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The effect of estradiol on TAZ in stromal cell line ST2 in osteoblast differentiation and ER+breast cancer cell

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The effect of estradiol on TAZ in stromal cell line ST2 in osteoblast differentiation and ER+breast cancer cell

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1

The effect of estradiol on TAZ in stromal
cell line ST2 in osteoblast differentiation
and ER+Breast Cancer Cell

By
Sara Alnassar
1

Mentor: Baruch Frenkel
1,2

A Thesis Presented to the
FACULTY OF THE USC GRADUA TE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2017

1 Department of Biochemistry & Molecular Biology, Keck School of Medicine, University
of Southern California, 90033 CA, USA
2 Departments of Orthopaedic Surgery

2

ACKNOWLEDGEMENTS
Completion of my master research project would not be accomplished without
the assistance and guidance provided from my mentor Dr. Baruch Frenkel, hence,
I would like to sincerely thank him for all his support throughout my research
project.
Also I would like to express my deepest appreciation for my second mentor Dr.
Judd Rice for taking over Dr. Baruch Frenkel role as a mentor during the difficult
times and for all his constant guidance, support and patience during last few
months of my research projects. He greatly helped with my defense preparation,
thesis writing and editing.
Also I would like to thank Dr. Nyam-Osor Chimgee for her assistance and
guidance in my second research project and all the members in Dr. Frenkel’s lab
Yonatan, Di, Jie, Gloy for being an amazing team members.
Moreover, sincere appreciation for Dr. Young-Kwon Hong for his valuable
encouragement and being a member of my defense committee.
I would Also like to acknowledge the support of both King Faisal Specialist
hospital and Research Centre and Ministry of Higher Education in Saudi Arabia
for granting me this opportunity to pursue my master degree and I’m honored to
be a recipient of their scholarship program.

3

I would like to express my deepest appreciation to the following:
To Dr. Futwan Almuhanna for all his support before and during my master
degree and for giving me this opportunity.
Also my sincere gratitude to Dr. Sultan Alsudairy for granting me the
opportunity to pursue my studies. Thank you Dr. Ranjit Parhar and all other
KFSHRC members who helped with my scholarship.
Last but not least, the deepest gratefulness to all my family members for all their
support and patience throughout my life.

4

Table of Contents
ACKOWLEDGEMENTS
.....................................................................................................
2

Abstract
..............................................................................................................................
7

Introduction
......................................................................................................................
9

First
Chapter-‐‑
The
effect
of
estradiol
on
TAZ
in
stromal
cell
line
ST2
in

osteoblast
differentiation………………………………………………………………………………………….9

Osteoporosis
..........................................................................................................................
9

Bone
remodelling
and
osteoprosis
.................................................................................
9

Estrogen
effects
on
bone
formation
and
osteoprosis
.............................................
11

RUNX2
role
in
Bone
maturation
and
osteoblastogenesis
.....................................
13

Osteoblast
differentiation
and
bone
formation
regulated
through
TAZ
..........
14

TAZ
induces
osteoblast
differentiation
by
activating

Runx2-‐‑dependant
Expression
of
osteocalcin
gene
.................................................
16

How
wnt
signaling
and
hippo
are
connected
............................................................
18

Results
..............................................................................................................................
20

E2
may
regulate
ostoblast
differentiation
through
the

regulation
of
TAZ
the
Upsstream
effector
of
RUNX2
...............................................
20

5

Indeed,
general
increase
of
TAZ
Expression
Corresponds
to

Estrogen
treatment
in
concentration
dependent
manner...............24

Different
regulation
of
TAZ
expression
by
SERMS
RAL
and
LAS…..27

Second
Chapter-‐‑
Estrogen-‐‑dependent
TAZ
activation
confers
cancer

Stem
cell
related-‐‑traits
and
increased
cell
proliferation
in
ER+BCC…....…….31

Estrogen-‐‑dependent
TAZ
activation
confers
cancer
stem
cell
related-‐‑traits

And
increased
cell
proliferation
in
ER+
BCC
.............................................................
31

Breast
cancer:
......................................................................................................................
31

Relationship
between
Runx1
and
TAZ
in
BCC
and
CSC

........................................
31

Results
..............................................................................................................................
36

RUNX1
knockdown
result
in
TAZ
upregulation
in
Mcf7/Rx1Dox

cells
Upon
dox
treatment
detected
by
western
blot
and
IF...................................36

Materials
and
Methods
................................................................................................
43

Reagents
................................................................................................................................
43

Cell
culture
............................................................................................................................
44

Western
blot
analysis
........................................................................................................
45

Immunofluorescence
........................................................................................................
46

Discussion
........................................................................................................................
47

6

Osteoblast
differentiation
and
bone
formation
regulated
through

TAZ
(
transcriptional
co-‐‑activator
with
PDZ-‐‑binding
motif)
...............................
47

Estrogen-‐‑dependent
TAZ
activation
confers
cancer
stem
cell
related-‐‑traits

and
increased
cell
proliferation
in
ER+
BCC:
.............................................................
50

TAZ
Incorporation
in
the
b-‐‑Catenin
Destruction
Complex
...................................
52

Future
direction
.............................................................................................................
55

Work
Cited
.......................................................................................................................
57

7

Abstract

Estrogens are female hormones that helps to maintain the process of bone
formation by various mechanisms. Estrogen deficiency leads to an imbalance in
bone remodeling process and affects osteoblast differentiation, hence causing
osteoporosis. Runx2 is an Osteoblast specific transcription factor that regulate
osteoblast differentiation process. TAZ is a coactivator transcription factor that
activates Runx2 and direct the differentiation of MSC into osteoblast by
inhibiting MSC differentiation into adipocytes. This project assayed the effects
of E2 on TAZ in ST2 mesenchymal stem cell (MSCs) that can differentiate into
osteoblast, hence understand more the underlying mechanism of MSC
osteoblastogenesis. We used St2 MSC to investigate if TAZ can be the link
between E2 and Runx2 in osteoblast differentiation process. We performed
western blot to observe the changes in protein level of TAZ after treating ST2
cells, which is cultured in CSS-E2 media, with various concentrations of
exogenous estrogen. Immunofluorescence technique was also performed to
further verify the initial observed results and determine the localization and
expression of TAZ in cytoplasm and nucleus. Results showed that E2 treatment at
physiological concentration of 10-10M and 10-9M increased the protein level of

8

TAZ in western blot of cytoplasm and nuclear protein extracts. Also same results
were observed in Immunofluorescence data demonstrating more insight into
molecular pathway and regulation process of osteoblast differentiation.
Understanding the underlying mechanism of osteoblast differentiation can help us
to treat and find a potential therapeutic target for osteoporosis.

