue types (48, 49). Among each tissue type shown, hematopoietic and lymphoid
tissues express the highest amount of ER-b mRNA.

57
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Abstract (if available)

Abstract Steroid hormones are lipophilic molecules synthesized in the adrenal cortex, the testes, and the ovaries. Within target cells, steroid hormones bind to steroid hormone receptors, which are intracellular transcription factors, to exert positive or negative effects on the expression of target genes. For example, estrogen regulates the growth and development of breast tissues through the action of estrogen receptor-α (ER-α) and progesterone receptor (PR). There are multiple subgroups of patients' breast cancers depending on their respective phenotypes. Hormonal intervention therapies, such as Tamoxifen, have demonstrated success in treating women with ER-α positive breast cancers. In triple-negative breast cancer (TNBC) patients, estrogen receptor-α, progesterone receptor, and HER2 expression is low or absent (table 1-1). There are currently no ""targeted therapies"" for TNBC. Uncovering mechanisms that drive this specific breast cancer subtype, and the subsequent development of targeted therapies which inhibit them, are priorities in cancer research. Estrogen receptor-β's (ER-β) function is a potential factor in the progression of breast cancer and, therefore, a potential therapeutic target for TNBC patients. For the following study, we investigate ER-β expression in TNBC patients. The hypothesis at the onset was if ER-β expression contributes to a proliferative phenotype, then using a validated antibody to quantify ER-β expression levels will reliably identify breast cancer cell lines and tissues which, upon estrogen stimulation, demonstrate an increase in proliferation. ER-β's expression is investigated by way of immunoblotting analysis combined with immunohistochemistry. With this in mind, Chapter 1 is an introduction and background on ER-β's characteristics, including its relevance to the field of cancer research. This chapter also explores the inconsistencies in published papers of the last two decades. Chapter 2 contains a description of results, as well as the methodology used. Our data simultaneously analyzes antibody specificity among various commercially available antibody reagents for their ability to detect a protein of ~59 kDa, the predicted molecular weight for ER-β. The most favorable antibody, called 4D2, is used to characterize ER-β expression in the context of breast cancer samples, both from cell lysates on an immunoblot, and from formalin-fixed, paraffin-embedded cells. Expression by mRNA is also evaluated by qRT-PCR in breast cancer cell lines and several lymphoma cell lines. Our conclusion is as follows

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Creator LaRue, Ryan (author)

Core Title Estrogen receptor-β characterization in breast cancer: development of a reliable assay for measuring expression

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 02/12/2018

Defense Date 12/21/2017

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag assay,beta,breast cancer,characterization,Estrogen,expression,OAI-PMH Harvest,receptor

Language English

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Advisor Press, Michael (committee chair), Lang, Julie (committee member), Stallcup, Michael (committee member)

Creator Email laruer14@gmail.com,rlarue@usc.edu

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