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Introduction
First chapter
the effect of estradiol on TAZ in stromal cell line ST2 in osteoblast
differentiation
Osteoporosis
Eight to nine million women in USA suffer from osteoporosis which is the
most common bone disorder in elderly women. Osteoporosis is a bone metabolic
condition in which the bone density is reduced compared to normal one which can
lead to weak bones and fractures. Bone loss process considerably increases after
menopause. Postmenopausal osteoporosis is attributable to insufficient calcium
intake, lack of excises and mainly and most common risk is hormonal changes and
loss of estrogen. [8]
Bone remodelling and osteoprosis
Bone is a complex connective tissue which can be constantly remodeled.
bone’s configuration and content are essential for their mechanical functions
which are essential for body support, tissue protection and calcium storage. Bone
composition consists of four types of cells: osteoblasts, osteocytes, bone lining
cells and osteoclasts. Osteoblasts which line the bone surface develop from
mesenchymal stem cells (MSC) and are essential for bone formation. [1,2]
Osteoclasts are differentiated cells derived from blood-borne monocytes stem
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cell. The processes of bone physiological remodeling require resorption of bone
matrix by osteoclast followed by formation of a new matrix by osteoblasts. This
remodeling process happens throughout the life and it’s essential for skeleton
maintenance and bone hemostasis.
However, an imbalance of bone remodeling process of bone formation and
resorption can lead to several bone-related issues. Bone loss, fractures and
osteoporosis can occur if osteoclast excessively breakdown bone with less
compensatory level of bone formation by osteoblast and vice versa. Thus, the
balance between bone formation and breakdown is highly controlled and
necessary for bone hemostasis. [5]

Figure A: bone remodeling process involves the process by which osteoclasts resorb old
bone and stimulate osteoblasts to make new bone.

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Estrogen’s effect on bone formation and osteoprosis
Estrogen which are known as female steroid hormones are produced by ovaries
and essential for reproductive organ and sexual development. Estrogen has a
complex effect on bone metabolism. Mainly It induce apoptosis of osteoclast
thus it inhibits bone resorption through binding to its intracellular receptors
expressed in bone cells. Estrogen has an essential effect on maintaining bone
mass by regulating bone forming osteoblast and bone-resorbing osteoclast,
therefore E2 deficiency result in increased osteoclast, and enhanced activity of
bone resorption process.
Many studies were conducted to illustrate the effect of E2 on MSCs
proliferation, differentiation process and apoptosis. Former studies conducted of
effect of E2 on ovariectomized (OVX) rats and SHAM showed that
Post-ovariectomy estrogen deficiency, results in bone loss, mainly affecting
trabecular bone. [4]
Estrogen regulates the expression of some factors, such as, β-growth factor
,
platelet-derived growth factor, and morphogenic bone proteins. These factors are
responsible for regulating the differentiation and metabolism of osteoblast, thus
E2 deficiency affects the process of bone formation. Moreover, estrogen
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deficiency leads to increased expression of interleukins 1 and 6, which increase
osteoplastic activity, which subsequently leads to impaired bone remodeling
processes [6,9]
Estrogen can regulate the coupling of osteoblast-osteoclast within BMU.
Estrogen exposure inhibits osteoblast apoptosis thus extending its life span and
keeping the equilibrium between bone resorption and bone formation [8].
This cellular mechanism of apoptosis resistance involves an inhibition of
caspase-3 activity, apoptosis-target genes suppression, such as apoptosis
regulator gene encoding the type 1 InsP3R. That estrogens-mediated suppression
of apoptosis specifically in osteoblast could illustrate the mechanism by which
estrogen therapies can help prevent and treat osteoporosis conducted in clinical
trials [7,8]
Recent evidence illustrated the function of Fas ligand (FasL) in osteoclast
apoptosis induced by estrogen treatment. Two mechanisms where characterized
in which Fasl affects osteoclast survival. an autocrine mechanism via
upregulation of Fasl in osteoclasts and in paracrine mechanisms via upregulation
of FasL in osteoblasts which result in pre-osteoclasts apoptosis. [7]. These
results demonstrate the protective effect of estrogen on bone by two different
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mechanisms that cause death apoptosis of pre-osteoclast and gives an insight into
a possible new therapeutically target for osteoporosis prevention and treatment.
[7 ,8]
It also shows that E2 stimulates the proliferation of MSCs and ER alpha
expression. However, ER beta can have an adverse effect either by directly
inhibiting cell proliferation, or by modulating the expression of Era. [10]
RUNX2 (Runt-related transcription factor 2) role in Bone maturation
and osteoblastogenesis
RUNX2 is the master regulator of bone formation and differentiation of
mesenchymal stem cell into osteoblast. [39]

https://www.researchgate.net/figure/224830071_fig1_Fig-1-Schematic-of-the-retinoblastoma-pathway-and-osteoblast-differentiation
Figure B: Schematic of osteoblast differentiation pathway

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Much of our understanding of underlying mechanism of osteoblast
differentiation comes from study that was conducted by Komori group. Komori
and his colleagues showed that Knockout mice for Runx2 resulted in Skeleton
deficiency and bone formation was completely abolished. This result indicated the
essential role of RUNX2 as a master regulator of this process. [39]
http://biochemistri.es/post/108227836201/q-with-reference-to-experimental-data-discuss
Figure C: Representative figure of the role of the transcription factor complex
Runx2 in the bone formation

Osteoblast differentiation and bone formation regulated through TAZ(
transcriptional co-activator with PDZ-binding motif)
hippo pathway is one of critical developmental pathways that regulate tissue
growth and organ development process. [11]. TAZ/YAP these molecules are
considered as a key regulator of hippo pathway through directly interacting with
TEAD which is involved in cell proliferation and differentiation pathway.
[18,19]
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TAZ and YAP shares sequence homology. TAZ serves as a key regulator of
transcription as it has the ability to bind and interact with various number of
transcription factor regulating embryogenesis, organs development and bone
formation and development. It also exhibits regulatory function on
transcriptional level of mesenchymal stem cell differentiation [14,15,16]. TAZ
can enhance osteoblastogensis as it serves as a coactivator of RUNX2 and
corepressor of PRARy. [20]. Previous studies conducted on zebrafish
elucidated that lack of TAZ resulted in ossification deficiency and lack of ventral
curvature [16]. Other studies on TAZ null mice showed that TAZ is essential for
skeletal development as the survived TAZ-null mice showed shorter skeleton
structure. [21]
Further studies conducted on transgenic mice to illustrated the effect of
TAZ overexpression in osteoblasts. Results were evident which showed an
increase in bone mass gain in osteoblast-specific TAZ overexpression in vivo.
Trabecular bone volume was increased approximately 26% up to the age of 32
weeks. Expression of Osteoblast specific transcription factor genes such as,
RUNX2, osterix were unregulated in calvarial cells of transgenic mice. Also
increased level of ALP and osteoclasin marker genes of osteoblast was observed.
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These evidences illustrate the importance and the critical role of TAZ in MSC
differentiation into osteoblast and bone formation and development in a
mammalian system. [35]

TAZ induces osteoblast differentiation by activating Runx2-dependant
expression of osteocalcin gene
TAZ can regulate osteoblast differentiation through RUNX2. As mentioned
above TAZ is a coactivator of osteoblast specific transcription factor Runx2. It
binds to RUNX2 via it’s WW Domain and this interaction is essential for
osteoblast differentiation and expression of Runx2 is essential for TAZ activity
in regulating osteoblast differentiation via the suppression of PPARγ
expression. [36]

Figure D: TAZ-dependent RUNX2 activation in MSC differentiation into osteoblast.
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Also other studies showed that TAZ can enhance TGF-β-mediated
signaling and TAZ overexpression result in recruitment of the transcriptional
regulators smad2,3 in the nucleus which then can regulate transcription. TAZ
mediated TGF-β -increased signaling may play role in the increase of osteoblast
differentiation as the addition of TGF-β inhibitor could greatly impede the
process of TAZ-mediated osteoblast differentiation. [36,37]
In conclusion, osteoblast-specific TAZ overexpression lead to substantial
bone mass gain in transgenic mice. Suggesting that TAZ has an essential key
regulator activity in both mesenchymal stem cell differentiation, and in bone
formation during the postnatal period, hence makes it an interesting potential
therapeutic target for osteoporosis treatment.

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How wnt signaling and hippo are connected
hippo pathway is one of critical developmental pathways that regulate tissue
growth and organ development process. Hippo signaling can integrate and
interact with other developmental pathways in order to regulate these processes.
However, coordination between these pathways needs to be more investigated.
Interaction between hippo and Wnt pathway is regulated via the interaction
between TAZ and DVL in the cytoplasm. TAZ is an integral member of hippo
pathway. Upon binding, TAZ has an inhibitory effect on phosphorylation of
DVL and hence leads to Wnt/ β-Catenin pathway restriction. On other hand
deficiency in TAZ expression leads to enhanced DVL2 phosphorylation,
increased nuclear and cytoplasmic accumulation of β-Catenin in Taz-null mice.
Furthermore, hippo pathway deficiency result in nuclear TAZ accumulation and
hence TAZ can’t bind to DVL. This leads to induction and activation of
Wnt-target genes. [11]

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previous studies showed that TAZ null mice developed polycystic kidneys
along with increased level of β-Catenin in both cytoplasm and nucleus.
Additionally, studies in Drosophila showed that Hippo signaling regulates the
expression of Wnt target genes. These evidence highlighting TAZ cytoplasmic
activity and function and its role in coordinating hippo signaling pathway with
Wnt signaling in osteoblast differentiation. [17]

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Results
E2 may regulate ostoblast differentiation through the regulation of
TAZ the upstream effector of RUNX2

Figure1 E2 upregulates the expression of TAZ in ST2 cells detected by western blot.
ST2 cell were treated with E2 for 24 hours with various concentrations from 10-10M up to
10-7M. Cells were harvested and whole cell, cytoplasmic and nuclear protein extracts were
collected after 24hours followed by western blot analysis.

E2
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of
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California
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As mentioned above Estrogen has a complex effect on bone metabolism. In
which it inhibits bone resorption in bone cells and has an essential effect on
maintaining bone mass by regulating bone forming osteoblast and
bone-resorbing osteoclast. Also E2 deficiency result in many bone-related issues
such as osteoporosis. Moreover, evidence of previous studies showed that E2
regulates MSCs proliferation, apoptosis and differentiation process. Thus, we
wanted to investigate the effect of Estradiol on the key effector of hippo signaling
pathway TAZ protein in ST2 mesenchymal cell. ST2 cells are bone-marrow
derived mesenchymal stem cells which can differentiate into osteoblast-like cells.
We wanted to investigate the effect of Estradiol on TAZ particularly which is the
key effector modulator of hippo pathway as previous studies illustrated TAZ
cytoplasmic activity, function and its role in coordinating hippo signaling
pathway with Wnt signaling in osteoblast differentiation.
TAZ serves as a key regulator of transcription as it has the ability to bind
TEAD which can regulate embryogenesis, organs development and bone
formation and development [14] [16] [18]. It also exhibits regulatory function on
transcriptional level of mesenchymal stem cell differentiation [16].
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of
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TAZ can function as a coactivator of RUNX2 and corepressor of PRARy., and
that’s how it can regulate osteoblastogensis [28].
Previous studies showed that lack of TAZ resulted in ossification deficiency
and lack of ventral curvature [16] and TAZ null mice showed shorter skeleton
structure. [20,29]. Furthermore, studies conducted on transgenic mice to
illustrated the effect of TAZ overexpression in osteoblasts. osteoblast-specific
TAZ overexpression resulted in bone mass gain in transgenic mice which sum up
the evidences that TAZ has an essential role in both mesenchymal stem cell
differentiation, and in bone formation during the postnatal period.
Thus in this experiment, ST2 cells first were incubated in CCS for two days to
deprive cells from estrogen exposure and any other types pf SERMS then cells
were treated with exogenous E2 with various range of concentrations from
10-10M up to 10-7 M in CCS for 24 hours. Both cytoplasmic and nuclear Protein
extracts were collected after 24 hours of E2 treatment. Then protein
concentrations were quantified and we run SDS-PAGEs of proteins of interest. We
used polyclonal anti-rabbit antibody to detect the level of cytoplasmic and nuclear
TAZ. Astonishingly, the level of TAZ was unregulated particularly at
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physiological level of estrogen in both cytoplasm and nuclear level upon Estradiol
treatment for 24 hours. Upon E2 treatment, apparently the upregulation effect on
TAZ was does-dependent: the protein level of TAZ at 10-9M seems to be the most
significant increase at both cytoplasmic and nuclear level, also there was a
noticeable increase at 10-10 M and 10-8 M. However, protein level decreased at
higher concentration of E2 at 10-7 with no noticeable changes compared to protein
level in control sample.

This western-blot data result is very consistent with our hypothesis and gave an
insight into how estrogen may has an effect on hippo signaling pathway via TAZ
regulation. To investigate more and test if similar results can be obtained,
Immunofluorescence was performed as the next test in order to visualize the effect
of E2 on TAZ expression level in vitro- cultured ST2 mesenchymal stem cell.

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Indeed, general increase of TAZ Expression Corresponds to
Estrogen treatment in concentration dependent manner:

Figure2 Representative immunofluorescence images from in vitro culture of
estrogen-treated ST2 cell obtained by laser confocal microscopy. ST2 cells were treated
with E2 for 24 hours (A-D) at different concentration starting from (10-10M up to 10-7M)
respectively. Cells were stained with TAZ antibody (green).

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Figure2 Representative immunofluorescence images from in vitro culture of
estrogen-treated ST2 cell obtained by laser confocal microscopy. ST2 cells were treated
with E2 for 24 hours (A-D) at different concentration starting from (10-10M up to 10-7M)
respectively. Cells were stained with TAZ antibody (green). Dapi(blue)

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To further verify whether or not estrogen has an effect on TAZ regulation
and expression in estrogen treated ST2 cells, Immunofluorescence were utilized
to detect TAZ expression in vitro cultured ST2 cell. ST2 cells were incubated
with CCS for 48 hours followed by exposure of exogenous E2 at different
concentration (10-10M up to 10-7M) for 24 hours. Subsequently cells were fixed
with 3.7% formaldehyde and permeabilization was done using 1% triton.
Fixed cells were incubated with primary antibody directed against TAZ (green)
overnight followed by an hour hour incubation with a fluorophore-conjugated
secondary antibody directed against first antibody. Images were visualized and
collected using laser confocal microscopy.
In immunofluorescence experiments, increase of TAZ protein expression was
observed at (10-10M up to 10-9M) of E2, with slight changes at 10-7M E2. The
fluorescence intensity of TAZ from ST2 cells treated with 10-9M and 10-10M
E2 (Figure 2(A–B)) respectively were significantly the highest. TAZ
fluorescence intensity at E2 concentration of 10-8M and 10-7M (Figure 2(C-D))
respectively were also slightly higher than control. These findings demonstrated
that exogenous exposure of E2 can further upregulates TAZ protein expression
in ST2 cells specifically at physiological level of E2 concentration
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Different regulation of TAZ expression by SERMS Ral and LAS

Figure 3: SERM/ Ral effect on TAZ expression in ST2 cells detected by IF. ST2 cells
incubated in CCS for 48 hours. After 48 hours cells were treated with Ral for 24 hours in CCS
with a various concentration starting from 10-10M up to 10-6M which represents (B to F)
respectively. Cells were fixed and IF was performed after 24 hours of Ral treatment.

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Figure 4: SERM/ Las effect on TAZ expression in ST2 cells detected by IF. ST2 cells
incubated in CCS for 48 hours. After 48 Hours cells were treated with Ral for 24 hours in CCS
with a various concentration starting from 10-10M up to 10-6M which represents (B to F)
respectively. Cells were fixed and IF was performed after 24 hours of Las treatment.

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SERMs are clinically approved for the treatment of and to prevent
osteoporosis which are used as a compensating treatment for estrogen deficiency
in postmenopausal women. We wanted to investigate the effect of SERMs on TAZ
expression, however, results showed different effect than results obtained from E2
treatment. It seems like that Ral and Las have an opposite effect on TAZ
expression than E2 on ST2 mesenchymal stem cells in a concentration dependent
manner.
In this experiment, different concentration of the SERMs were used ranging
from 10-7M up to 10-10M, similar to the concentration we used in E2 experiment.
same experiment of E2 treatment was applied but with different SERMs RAL and
LAS.
In this experiment Immunofluorescence were conducted to visualize TAZ
expression in vitro cultured ST2 mesenchymal cell. ST2 cells were incubated
with CCS for 48 hours followed by exposure of exogenous SERMs both LAS
and RAL at various concentration ranging from 10-10M up to 10-7M for 24
hours. Subsequently cells were fixed with 3.7% formaldehyde and cells were
permeabilized by 1% triton.
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Fixed cells were incubated with primary antibody directed against TAZ (green)
overnight followed by an hour hour incubation with a fluorophore-conjugated
secondary antibody directed against first antibody. Images were visualized and
collected using laser confocal microscopy.
In immunofluorescence protein expression experiments, there was a significant
decrease of TAZ protein expression at (10-10M) concentration of RAL
treatment, with no substantial changes at other different concentration. These
results show opposite effect of RAL treatment compared to E2 treatment which
showed an increase in TAZ expression. Moreover, the fluorescence intensity of
TAZ from ST2 cells treated with 10-10M up to 10-8M LAS (Figure 4(B-D))
respectively were significantly lower than control. TAZ fluorescence intensity at
LAS concentration of 10-7M (Figure 2E) were also slightly lower than control.
These findings demonstrated that exogenous exposure of SERMs can have an
inhibitory effect on TAZ protein expression in ST2 cells specifically around
10-10M, 10-9M concentration where E2 showed significant regulation effect on
TAZ expression around same concentration.
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Second chapter
Estrogen-dependent TAZ activation confers cancer stem cell
related-traits and increased cell proliferation in ER+ BCC
Breast Cancer
Breast cancer is considered almost third most common cancer in the world and the
most frequent type of cancer among women. It is the second type of cancer with
highest incidence of cancer death in women after lung cancer. ER positive breast
cancer accounts for almost 80% of all breast cancer cases which indicates that
cancer cell growth and proliferation is attributable to estrogen response. [39,40]
Relationship between Runx1 and TAZ in breast cancer and cancer stem
cell
The RUNX1 transcription factor is component of RUNX gene family which
encodes a Runt-related transcription factor. RUNX1 is essential TF that is
involved in hematopoietic cell types development and bone formation; and it’s
sufficient to induce oncogenic phenotype transformation to acute myelogenous
leukemia. However, it has an opposing role in breast cancer ER positive cells. [41]
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Runx1 functions as a tumor suppressor in breast cancer ER positive and it’s
the target factor in ERα-mediated downstream transcriptional activation. Recent
studies showed that RUNX1 inhibits the ERα-mediated inhibitory pathway of
AXIN1, thus it’s loss in breast cancer allow ERα-mediated inhibition mechanism
of AXIN1, which result in n upregulation of β-catenin signaling pathway. Thus
The mechanism in which RUNX1 antagonizes oestrogen-dependant suppression
of AXIN1 expression, illustrated RUNX1 function as a tumor suppressor, [25]
β-Catenin is a key target of Wnt signaling pathway and it’s part of destruction
complex thus it plays essential roles in cancer. Axin1 is a component of The
β-catenin destruction complex along with other proteins and it’s essential for the
integrity of the complex. [26,27]
loss of RUNX1 cause deregulation of β-catenin which subsequently promotes
cell growth and and induction of stem cell markers. β-catenin deregulation
through RNUX1 knockdown is associated with cancer cell proliferation and
cancer stem cells [28]. Furthermore, RUNX1 silencing in breast cancer is
associated with high expression of stem cell markers such as, SOX2 and other
markers [29,30] and SOX2 mRNA levels was normalized upon RUNX1
re-induction. Thus, RUNX1-depedent aberrant expression of β-catenin
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particularly in ER+ breast cancer may promotes the growth of stem cell like
population, expression of stem cell markers and cell proliferation in cancer. [25]
so the further investigation about the underlying mechanism of how RUNX1 loss
can be associated with induction of cancer stem cell markers. This mechanism
can be regulated by the association between RUNX1, AXIN1 and TAZ/YAP the
key effector of hippo signaling pathway.
Many studies illustrated that TAZ plays an important role in cell proliferation
and promotes stemness traits such as regeneration, and tumorigenesis. [33]

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Recent studies showed that TAZ activity is linked with the expression of
Breast CSCs biological properties thus it’s important for tumor initiation, tumor
heterogeneity and for self-renewal characteristics in breast cancer cells. [31,32]

http://www.sciencedirect.com.libproxy1.usc.edu/science/article/pii/S009286741400734X
Figure E: YAP/TAZ Incorporation in the β-Catenin Destruction Complex

YAP/TAZ are components of b-Catenin Destruction Complex. The Hippo key
regulator YAP/TAZ have both negative and positive effect on Wnt signaling
pathway. Upon activation of Wnt signaling, YAP/TAZ become dislocated from
the complex and accumulate in the nucleus and activate down stream pathway
and target genes.
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β-catenin is inactivated by β-TrCP recruitment; this recruitment process is
facilitated by TAZ/YAP. Therefore, dislocation of TAZ/YAP from the complex
is associated with Wnt/β-catenin signaling activation. To sum, YAP/TAZ loss is
essential to sustain the proliferation state of ES cells and inhibition of ES cell
differentiation. [34]

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Results
RUNX1 knockdown result in TAZ upregulation in Mcf7/Rx1Dox cells
upon Dox treatment detected by western blot and IF

Figure 5: upregulation of TAZ expression in Mcf7/Rx1Dox cells upon dox treatment
detected by western blot. MCF7/RX1Dox cells were cultured in DMEM media. Control
treated with DMSO, Dox-treated cell was treated with Dox (200ng/mL) for 48 hours. After
48hours, IWR was added to the third Dox treated sample for 6 hours. Subsequently, cytoplasmic
and nuclear protein extract were collected. RUNX1 and TAZ expression were detected in
western blot using both AB against TAZ and Runx1.

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We want to investigate the relationship between TAZ the key effector
modulator of hippo pathway and RUNX1 tumor suppressor in MCF7/RX1Dox
cell.
RUNX1 is a tumor suppressor and Previous studies showed that loss of
RUNX1 in ER+ mammary epithelial cells leads to β-catenin upregulation,
mitosis dysregulation and stimulation of stem cell markers and cell proliferation
[25]. Recent studies also illustrated how RUNX1 can regulates AXIN 1 in
estrogen –dependent manner in ER+ breast cancer cells. It was shown that
RUNX1 is a positive regulator of AXIN1 only in the presence of estrogen as it
antagonizes AXIN1 suppression mechanism by estrogen. AXIN1 is part of
β-catenin destruction complex, hence, loss of RUNX1 leads to decrease in
AXIN1 and increase in β-catenin and stem cell marker in the presence of
estrogen. [25]
Our work begins to investigate operative downstream mechanisms that links
this pathway with stem cell markers and cell proliferation mechanisms
particularly hippo pathway signaling according to results that were obtained
from previous studies.
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Thus MCF7/RX1Dox cells first were cultured in DMEMF12, and treated with
Dox (200ng/mL) for 48 hours. In IWR treated samples, cells were treated with
IWR for 6 hours. IWR compounds was used as it functions to stabilize Axin1
proteins through a direct binding. Axin which is a component of the b-catenin
destruction complex thus IWR helps to inhibit Wnt signaling response via
AXIN1 stabilization. Subsequently, cytoplasmic and nuclear protein extract were
collected after DOX and IWR treatment. RUNX1 and TAZ expression were
detected in western blot using both AB against TAZ and RUNX1.
Then protein concentrations were quantified and we run SDS-PAGEs of
proteins of interest. We used polyclonal anti-rabbit antibody to detect the
cytoplasmic and nuclear level of both protein TAZ and RUNX1. Cell treated with
DOX showed knock down effect of RUNX1. Astonishingly, the level of TAZ
was significantly unregulated when RUNX1 expression decreases in both
cytoplasm and nuclear level upon DOX treatment. Moreover, treatment of IWR,
which is used to stabilize AXIN1 and hence inhibits Wnt signaling response,
showed decrease of TAZ expression level compared to its level after Dox
treatment in both cytoplasmic and nuclear extracts.
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To sum up, expression level of TAZ was RUNX1-dependent: the protein level of
TAZ increased substantially when there was a decrease in RUNX1 level by DOX
treatment followed by a noticeable decrease in TAZ expression upon IWR
treatment.

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Figure 6 upregulation of TAZ expression in Mcf7/Rx1Dox cells upon dox treatment
detected by IF. Representative immunofluorescence images from in vitro culture of
Dox-treated MCF7/RX1Dox cell obtained by laser confocal microscopy. Cells were treated
with DOX (200ng/ml) for 48 hours (B and D). Subsequently cells were fixed and IF was
performed. Cells were stained with RUNX1 and TAZ antibody (green).

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To further verify western blot results of the relationship between TAZ
and RUNX1 on MCF7/RX1dox cells, Immunofluorescence were performed to
detect TAZ expression in vitro cultured MCF7/RX1Dox treated with dox and IWR.
cells were cultured in DMEM media and treated with DOX (200ng/ml) for 48
hours. (B and D). Subsequently cells were fixed with 3.7% formaldehyde and
permeabilization was done using 1% triton. Fixed cells were incubated with
primary antibody directed against TAZ and RUNX1 (green) overnight followed
by an hour hour incubation with a fluorophore-conjugated secondary antibody
directed against first antibody. Images were visualized and collected using laser
confocal microscopy.

In immunofluorescence protein expression experiments, increase of TAZ
protein expression was observed in DOX treated cells when RUNX1 knock
down occurs. The fluorescence intensity of TAZ in MCF7/RnxDox cells treated
treated with dox (Figure5-D) was substantially increased compared to control
sample (figure5_C). These results are consistent with the significant decrease of
RUNX1 expression in dox-treated cells observed in IF images (figure
5c,d)compared to the control untreated cells. These findings supported that initial
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results obtained from western blot analysis and it highlights the relationship
between TAZ which is the effector modulator of hippo pathway with the tumor
suppressor RUNX1. It’s clearly shown that Runx1 knock down results in
upregulation of TAZ expression level.

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Materials and Methods
Reagents
Table 1. Reagents
Materials Sources

Bio-Rad Protein Assay Kit Bio-Rad Laboratories, Hercules,
CA
Charcol stripped fetal bovine
serum (CSS)
Gemini, West Sacramento, CA
Doxycycline (Dox)
Estradiol (E2)
Calbiochem, La Jolla, CA
Sigma-Aldrich, St. Louis, MO
Fetal bovine serum Gemini BioProducts, West
Sacramento, CA
Formadehyde 37% Solution J.T. Baker, Phillipsburg, NJ

Mouse monoclonal primary
antibody
Sigma-Aldrich, St. Louis, MO
Protease inhibitor cocktail
Raloxifene hydrochloride
RPMI1640
RPMI1640 without phenol red

Sigma-Aldrich, St. Louis, MO
Sigma-Aldrich, St. Louis, MO
Gibco, Invitrogen, Carlsbad, CA
Gibco, Invitrogen, Carlsbad, CA

Typsin (10X) Gibco, Invitrogen, Carlsbad, CA
aMEM

Facility core, USC

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Cell culture
ST2 cells are bone-marrow derived mesenchymal stem cells which can
differentiate into osteoblast-like cells. ST2 cells were maintained in RPMI1640
(Mediatech) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin. MCF7 cells were maintained in DMEM supplemented
with 10% FBS and 1% P/S.
In experiments in which ST2 cells were treated with Estrodial, cells were
cultured in RPMI without phenol red , supplemented with 10% CSS charcoal
stripped fetal bovine serum (CSS) and 1% P/S. E2 stock solutions with different
concentration ( 10-7M to 10-3M) were prepared by diluting E2 with ethanol.
St2 cell cultured in 6 well plates were treated with Estradiol of different
concentration starting from 10-7 up to 10-3 at a constant volume of 2ul/2ml to
reach final concentration of 10-10M to 10-7M. ethanol were added to control
sample with same volume of 2ul/2ml.

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Western blot analysis
For western blot analysis of TAZ expression, estrogen treated ST2 cells were
harvested and protein extract were prepared using RIPA cell lysis buffer
(ThermoFisher Scientific) with (1:100) dilution of Protease inhibitor cocktail
(Sigma-Aldrich). cytoplasm and nuclear extracts obtained using NE-PER kit
(ThermoFisher Scientific) according to the manufacturer’s instructions. Protein
concentrations measured using Bio-Rad Protein Assay Kit. Protein extracts (20 ug
of cell lysate). cell lysate was separated by SDS-PAGE (polyacrylamide gel
electrophoresis) and transferred onto AmershamHybond-P PVDF membranes
(GE Healthcare) and subsequently blocking with 5% non-fat dry milk. Protein of
interest was detected with monoclonal antibodies obtained from Cell Signaling
Technology, Inc. 1:1,000 dilution of rabbit anti-RUNX1 (#8529), and rabbit
anti-TAZ (#4883.) were used. Mouse anti-GAPDH (#97166) antibodies were
purchased from Santa Cruz Biotechnology and 1:200 dilutions was used. Goat
anti rabbit secondary antibody (SC-2005, Santa Cruz). HRP-conjugated donkey
anti-mouse (sc-2314) and goat anti-rabbit (sc-2004) antibodies were purchased
from Santa Cruz Biotechnology and 1:2,000 dilutions were used.

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ECL2 western blotting detection system was used for Immunodetection from
(ThermoFisher Scientific).
Immunofluorescence
Cells were plated on aseptic glass cover slip over night. Cover slip were
immersed in 90% ethanol for few minutes and flamed. Cell then were plated on
aseptic glass cover slip in 6 well plates over night with a density to reach full
confluency on the next day. followed by Estradiol treatment method mentioned
above. Treated cells washed with 1%PBS and fixed with 3.7% formaldehyde for
20 minutes in room temperature then washed with 1% PBS 3x. cells were
permeabilized with 1% triton for 5 minutes at room temperature then washed 3x
with 1% PBS. Cover slip containing cell were incubated with 1%PSA diluted in
T-PBS for half an hour then probed with rabbit anti-TAZ (#4883) with 1:150 ul
volume over night. Fixed cell were washed with 1%PBS 3x and coated with
fluorescent anti rabbit secondary Antibody for an hour at room temperature then
washed 1%PBS 3x and left to dry. A drop of VECTASHIELD Mounting
Medium was added to microscopic glass slide and the cover slip were placed on
top of it.

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Discussion
Osteoblast differentiation and bone formation regulated through TAZ(
transcriptional co-activator with PDZ-binding motif)
Using st2 cells which are bone-marrow derived mesenchymal stem cells that
can differentiate into osteoblast-like cells. we demonstrated that
osteoblast-specific overexpression of TAZ achieved upon exogenous exposure of
estradiol mainly at physiological concentration.
Generally various previous studies proved that E2 and SERM both an essential
effect on maintaining bone mass by regulating bone forming osteoblast and
bone-resorbing osteoclast, thus E2 deficiency result in osteoporosis. Estrogen
induction suppresses bone resorption and thus can maintain bone mass. It was
shown that exogenous induction of estrogen result in increased bone formation
in ovary-intact rats. Ovariectomized rats were treated with E2 showed an
increase in trabecular bone volume in a dose dependent manner. Also further
results illustrated that estrogen can maintain bone volume by two mechanisms
through both suppression of bone resorption, and via stimulating bone formation.
[12,13]

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Osteoblasts cells are differentiated from mesenchymal stem cell. It was shown
that Differentiation to osteoblasts is regulated by TAZ.
TAZ which is the key effector regulator of hippo pathway is a transcriptional
coactivator with a PDZ-binding motif which regulates differentiation of
mesenchymal cells into osteoblasts and inhibits MSC differentiation into
adipocytes [14,15]. Previous studies indicated that TAZ can regulate osteoblast
differentiation as it functions as a transcription coactivator for RUNX2. RUNX2
is a transcription factor that activate the expression of osteoblast-specific gene.
[16]
In our experiment to address the increased expression level of TAZ by E2 in vitro
cultured cell ST2. we first treated ST2 cells with exogenous E2 at different
concentration from 10-10 concentration to address any slight effect of E2 on TAZ,
10-9M of E2 observe the effect of E2 at physiological level on TAZ and we used
10-8M and 10-7M of E2 to address the highest concentration effects of exogenous
E2 on TAZ level. We run western blot analysis for cytoplasm and nuclear extracts
to address the effect of E2 on TAZ in both cytoplasm and nuclear compartments.
TAZ upon activation translocate and accumulate into the nucleus. Indeed, in our
results it was shown that E2 increased TAZ expression level in ST2 in
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concentration dependent manner with highest effect observed at physiological
level of estrogen in nuclear and cytoplasmic extracts. Experiments was done in
duplicate independently and same results were obtained. However, western blot
results of whole cell extract did not show noticeable effect of E2 on TAZ. To
further address the effect of E2 on cytoplasmic and nuclear TAZ level,
Immunofluorescence experiment was performed to visualize TAZ
compartmentalization and the level of TAZ in cytoplasm and nucleus. It showed
that both cytoplasm and nuclear TAZ level was substantially increased around
10-10M and 10-9M.
These results are consistent with all previous studies and research that was done
on the effect of E2 on bone formation, and TAZ role as a coactivator of Runx2
Transcription factor of osteoblast-specific gene. These results could give an
insight on how estrogen can regulate bone formation through upregulation of TAZ
expression level, hence activating RUNX2 which subsequently activates the
transcription of osteoblast genes.

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Estrogen-dependent TAZ activation confers cancer stem cell
related-traits and increased cell proliferation in ER+ BCC:
ERα is essential for the growth and differentiation of mammary epithelial cell
during puberty and pregnancy, thus its mutation can cause breast carcinogenesis.
Runx1 is the target factor in ERα--mediated downstream transcriptional
activation. Recent studies showed that RUNX1 loss in breast cancer facilitates
ERα-mediated inhibition mechanism of AXIN1, resulting in upregulation of
β-catenin signaling pathway. Thus The mechanism in which RUNX1
antagonizes estrogen-dependent suppression of AXIN1 expression, illustrated
RUNX1 function as a tumor suppressor [25]

β-Catenin is a key effector of Wnt signaling pathway and β-Catenin is part of
destruction complex thus it plays essential roles in cancer. Activation of Wnt,
results in destruction of β-catenin destruction complex, allowing β-catenin to
activate downstream LEF/TCF target genes. The β-catenin destruction complex
consists of the scaffold proteins AXIN1, adenomatous polyposis coli APC, and
glycogen synthase kinase 3α/β (GSK3α/β), which phosphorylate β-catenin.
phosphorylation of β-catenin result in its degradation [26,27]
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Moreover, it was shown that loss of RUNX1 cause deregulation of β-catenin
which subsequently stimulates cell growth and stem cell markers. β-catenin
deregulation is associated with cancer cell proliferation and cancer stem cells
[28]. Recent study showed that RUNX1 knockdown promotes MCF7 breast
cancer cell proliferation [25]. Moreover, by promoting the restoration of
RUNX1 by Dox inducible mechanism in MCF7/shRx13′-UTR cells, they gained
their normal growth characteristic. Furthermore, high expression of stem cell
markers such as, SOX2 and other markers upon RUNX silencing was detected
[29,30]. SOX2 mRNA levels was back to normal upon RUNX1 re-expression.
Same results were obtained in T47D breast cancer cells. Moreover, SOX2
mRNA expression level was substantially increased in biopsies obtained from
RUNX1-mutant compared to its expression in RUNX1-WT human primary
breast tumors in previous clinical studies. Thus, RUNX1-depedent aberrant
expression of β-catenin particularly in ER+ breast cancer may enhance the
growth of stem cell like population and cell proliferation in cancer. [25]

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Many studies illustrated that TAZ plays an important role in cell proliferation
and particularly inducing stemness traits such as regeneration, and
tumorigenesis. [33]
Recent studies showed that TAZ activity Is associated with the main
Biological characteristics of Breast CSCs. It was shown that endogenous TAZ
level is essential to induce CSC-associated traits thus it’s important for tumor
initiation, tumor heterogeneity and for self-renewal characteristics in breast
cancer cells. Experiments results showed that TAZ induction can turn benign
tumors into aggressive phenotype tumor. [31-32]
TAZ Incorporation in the b-Catenin Destruction Complex
The Hippo key regulator YAP//TAZ have both negative and positive effect on
Wnt signaling pathway. In this paper evidence provided showed that both YAP
and TAZ are components of the β-catenin destruction complex. Upon activation
of Wnt signaling, YAP/TAZ become dislocated from the complex, and results in
nuclear accumulation of TAZ and YAP and subsequent activation of down
stream pathway. β-catenin is inactivated by β-TrCP recruitment facilitated by
TAZ/YAP.
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So dislocation of TAZ/YAP from the complex is associated with Wnt/β-catenin
signaling activation. To sum, YAP/TAZ loss is essential to sustain the
proliferation state of ES cells and with suppression of ES cell differentiation.
[34]

In our experiments we wanted to investigate further how RUNX1 loss in
ER-positive cells is associated with the induction of the stem cell marks in Breast
Cancer.
Upon RUNX1 knock down in DOX- treated MCF7/RX1Dox, TAZ expression
was elevated substantially in both western blot and immune florescence
experiments (figure5,6). IWR used to stabilize Axin1 proteins through a direct
binding, thus results in Runx1 repression. Runx1 re-expression normalized TAZ
expression. To sum up, these results could illustrate how RUNX1 loss leads to
induction of stem cell markers through axin1 and TAZ which is an integral
component of hippo pathway. TAZ is part B-catenin destruction complex and a
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key effector of hippo pathway which regulates stem cell self renewal and
differentiation state [34]. As mentioned above axin1 is part of the β-catenin
destruction complex [34] and Runx1 is a positive regulator of AXIN1 [25]in ER
positive cells. Hence, loss of RUNX1 results in the E2-dependent inhibition of
AXIN1 [25], therefore, this result in deregulation of β-catenin destruction
complex and dislocation of YAP/TAZ from the complex. Dislodgment of both
YAP/TAZ from the destruction complex leads to nuclear accumulation of
YAP/TAZ and hence activation of downstream pathway. YAP/TAZ are
coactivator transcription factor that activate TEAD target genes and this could
lead to expansion of cancer stem cell.

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Future direction
1-Hypothesis that TAZ regulates RUNX2 in osteoblast differentiation in
estrogen dependent manner
• Diferrent cell lines can to be used to further illustrate the
hypothesis. We can repeat experiment on human cell such as SM30,
which is human mesenchymal stem cells.
• We can run western blot to look for Runx2 protein level after
treating ST2 with different estrogen concentration.
• To further emphasize the direct relationship betweenE2, TAZ and
RUNX2 in osteoblast differentiation, we can Knock down TAZ and
look for RUNX2 Target gene expression after treating cells with
different concentration of E2.
• We only looked at the effect of different serums on TAZ in ST2
cells, we can can further test the effect of different serums such as
RAL, lAS on TAZ level in ST2 by western blot
• Further we can Induce endogenous mutational in which TAZ is
constitutively active and look for activation of target genes and its
effect on osteoblast differentiation pathway

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2-To further test the hypothesis that TAZ oncogenic function in ER+ BCC is
negatively regulated by RUNX1
• We can Repeat experiment in different ER+ BCC such as T47D.
• In our experiment cells were cultured in normal complete medium, to
further investigate the relationship between TAZ and RUNX1 in ER+
BCC we can Run
o Same experiment in CCS to exclude all serums and estrogens effect
and treat cells with, DOX +E –E to further illustrate that it’s
estrogen dependent regulation mechanism.

• Also we can knock down TAZ by shRNA, and run same experiment to
look for the expression of stem cell markers to further proof the
relationship between RUNX1 and TAZ in ER+ BCC.

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89:755–764.

Abstract (if available)

Abstract Estrogens are female hormones that helps to maintain the process of bone formation by various mechanisms. Estrogen deficiency leads to an imbalance in bone remodeling process and affects osteoblast differentiation, hence causing osteoporosis. Runx2 is an Osteoblast specific transcription factor that regulate osteoblast differentiation process. TAZ is a coactivator transcription factor that activates Runx2 and direct the differentiation of MSC into osteoblast by inhibiting MSC differentiation into adipocytes. This project assayed the effects of E2 on TAZ in ST2 mesenchymal stem cell (MSCs) that can differentiate into osteoblast, hence understand more the underlying mechanism of MSC osteoblastogenesis. We used St2 MSC to investigate if TAZ can be the link between E2 and Runx2 in osteoblast differentiation process. We performed western blot to observe the changes in protein level of TAZ after treating ST2 cells, which is cultured in CSS-E2 media, with various concentrations of exogenous estrogen. Immunofluorescence technique was also performed to further verify the initial observed results and determine the localization and expression of TAZ in cytoplasm and nucleus. Results showed that E2 treatment at physiological concentration of 10-10M and 10-9M increased the protein level of TAZ in western blot of cytoplasm and nuclear protein extracts. Also same results were observed in Immunofluorescence data demonstrating more insight into molecular pathway and regulation process of osteoblast differentiation. Understanding the underlying mechanism of osteoblast differentiation can help us to treat and find a potential therapeutic target for osteoporosis.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Alnassar, Sara (author)

Core Title The effect of estradiol on TAZ in stromal cell line ST2 in osteoblast differentiation and ER+breast cancer cell

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/31/2017

Defense Date 06/12/2017

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

Tag bone metabolism,ER breast cancer,estradiol,OAI-PMH Harvest,osteoblast differentiation,ST2

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Rice, Judd (committee member)

Creator Email n_aln@hotmail.co.uk,salnassa@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-419308

Unique identifier UC11263273

Identifier etd-AlnassarSa-5660.pdf (filename),usctheses-c40-419308 (legacy record id)

Legacy Identifier etd-AlnassarSa-5660.pdf

Dmrecord 419308

Document Type Thesis

Rights Alnassar, Sara

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA