m 4-h i a rhabdomyosarcoma cells...rhabdomyosarcoma (rms) is the most common pediatric soft tissue...

MECHANISMS OF 4-HYDROXYTAMOXIFEN-INDUCED APOPTOSIS IN

RHABDOMYOSARCOMA CELLS

by

Kevin Min Chen

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Medical Biophysics

University of Toronto

© Copyright by Kevin Min Chen (2011)

Mechanisms of 4-hydroxytamoxifen-induced apoptosis in rhabdomyosarcoma cells

Kevin Min Chen

Master of Science

Department of Medical Biophysics University of Toronto

2011

ABSTRACT

Rhabdomyosarcoma (RMS) is a malignant soft-tissue sarcoma in children, accounting

for about 40% of pediatric soft-tissue tumours. Five-year survival for metastatic RMS is

only about 25%. Furthermore, there has been no significant improvement in RMS

survival since 1975, pointing to a need for improved therapy.

Previous work in our lab has shown that 4-hydroxytamoxifen (4OHT) leads to

increased apoptosis and decreased viability in RMS cells. Expanding on this work, the

current project aims to elucidate the mechanisms behind 4OHT-induced apoptosis in

RMS cells, focusing on the roles of estrogen receptors (ER) and MAP kinases (MAPK).

We found that: 1) 4OHT-induced apoptotic signaling was associated with increased

MAPK phosphorylation, 2) Inhibition of MAPK protected cells against 4OHT, 3)

Inhibition of ER also protected against 4OHT, and 4) ER inhibition blocked 4OHT-

associated MAPK phosphorylation.

ii

ACKNOWLEDGEMENTS

This work would not have been possible without the generous support and assistance

received from the individuals and organizations below:

Funding was provided in part by a Frederick Banting and Charles Best Canada

Graduate Scholarship (CGS) - Master's Award, from the Canadian Institutes of

Health Research (CIHR).

Much gratitude goes to Dr. David Malkin for his guidance, and for cultivating a

healthy and stimulating research environment. By combining clinical expertise

with scientific knowledge, by constantly drawing links between the lab and the

clinic, Dr. Malkin has taught his students to see the “bigger picture” in the

complex world of medical research.

As members of my advisory committee, Drs. Jane Aubin and Meredith Irwin have

provided valuable insight and advice that shaped the outcome of this project.

I am also indebted to members of the Malkin lab (in particular Dr. Adam Durbin),

as well as Dr. Uri Tabori and members of the Tabori lab, for thoughtful feedback

during every stage of my work.

Finally, much gratitude is owed to my family and friends, including my

grandfather and grandmother (both of whom passed away in June 2011). They

have provided invaluable support (social, mental, and sometimes even scientific)

during the ups and downs of this project.

iii

TABLE OF CONTENTS

Abstract ...................................................................................................................................... ii Acknowledgements .................................................................................................................... iii List of Figures ............................................................................................................................ v List of Abbreviations .................................................................................................................. vi Chapter 1: Introduction and Background .................................................................................... 1

1.1 Rhabdomyosarcoma ..................................................................................................... 1 1.1.1 Overview ............................................................................................................. 1 1.1.2 Embryonal rhabdomyosarcoma .......................................................................... 5 1.1.3 Alveolar rhabdomyosarcoma .............................................................................. 5

1.2 Estrogen Signaling in Rhabdomyosarcoma ................................................................. 8 1.2.1 Estrogen and its receptors ................................................................................... 8 1.2.2 Linking estrogen to rhabdomyosarcoma ............................................................. 11

1.3 Selective Estrogen Receptor Modulators ..................................................................... 13 1.3.1 Overview ............................................................................................................. 13 1.3.2 Tamoxifen ........................................................................................................... 16

1.4 Mechanisms of Tamoxifen and 4-Hydroxytamoxifen ................................................. 18 1.4.1 Estrogen receptors ............................................................................................... 18 1.4.2 Caspases .............................................................................................................. 19 1.4.3 Microsomal antiestrogen binding site ................................................................. 20 1.4.4 Calcium signaling ............................................................................................... 21 1.4.5 Mitochondria ....................................................................................................... 21 1.4.6 Transforming growth factor β ............................................................................. 22 1.4.7 Protein kinase C .................................................................................................. 23 1.4.8 Mitogen-activated protein kinases ...................................................................... 24 1.4.9 Other proposed mechanisms ............................................................................... 25

1.5 Rationale and Hypothesis ............................................................................................ 28 Chapter 2: Materials and Methods .............................................................................................. 29 Chapter 3: Results ....................................................................................................................... 33

3.1 Effects of 4OHT on RMS cell lines ............................................................................. 33 3.1.1 4OHT leads to loss of cell viability .................................................................... 33 3.1.2 4OHT induces apoptotic signaling ..................................................................... 35

3.2 Involvement of MAPK in the effects of 4OHT ........................................................... 36 3.2.1 4OHT-induced apoptotic signaling is associated with increased MAPK

phosphorylation .................................................................................................. 36 3.2.2 MAPK inhibition protects against 4OHT-induced loss of viability ................... 36 3.2.3 MAPK inhibition blocks 4OHT-induced apoptotic signaling ............................ 37

3.3 Involvement of estrogen receptors in the effects of 4OHT ......................................... 41 3.3.1 ER inhibition protects against 4OHT-induced loss of viability .......................... 41 3.3.2 ER inhibition blocks 4OHT-induced apoptotic signaling ................................... 43

3.4 Linking the MAPKs with ERs ..................................................................................... 45 Chapter 4: Discussion and Future Directions ............................................................................. 47

4.1 Involvement of Caspases ............................................................................................. 47 4.2 Involvement of MAPKs ............................................................................................... 49 4.3 Involvement of ERs ..................................................................................................... 50 4.4 Concluding Remarks .................................................................................................... 56

References ................................................................................................................................... 57

iv

LIST OF FIGURES

Figure 1: Comparison of survival for non-metastatic and metastatic RMS

Figure 2: Recent trends (1976-2000) in survival of RMS patients

Figure 3: Proportion of RMS subtypes

Figure 4: Cholesterol and the steroid hormones

Figure 5: Synthetic estrogen receptor modulators

Figure 6: 4OHT leads to loss of viability in RMS cell lines

Figure 7: 4OHT induces apoptotic signaling in RMS cell lines.

Figure 8: 4OHT leads to increased MAPK phosphorylation in RMS cell lines.

Figure 9: MAPK inhibition protects against 4OHT-induced loss of viability

Figure 10: MAPK inhibition protects against 4OHT-induced apoptotic signaling

Figure 11: The ER inhibitor fulvestrant protects against 4OHT-induced loss of viability

Figure 12: Visible changes in cell appearance associated with 4OHT-induced cell death

can be partially blocked with the ER inhibitor fulvestrant

Figure 13: The ER inhibitor fulvestrant protects against 4OHT-induced apoptotic

signaling

Figure 14: The ER inhibitor fulvestrant blocks 4OHT-induced JNK phosphorylation but

does not affect basal phospho-JNK levels

Figure 15: Model of 4OHT activity in RMS cells.

v

LIST OF ABBREVIATIONS

4OHT 4-hydroxytamoxifen

AEBS microsomal antiestrogen binding site

AIF apoptosis-inducing factor

ARMS alveolar rhabdomyosarcoma

ASK apoptotic signaling kinase

cAMP 3',5'-cyclic adenosine monophosphate

E2 17-β-estradiol

EGF epidermal growth factor

ER estrogen receptor

ERα estrogen receptor α isoform

ERβ estrogen receptor β isoform

ERE estrogen response element

ERK extracellular signal-regulated kinase

ERMS embryonal rhabdomyosarcoma

FOXO1A forkhead box O1A

IAP inhibitor of apoptosis

IGF insulin-like growth factor

JNK c-Jun N-terminal kinase

MAP2K mitogen-activated protein kinase kinase

MAP3K mitogen-activated protein kinase kinase kinase

MAPK mitogen-activated protein kinase

MEK MAPK/ERK kinase

MLK mixed lineage kinase

MMP-9 matrix metalloproteinase-9

vi

MNAR modulator of nongenomic activity of estrogen receptor

MTP mitochondrial transmembrane potential

PARP poly(ADP-ribose) polymerase

PAX3 paired box gene 3

PAX7 paired box gene 7

PI3K phosphatidylinositol-3-OH kinase

PIP2 phosphatidylinositol 4,5-bisphosphate

PKC protein kinase C

RMS rhabdomyosarcoma

SERM selective estrogen receptor modulator

TAM tamoxifen

TGFβ transforming growth factor β

vii

CHAPTER 1: INTRODUCTION AND BACKGROUND

1.1 Rhabdomyosarcoma

1.1.1 Overview

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma [1], with

an annual incidence of 4.5 per million in children ages 19 and under [2]. It accounts for 40%

of all soft tissue sarcomas [2] and 10% of all solid tumours [3, 4] in children. RMS is thought

to arise from immature mesenchymal cells committed to the skeletal muscle lineage [1].

However, primary RMS can present almost anywhere in the body, even in tissues where

skeletal muscle is not normally found [5-7]. The most common sites of occurrence are the

head and neck (including orbital and parameningeal regions), genitourinary tract, and

extremities [1].

Although most cases of RMS are thought to occur sporadically, various familial

syndromes have been linked to RMS. These include Li-Fraumeni Syndrome, Costello

syndrome, neurofibromatosis, and Beckwith-Wiedemann Syndrome [2]. Li-Fraumeni

Syndrome is an autosomal dominant disorder associated with mutations in p53, leading to

elevated risk of many early-onset cancers, including sarcomas, acute leukemia, pre-

menopausal breast carcinoma, brain tumours, and adrenocortical carcinoma [8].

Neurofibromatosis is characterized by mutations in neurofibromin 1 (NF1) or 2 (NF2), both

of which are tumour suppressors [9]. The NF1 mutation results in a 20-fold increased risk of

RMS relative to the general population [2]. Costello syndrome, caused by mutations in

HRAS, is characterized by mental and physical developmental defects, as well as increased

risk of various malignancies (including rhabdomyosarcoma) [10]. Beckwith-Wiedemann

1

Syndrome, caused by genetic errors at chromosome region 11p15, is associated with

overgrowth, abdominal wall defects, and increased risk of tumours such as

rhabdomyosarcoma [11].

RMS is diagnosed based on histological examination by light or electron microscope, as

well as immunohistochemical detection of muscle-specific protein markers (including actin,

myosin heavy chain, desmin, myoglobin, Z-band protein and MyoD) [12].

Treatment modalities include chemotherapy, surgery, and radiation. Unfortunately, most

RMS tumours occur in the head and neck region or the genitourinary tract, and large surgical

resections in these sensitive sites are often not feasible [13]. The most common

chemotherapy regimen for RMS is the “VAC” combination, comprised of vincristine (an

inhibitor of microtubule polymerization), actinomycin D (a DNA intercalator), and

cyclophosphamide (a DNA alkylator) [14]. Ifosfamide (a DNA alkylator) and etoposide (a

topoisomerase II inhibitor) are also frequently used, as part of the “VAI” (vincristine,

actinomycin D, and ifosfomide) and “VIE” (vincristine, ifosfomide, and etoposide)

combinations [14]. Doxorubicin (a DNA intercalator) is also commonly used, often in

combination with vincristine and cyclophosphamide [15, 16].

With conventional multimodal therapy, patients with non-metastatic RMS have five-year

failure-free survival of approximately 75% [14]. However, for metastatic RMS patients

(which comprise 15% of cases) this figure drops to 25% [17]. Even more discouraging is a

recent study that showed little or no improvement in overall RMS survival from 1976 to

2000 [2].

2

n = 692

Non-metastatic

Failure-free survival

Years

Metastatic

n = 127

Failure-free survival

Years
Figure 1: Comparison of survival for non-metastatic and metastatic RMS. Although non-metastaticRMS has a decent outlook with five-year survival around 75% (with minor differences depending on the chemotherapy regime used), metastatic RMS yields a five-year survival rate of only 25%.
Figures modified from: 1) Crist W.M., et al., Intergroup Rhabdomyosarcoma Study-IV: Results for Patients With Nonmetastatic Disease. Journal of Clinical Oncology, 19(12), 2001: 3091-3102. 2) Breneman J.C., et al., Prognostic Factors and Clinical Outcomes in Children and Adolescents With Metastatic Rhabdomyosarcoma—A Report From the Intergroup Rhabdomyosarcoma Study IV. Journal of Clinical Oncology, 21(1), 2003: 78-84. Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved.

3

Most cases of RMS fall into one of two subtypes: embryonal and alveolar. They can be

distinguished at the molecular, genetic, and histological levels, and are associated with

different sites of occurrence and clinical outcomes. There are also several rarer subtypes. The

definitions of these other variants are more vague and controversial, with many different

classification systems in use [18].

Figure 2: Recent trends (1976-2000) in survival of RMS patients. There is a small increase in 4-year survival over the past few decades, but the improvement is not consistent (with large fluctuations). Furthermore, the difference in survival between 1976-1980 and 1996-2000 is not statistically significant. Reprinted with permission from Reference #2.

4

1.1.2 Embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) accounts for about two thirds of RMS cases [2].

It occurs most commonly in children during the first few years of life [2, 19], and tends to

arise in the head and neck region [20]. ERMS is associated with a more favourable prognosis

compared to alveolar RMS, with 10-year survival around 75% [13].

At a molecular level, ERMS is characterized by a loss of heterozygosity at chromosome

locus 11p15.5 [21, 22]. The exact implications of this LOH are unclear, but the region

contains several putative tumour suppressors, including GOK [23, 24], WT2 [25], and p57kip2

[25].

Loss of imprinting (LOI) is also observed at 11p15.5 [12, 26]. Interestingly, the pro-

proliferative insulin-like growth factor II (IGF-II) gene is maternally imprinted (paternally

expressed), while the putative tumour suppressor p57kip2 is imprinted in the other direction

[22]. Therefore, LOH (resulting from loss of the maternal allele and duplication of the

paternal allele) leads to overexpression of IGF-II but loss of p57kip2 expression, both of which

are changes that are thought to possibly contribute to tumourigenesis.

1.1.3 Alveolar rhabdomyosarcoma

The other major RMS subtype is alveolar rhabdomyosarcoma (ARMS), named for its

histological appearance, which bears some resemblance to pulmonary alveoli. ARMS

accounts for about a quarter of RMS cases, and is distributed evenly from ages 0 to 19 [2]. It

occurs more commonly in the extremities [20], and is associated with poorer clinical

outcomes compared to ERMS, including increased incidence of metastasis and higher

mortality [20]. 10-year survival for ARMS is only 20% (compared to 75% for ERMS) [13].

5

ARMS is usually associated with one of two chromosomal translocations, each fusing

together two transcription factors. The most common one, t(2;13)(q35;q14), fuses the DNA-

binding domain of paired box 3 (PAX3) with the transactivation domain of forkhead box

O1A (FKHR or FOXO1A) [12, 20, 27]. The resulting fusion protein is a potent transcription

factor that can disrupt the regulation of PTEN [28], VEGF [29], and IGF-I [30]. Less

frequently found is t(1;3)(q36;q14), which fuses the DNA-binding domain of PAX7 with the

transactivation domain of FKHR [31]. ARMS with t(1;13) translocations appear to be

clinically less aggressive than those with t(2;13). Translocation-negative ARMS are also

reported and may confer an intermediate biological behavior between the more common

subtypes.

6

Embryonal66%

Alveolar26%

Spindle cell1%

Mixed2%

Embryonal sarcoma

3%

Pleomorphic2%

Figure 3: Proportion of RMS subtypes. A survey of 848 patients aged 0-19, with RMS diagnosed between 1975 and 2005. Data taken from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) Program (Reference #2).

7

1.2 Estrogen Signaling in Rhabdomyosarcoma

1.2.1 Estrogen and its receptors

Estrogen is a steroid hormone (in fact, a group of three related steroid hormones: 17β-

estradiol, estrone, and estriol), in the same class of compounds as progesterone, testosterone,

glucocorticoids, and mineralocorticoids. Steroid hormones share the characteristic four-ring

carbon skeleton of cholesterol, the lipid precursor from which they are derived. They span an

wide range of biological functions, from metabolism and electrolyte homeostasis, to

functions in the reproductive, immune, and nervous systems. However, their basic mode of

action is similar: the hormone diffuses into a target cell, binds to a specific intracellular

receptor (a member of the steroid hormone superfamily of receptors), and together the

hormone-receptor complex acts as a transcription factor [32].

Estrogen has wide-ranging functions, including roles in reproduction (including the

buildup of the luteal surge during the menstrual cycle), development of breast tissue [33, 34]

and other secondary sexual characteristics [35], maintenance and protection of the

cardiovascular system in both men and women [36, 37], and preservation of bone integrity in

post-menopausal women [34, 38-40].

8

OH

O

OH

OH

OH

OH

OH

O

OH

OOH

O

C

cholesterol testosterone progesterone

17β-estradiol estrone estriol

O

O

OH

O OH

O

O

OH

OH

OH

aldosterone cortisol

Figure 4: Cholesterol and the steroid hormones. Steroid hormones share in common the characteristic four-ring structure of cholesterol, the precursor from which they are biosynthesized. The estrogens (17β-estradiol, estrone, and estriol) are the major female sex hormones. Progestogens (such as progesterone) are primarily involved in pregnancy. Androgens (such as testosterone) are the major male sex hormones. Glucocorticoids (such as cortisol) are involved in glucose metabolism. Mineral corticoids or mineralocorticoids (such as aldosterone) are involved in electrolyte resorption and balance.

9

Estrogen signaling begins with the synthesis of estrogen in endocrine cells. From there,

they travel through the bloodstream to target cells, enter by simple or facilitated diffusion,

and bind to estrogen receptors (ER) in the cytoplasm or nucleus. At this point, estrogen

signaling branches into two paths: genomic and nongenomic signaling.

In genomic signaling, the estrogen-bound receptor dimerizes and acts as a transcription

factor for the target cell. The dimer binds to specific recognition sites on DNA known as

estrogen response elements (ERE) [41-44], and recruits transcriptional coactivators or

corepressors upon binding [43, 45, 46].

The nongenomic mechanism is a more recent model in which the estrogen-bound ER

feeds directly into other signaling pathways, including the ERK, p38, Protein Kinase C, and

PI3K/Akt pathways [47-50]. For example, the estrogen-ER complex can interact with Src

and Sos [51-53] to activate the ERK pathway. Nongenomic signaling tends to occur more

rapidly, and can produce responses within seconds or minutes [43]. Although ERs reside

predominantly in the cytoplasm and nucleus, a very small proportion is found attached to the

plasma membrane, which may further contribute to nongenomic ER signaling.

Although the genomic and nongenomic pathways described above involve estrogen as

the inducing ligand, it should be noted that many estrogen-like molecules (including

synthetic compounds such as tamoxifen) could also bind to the ER and produce genomic and

nongenomic responses. More details will be discussed in Chapter 1.3 (“Selective Estrogen

Receptor Modulators”).

Estrogen receptors are modular proteins comprised of several functional elements,

including sites for DNA-binding, ligand-binding, coactivator-binding, and dimerization [42,

54, 55]. There are also two transcriptional activation elements: activation function 1 (AF-1),

10

which is ligand-independent, and activation function 2 (AF-2), which is ligand-dependent [42,

56, 57]. Although AF-1 provides some transactivation function to ER that is not bound to

ligand, full activity is only achieved by the synergistic combination of both AF-1 and AF-2 in

a ligand-bound ER [42, 57].

There are two known ER isoforms in humans: estrogen receptors α (ERα or ESR1) and β

(ERβ or ESR2), cloned in 1985 [58] and 1996 respectively [59, 60]. Both ERs share the basic

functional elements described previously. Sequence similarities between ERα and ERβ [42,

60] in the DNA-binding, ligand-binding, and dimerization regions enable both receptors to

recognize similar sites on DNA [42, 61], to bind most ligands with somewhat comparable

affinities [62], and to heterodimerize with one another [42, 63]. The remaining regions are

more distinct [42, 60], and as a whole ERα and ERβ often elicit different biological responses

[64, 65].

More recently a membrane-bound estrogen receptor, structurally unrelated to ERα or

ERβ, have garnered increasing attention. First cloned in 1996 [66], GPR30 is a G protein-

coupled receptor (GPCR) shown to bind 17β-estradiol, tamoxifen, fulvestrant, and a number

of other classical ER ligands [67]. It has been shown to activate several membrane-initiated

signaling pathways, including ERK, cAMP, and PI3K [67]. However, much less is known

about this newer estrogen receptor compared to the “classical” receptors ERα and ERβ.

1.2.2 Linking estrogen to rhabdomyosarcoma

Although there is a wealth of research on paracrine and autocrine signaling in RMS,

including pathways involving IGF [22, 68-71], VEGF [72-74], and EGF [75-78], endocrine

11

signaling is a newer area of exploration. However, there are several clues that point to

estrogen signaling as a potentially important player in the biology of RMS.

RMS incidence rate shows a bimodal distribution with respect to age of onset, peaking

shortly after birth and during puberty [2]. It is intriguing to consider that both peaks

correspond to periods of prolonged estrogen exposure.

Another clue comes from work on desmoid tumors, a rare non-metastatic but locally

aggressive fibrous tumour. 80% of desmoid tumour patients are women, mostly in their

childbearing years (ages 20 to 40) [79]. Therefore, it had been speculated that elevated

hormone levels might be involved in desmoid tumourigenesis. Although the mechanisms are

still unclear, many studies have shown that tamoxifen (a modulator of estrogen receptors)

leads to reduced size and local invasiveness of desmoids [80-82]. Today, tamoxifen is a

routine treatment option for desmoid tumors [83-85]. Given the success of tamoxifen in

desmoid tumours, our lab had previously postulated that it might be worthwhile to explore

the effects of this agent in related tumours of similar ontogeny, such as sarcomas [4].

There have been isolated case studies that suggest a possible connection between

estrogen and RMS [86, 87]. More recently, experimental results from our group have pointed

in the same direction [4]. It was shown that RMS primary tumours and cell lines express ERβ

(though not ERα). In RMS cell lines, 17-β-estradiol (E2) stimulates cell proliferation, while

4-hydroxytamoxifen (an active metabolite of tamoxifen) inhibits cell proliferation and

activates apoptotic signaling. Furthermore, removal of steroids from the culture medium

sensitizes the cells to 4-hydroxytamoxifen, and supplementing E2 back into the medium

partially reverses this effect.

12

Very little is understood about endocrine signaling in RMS, and given the possible links

between estrogen and RMS, as well as the recent evidence pointing to the efficacy of 4-

hydroxytamoxifen in RMS cell lines, it may be worthwhile to explore estrogen signaling as a

potential therapeutic target in RMS. In this study, we explore the mechanisms behind 4-

hydroxytamoxifen-induced apoptosis in RMS cell lines.

1.3 Selective Estrogen Receptor Modulators

1.3.1 Overview

Classically, most ER modulators can be considered agonists or antagonists, depending on

their activity relative to that of 17-β-estradiol (E2). Agonists (such as diethylstilbestrol)

mimic E2 by activating the ER in the same manner as E2. On the other hand, antagonists

(such as fulvestrant) produce no biological response; they have affinity for the receptor, but

no efficacy.

Over time, it became clear that this classic model is oversimplified. Many ER modulators

were found to have mixed activity. For example, tamoxifen (TAM), initially classified as an

antagonist or antiestrogen, actually exhibits both agonistic and antagonistic properties. It acts

as an antagonist in the breast, but an agonist in the uterus and bone [88, 89]. These “mixed-

activity” compounds are known as selective estrogen receptor modulators (SERM).

A key explanation for this mixed activity involves the differential expression of

transcriptional coregulators in various tissues. Recall that in the genomic model of ER

signaling, the dimerized complex binds to DNA and recruits coactivators or corepressors to

regulate transcription. These coregulators are expressed at different levels and proportions in

different tissues. SERMs may take advantage of these tissue-specific properties to produce

13

tissue-specific responses [90, 91]. For example, TAM acts as an antagonist in the breast by

recruiting corepressors (preferentially over coactivators) to the DNA, thus blocking

transcription of target genes. However, in the uterus (where TAM acts as an agonist), there is

a higher concentration of the coactivator Src-1 [90]. The local abundance of coactivators

effectively overwhelms the corepressors, allowing TAM to function as an agonist in this

environment.

Apart from this agonist-antagonist dichotomy, there is a further level of complexity in

this system. Instead of blocking the E2 pathway (like an antagonist) or retracing the same

pathway followed by E2 (like an agonist), SERMs can also redirect ERs into new genomic

pathways [89, 92]. For example, TAM has been shown to induce expression of maspin (a

tumor suppressor downregulated in many breast tumors) by binding onto ERα and following

a genomic pathway [93]. E2 itself, however, does not regulate maspin expression. In this case,

TAM is neither mimicking E2 nor opposing it, but rather, initiating an entirely new pathway

of its own.

Furthermore, ERα and ERβ often produce different responses to the same ligand. For

example, TAM is a partial agonist for ERα, but an antagonist (usually) for ERβ [54, 65, 94-

97]. Therefore, differential expression of ERα and ERβ in different tissues adds further

complexity to the mixed properties of SERMs.

14

ON

OH

O

N

lasofoxifene

ON

OH

4-hydroxytamoxifen tamoxifen

O

S

OH

OH

ON

raloxifene

OH

OH

OH

OH

(CH2)9

SO

CF2

CF3

fulvestrant

diethystilbestrol

Figure 5: Synthetic estrogen receptor modulators. Traditionally most estrogen receptor modulators were classified as either agonists (such as diethystilbestrol) or antagonists (such as fulvestrant). However, many compounds were found to have mixed agonistic/antagonistic activity depending on the tissue. These are known as selective estrogen receptor modulators (SERM). Tamoxifen (and its metabolite 4-hydroxytamoxifen) is a first-generation SERM used primarily to treat and prevent breast cancer. Raloxifene, a second-generation SERM, is used to prevent breast cancer and osteoporosis. Lasofoxifene is a third-generation SERM used to prevent and treat osteoporosis.

15

Prior to the development of SERMs, endocrine therapy faced a long-standing dilemma:

the benefits of estrogen supplementation (such as cardioprotection and preservation of bone

mineral density) must be balanced against the drawbacks (such as increased risk for breast

and uterine cancer). Therefore, a good ER modulator should work in opposing ways--namely,

to mimic estrogen in the cardiac vasculature and bone, but not in the breast and uterus. The

advent of SERMs opened up the possibility of new compounds that might offer such specific

activity profiles.

Since the development of the first generation of SERMs like TAM, newer waves of

agents have emerged. Second-generation SERMs (such as raloxifene, used for prevention

and treatment of osteoporosis, and prevention of breast cancer) were designed to improve

upon TAM by minimizing its associated risk of uterine cancer [98]. These first two

generations of SERMs were mostly developed by modifications on a common triphenyl

structure. The third generation (and beyond) of SERM development has expanded beyond

this structural template, producing entirely different compounds with further refined tissue-

specific activity profiles [98]. For example, lasofoxifene is a third-generation SERM with

highly agonistic activity in the bone, but is antagonistic in both the breast and uterus, making

it suitable for prevention and treatment of osteoporosis [99].

1.3.2 Tamoxifen

The story of tamoxifen began as an attempt to create a “morning-after” pill, following the

discovery of several triphenyl compounds with contraceptive effects in laboratory animals

[100, 101]. First synthesized in 1962, tamoxifen (initially known as ICI46474) was a failed

16

contraceptive (in fact, it was slightly profertile) recycled into one of the most widely used

anti-cancer agents [101].

Today, apart from its use for the treatment and prevention of breast cancer [102], TAM is

also used to treat a number of other malignancies, including desmoid [84], hepatocellular

[103, 104], ovarian [105, 106], pancreatic [107, 108], and renal cell carcinomas [109, 110],

as well as melanoma and glioma [111]. As a beneficial side effect, TAM helps prevent

osteoporosis in post-menopausal women, evidenced by decreased incidence of osteoporotic

fractures [112] and increased bone mineral density [113]. TAM may also provide some

protection against coronary vascular disease by lowering cholesterol [114]. Negative side

effects of TAM include elevated risk of uterine cancer, thrombosis, stroke, and cataracts [115,

116].

TAM has a prolonged half-life, which offers an advantage against late-stage tumours

[102]. For example, TAM and raloxifene are structurally related, and equally effective in

preventing breast cancer [117], yet TAM is superior to raloxifene in its ability to combat the

tumour itself [102]. With a much shorter half-life, raloxifene is rapidly cleared from the body.

Therefore, although raloxifene may have some efficacy in cancer prevention, it does not

accumulate in sufficient concentrations to overcome late-stage breast tumours [102].

In the body, TAM is converted (mainly by liver enzymes) into several active metabolites,

including 4-hydroxytamoxifen, desmethyltamoxifen, α-hydroxytamoxifen, and tamoxifen N-

oxide [118, 119]. 4-hydroxytamoxifen is among the most commonly studied, and exhibits

affinities up to 100 times stronger than TAM itself [119].

17

1.4 Mechanisms of Tamoxifen and 4-Hydroxytamoxifen

Tamoxifen (TAM) and 4-hydroxytamoxifen (4OHT) have been associated with a number

of molecular targets. The following is an overview of some potential mechanisms proposed

for TAM or 4OHT. These include direct binding targets (such as ERs, AEBS, and PKC), as

well as downstream targets (such as MAPKs, TGF-β, and survivin). Some of these may be

linked in the same pathway, whereas others belong to separate independent pathways.

1.4.1 Estrogen receptors

TAM was designed to modulate estrogen activity. Not surprisingly, estrogen receptors

(ER) have been studied intensely in the search for the mechanisms of TAM. Evidence of ER

involvement comes from many levels of evaluation, including structural studies, binding

assays, molecular and functional data taken in vitro and in vivo, and clinical data.

TAM and 4OHT demonstrate competitive binding with estrogen [120-122]. Crystal

structures of ER-4OHT complexes confirm a direct interaction [123, 124].

Many effects of TAM and 4OHT are ER-dependent. For example, mutations in ERα alter

its response to 4OHT [125, 126]. On a more functional level, 4OHT-resistant ERα-negative

HeLa cells become sensitized to 4OHT upon stable transfection of ERα [127]. In

myoepithelial cells, TAM upregulates nitric oxide (which is growth-suppressive [128]) in an

ERβ-dependent manner [129]. Breast cancer cells kill lymphocytes by expressing FasL, and

TAM inhibits this FasL expression in an ER-dependent manner [130]. ERs have also been

shown to recognize novel binding sites on DNA when activated by TAM or 4OHT. For

example, binding of 4OHT to ER leads to transactivation of quinone reductase via

18

recognition of an electrophile response element (not the estrogen response element

traditionally associated with ER signaling) [131].

Patients with ER-positive breast cancer respond better to tamoxifen compared to those

with ER-negative tumours. Traditionally this was observed with respect to ERα status [132],

but recently ERβ expression was also shown to be associated with more favourable prognosis

[133].

Despite the great volume of evidence supporting a role for ERs, the downstream events

are not as clear. Furthermore, it has long been observed that estrogen cannot fully protect

against micromolar concentrations of TAM in vitro, suggesting the existence of non-ER

targets [134]. Therefore, ERs are only the beginning of a subset of pathways.

The membrane-bound ER, GPR30, is also known to bind TAM and 4OHT, but little or

no evidence exists to link it to apoptosis. In fact, quite the opposite, GPR30 has been

implicated in TAM resistance [135], and in the progression of thyroid [136] and endometrial

[137] cancers.

1.4.2 Caspases

Cell death following TAM or 4OHT treatment has been consistently linked to activation

of caspases [4, 138]. For example, treatment of MDA-MB-231 and BT-20 breast cancer cells

with TAM or 4OHT leads to increased activity of caspase-3, -8, and -9, followed by the

appearance of apoptotic morphology [138]. Pretreatment with a caspase inhibitor blunted the

apoptotic response. Other studies have shown cleavage of PARP (a caspase-3 substrate)

following TAM or 4OHT treatment [139]. In N-nitroso-N-methylurea-induced rat mammary

19

tumours, TAM leads to elevated caspase activity and a 10-fold increase in tumour apoptotic

index [140].

The evidence for caspase involvement is overwhelming, but the upstream events leading

to caspases activation are not as well elucidated.

1.4.3 Microsomal antiestrogen binding site

The microsomal antiestrogen binding site (AEBS) is a membrane-associated complex

with high affinity for SERMs bearing a cationic aminoethoxy side chain (including TAM and

4OHT), but no affinity for estrogen or non-cationic SERMs [141]. It is comprised of two

enzymes (3β-hydroxysterol-δ8-δ7-isomerase and 3β-hydroxysterol-δ7-reductase) involved in

cholesterol metabolism [142]. Binding of TAM to the AEBS inhibits its enzymatic functions,

leading to accumulation of sterol substrates that, in turn, can induce cell cycle arrest and

apoptosis [141, 143].

Although the mechanisms leading to apoptosis are unclear, accumulation of these sterols

results in them becoming auto-oxidated into oxysterols that have been shown to depolarize

the mitochondrial membrane [141]. Mitochondrial depolarization may trigger apoptotic

release of cytochrome c.

While there is overwhelming evidence suggesting that TAM physically binds onto the

AEBS, the downstream events leading to apoptosis, and the relative importance of this

pathway to apoptosis, are less obvious.

20

1.4.4 Calcium signaling

TAM triggers a rapid increase of intracellular calcium [144]. Chelation of calcium blocks

TAM-induced apoptosis in HepG2 human hepatoblastoma cells [145]. TAM-induced

calcium also leads to generation of reactive oxygen species (ROS) in HepG2 cells, followed

by apoptosis [146]. Inhibition of ROS protected against apoptosis, and chelation of calcium

abrogated ROS generation. In MCF-7 and glioma cells, TAM leads to expansion of calcium

waves and sensitized the cells to calcium ionophore-induced cell death [147]. More recently,

Bollig et al. demonstrated an ER-independent mechanism for 4OHT, involving calcium

release from the endoplasmic reticulum and upregulation of protein phosphatase 1α (PP1α)

[148]. siRNA knockdown of PP1α inhibited calcium release and conferred protection against

TAM.

TAM and 4OHT may also modulate calcium signaling by binding calmodulin, thus

inhibiting calmodulin-dependent enzymes such as cAMP phoshodiesterase [149]. Finally,

TAM has been shown to antagonize calcium-dependent induction of the mitochondrial

permeability transition, a mechanism that is commonly implicated in chemical-induced

apoptosis [150].

1.4.5 Mitochondria

Many drugs collapse the mitochondrial transmembrane potential (MTP) [151], triggering

the release of cytochrome c into the cytoplasm [152]. Cytochrome c, in conjunction with

apoptosis-inducing factor (AIF), initiates the intrinsic apoptotic pathway by activating

caspase-9 [153, 154]. TAM and 4OHT may have a similar effect.

21

Incubation of TAM with isolated rat liver mitochondria leads to complete collapse of the

MTP [155]. In human mammary epithelial cells, TAM treatment leads to MTP collapse and

mitochondria condensation alongside caspase activation [156]. TAM-resistant cells obtained

by repeated passage contained increased mitochondrial mass, and resisted MTP collapse

[156].

TAM treatment in MCF-7 and MDA-MB-231 breast cancer cells under serum-free

conditions led to rapid apoptosis within an hour, and is associated with MTP collapse and

release of cytochrome c from the mitochondria [157]. Inhibition of mitochondrial

permeability transition protected against TAM-induced apoptosis. Cell death was also

associated with elevated reactive oxygen species (ROS). Inhibition of NADPH oxidase

protected against apoptosis, but did not prevent cytochrome c release, suggesting that

cytochrome c release precedes ROS production.

In a recent study, TAM led to mitochondrial depolarization, and translocation of

cytochrome c and AIF from the mitochondria [141]. They also detected an increase in the

ratio of Bax (pro-apoptotic) to Bcl-2 (anti-apoptotic) expression, shifting the balance towards

apoptosis.

1.4.6 Transforming growth factor β

Transforming growth factor β (TGF-β) is a cytokine with seemingly contradictory roles.

In primary cells and cell lines TGF-β is growth-inhibitory and pro-apoptotic, but in late-stage

tumours it also stimulates angiogenesis and inhibits antitumour immune responses [158]. The

net effect of TGF-β is unclear, but there seems to be a consistent association between TAM

and TGF-β.

22

In some studies, TAM leads to increased TGF-β activity in breast cancer cells, and anti-

TGF-β1 antibody inhibits TAM-induced apoptosis [159-161]. Moreover, TAM results in

elevated secretion of TGF-β in TAM-sensitive cells, but not in TAM-resistant variants [158,

161]. In advanced metastatic breast cancer patients, those who respond to TAM tend to have

elevated TGF-β2, whereas non-responsive patients show no changes in TGF-β2 expression

[158, 161, 162].

However, there are also studies pointing in the opposite direction, showing a decrease in

TGF-β following TAM treatment [163, 164]. This may be, in part, due to the timescale

involved. For example, Perry et al. reported a slight decrease in TGF-β protein levels within

6 hours, followed by a significant increase after 12 hours [159].

1.4.7 Protein kinase C

It is well established that TAM and 4OHT can bind, translocate, and modulate protein

kinase C (PKC) activity [165]. PKC inhibition has been heavily studied as an avenue for anti-

manic therapy, and TAM has consistently shown to be a promising anti-manic agent [166,

167]. However, the relevance of PKC in the context of TAM-induced apoptosis is still being

investigated.

TAM is often used to treat glioma. Given the extreme overexpression of PKC (orders of

magnitude higher than normal) and the efficacy of PKC inhibitors in glioma cells, it is

plausible that TAM works in part by targeting PKC [168].

However, there is contradictory evidence regarding whether TAM activates or inhibits

PKC, depending on the system used, though more studies tend to point to TAM as an

inhibitor [169-173]. For example, Ahn et al. proposed a mechanism in MCF-7 breast cancer

23

cells involving activation of PKC [169]. Gundimeda et al., on the other hand, showed that

TAM and 4OHT are PKC inhibitory in MBA-MB-231 breast cancer cells [174].

This is further complicated by the fact that PKC modulators sometimes exhibit opposing

effects: the same agent may be pro-apoptotic in one cell type, but anti-apoptotic in another

[175].

1.4.8 Mitogen-activated protein kinases

Mitogen-activated protein kinases (MAPK) are serine-threonine kinases involved in a

wide range of cellular responses, including proliferation, differentiation, and apoptosis. They

can be activated by a number of stimuli, including growth factors, radiation, cytokines, and

oxidative stress, ultimately leading to phosphorylation of transcription factors, and

subsequent transcriptional regulation. MAPK signaling cascades share in common the

sequential phosphorylation of three central kinases: MAP3K (MAP kinase kinase kinase),

MAP2K (MAP kinase kinase), and MAPK itself. By far the most commonly studied MAPKs

are c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). All

three have been shown to have a possible role in TAM- and 4OHT-induced cell death.

When breast cancer cells are treated with TAM, JNK phosphorylation was observed

alongside caspase cleavage and apoptosis [138]. A dominant-negative JNK1 mutant partially

blocks TAM-induced apoptosis, suggesting a causal role for JNK1 in mediating TAM

activity.

Early evidence for p38 involvement was shown in MCF-7 cells, where 4OHT activates

p38 alongside apoptosis, and inhibition of p38 protects against 4OHT [127]. The same

pattern was observed in HeLa cells stably transfected with ERα. One of the downstream

24

transcriptional targets of p38 is thought to be TGF-β. Both are activated in response to 4OHT,

and inhibition of p38 also abolishes TGF-β activation [161, 176].

Studies have also shown an involvement for ERK. For example, treatment of MCF-7

breast cancer cells with TAM leads to rapid ERK activation, and inhibition of ERK using

PD98059 protected against TAM-induced apoptosis [177]. Inhibition of epidermal growth

factor receptor (EGFR) antagonizes both ERK activation and apoptosis, consistent with a

nongenomic ER pathway. In another study, 4OHT leads to ERK activation and apoptosis in

HeLa cells stably transfected with human ERα [92]. Inhibition of ERK using PD98059

protects against apoptosis, while activation of ERK using epidermal growth factor (EGF)

enhances apoptosis.

1.4.9 Other proposed mechanisms

TAM inhibits glucosylceramide synthase, leading to accumulation of its substrate,

ceramide [178, 179]. Ceramide, in turn, has been commonly linked to apoptosis in many

systems [180, 181].

TAM also upregulates c-myc mRNA and protein, and downregulation of c-myc using an

antisense oligonucleotide protects MDA-231 breast cancer cells against TAM (whereas a

nonspecific oligomer had no effect) [182]. While this particular study shows strong evidence

for c-myc involvement in TAM-induced apoptosis, in general c-myc has seemingly

contradictory and multi-faceted roles. It is involved in both proliferation and apoptosis. Its

effects are global; c-myc is believed to regulate 15% of all genes [183]. Despite its many

roles in driving apoptosis [184], it is also a proto-oncogene, and found to be upregulated in

many cancers [185, 186]. Overexpression of c-myc is also a basis of tumourigenesis in many

25

animal models [187, 188]. The downstream effects of TAM-induced c-myc will need to be

established to complete this potential pathway.

TAM is a hydrophobic compound that can partition into biomembranes, which may have

wide-ranging consequences, including disruption of membrane fluidity [111, 189]. The

details are unclear, but conceivably, changes in membrane fluidity may trigger the release of

membrane-anchored second messengers leading to downstream signaling events [111, 150].

TAM and 4OHT have also been shown to inhibit a number of potassium channels by

partitioning into the membrane and disrupting interactions between the channels and

phosphatidylinositol 4,5-bisphosphate (PIP2) [190, 191].

Insulin-like growth factors (IGF) are mitogens for breast cancer, which express the IGF-I

receptor (IGF-IR) [134]. TAM has been shown to disrupt IGF signaling by inhibiting IGF-I-

induced phosphorylation of IGF-IR, and by blocking downstream phosphatidylinositol 3-

kinase (PI3K) signaling [134, 192]. TAM treatment in vivo is also associated with decreased

serum IGF-I [134].

Survivin belongs to the inhibitor of apoptosis (IAP) family of proteins, and functions as a

negative regulator of apoptosis. TAM treatment is associated with decreased survivin

expression, thus promoting apoptosis [103, 104]. In human breast cancer cells, inhibition of

survivin enhances TAM-induced apoptosis, and overexpression of survivin confers resistance

against TAM [193].

Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of extracellular

matrix, and has been shown to have anti-angiogenic properties. Transduction of the MMP-9

gene leads to tumour regression in vivo [194]. Several studies have shown that TAM leads to

26

increased MMP-9 activity in vitro and in vivo, correlated with decreased tumour growth and

angiogenesis [164, 194, 195].

Interestingly, due to the positive charge of TAM (imparted by an amino group), it is able

to electrophoretically accumulate into the negatively charged mitochondrial matrix (40-fold

more concentrated) [196]. There, it inhibits topoisomerases and downregulates the

transcription of mitochondrial genes. Over time, it also leads to degradation of mitochondrial

DNA. Although the authors of this study did not discuss their finding in the context of

apoptosis, the idea of TAM eliciting global effects on mitochondrial genes is a novel and

overlooked mechanism, and the implications are relatively unexplored.

Certain actions of TAM are, so far, not linked to apoptosis. For example, estrogen

epoxidation is one of the reactions involved in breast tumourigenesis, and TAM competes

with estrogen for epoxidation [197]. This was proposed as a mechanism for cancer

prevention rather than cancer treatment. Estrogen-related receptors (ERR) are known to bind

TAM and 4OHT, but little or no evidence exists to link them to apoptosis. In fact, ERRγ has

instead been linked to TAM resistance [198].

27

1.5 Rationale and Hypothesis

Greater understanding of the mechanisms behind TAM- or 4OHT-induced apoptosis may

help identify new therapeutic targets, or lead to improved combinatorial drug formulations

for the treatment of RMS. In addition, we become better equipped to understand the

mechanisms of TAM resistance once we understand the mechanisms of its anti-cancer effects.

Despite the wealth of research on the mechanisms of TAM and 4OHT, more work is

needed to help connect these scattered leads, and many gaps are yet to be filled before the

many pieces of data can be fit into a coherent network. Furthermore, the effect of 4OHT in

RMS is a new area of research, and none of the mechanistic studies for TAM or 4OHT

described in Chapter 1.4 have been applied to RMS.

In this study, we explore the relevance of ER, MAPK, and caspase signaling during

4OHT-induced cell death in RMS. We hypothesize that 4OHT-induced apoptotic signaling is

mediated by a causal sequence from ERs to MAPKs to caspases.

28

CHAPTER 2: MATERIALS AND METHODS

Cell cultures

Cell lines (from Dr. A. Thomas Look of Dana-Farber Cancer Institute, Boston, MA) were

derived from embryonal (RD) and alveolar (RH4, RH30) RMS tumours. Cells were

maintained in Dulbecco's Modified Eagle Medium (DMEM; Wisent, Cat #: 319-005-CL)

with 10% fetal bovine serum (FBS) (Wisent, Cat #: 080450). Drug treatments were carried

out under serum-free conditions, using either DMEM or phenol red-free DMEM (prfDMEM)

(Wisent, Cat #: 319-051-CL).

Drug treatments

RMS cells were plated at a density of 104 cells/well in 96-well plates for MTT assay, or

at 3×105 cells/well in 6-well plates for protein extraction. They were allowed to adhere and

recover for at least 6 hr in DMEM + 10% FBS, then serum-starved overnight in DMEM or

prfDMEM. After overnight serum-starvation, the cells were subject to the appropriate drug

treatments (for details, see captions for Figures 6 to 14), and undergo either the MTT assay,

or protein extraction for western blot.

The cell plating numbers for MTT assay (104 cells/well) and western blot (3×105

cells/well) result in similar cell densities, as the area of each well in the 6-well plate is

approximately 30 times that of a well in the 96-well plate.

Z-4-hydroxytamoxifen (4OHT) was purchased from Sigma (H7904-5MG). The MAPK

inhibitors SP600125, SB203580, and PD98059 were purchased from Calbiochem, and

reconstituted in dimethyl sulfoxide (DMSO). Fulvestrant (ICI182780) was purchased from

29

Tocris Bioscience, and reconstituted in ethanol. Vehicle controls ensured that concentrations

of ethanol and/or DMSO in the medium remained constant within each set of treatments.

MTT viability assays

Cells were plated and treated in 96-well plates (as described above) in triplicates.

Following the appropriate drug treatment, the cells were incubated in 100 µL/well of medium

containing 10% TACS MTT Reagent (R&D Systems). After 3 hr, 100 µL of detergent

solution (provided with the assay kit) was added to each well, and incubated for 4 hr to

overnight at 37°C in the dark. Absorbance was measured at 570 and 650 nm, and the

difference (A570 minus A650) was recorded as a raw measure of overall cell viability. These

raw values were normalized to the vehicle average to yield percent cell viability. Data from

each triplicate set were averaged, and the average values from 3 experiments (involving 3

independently passaged populations of cells) were used for statistical analysis. Results are

shown as mean (from the 3 replicates × 3 experiments) +/− SEM (from the 3 experiments).

This assay measures the ability of the cells to metabolize the tetrazolium MTT reagent (3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), as an indication of their

combined metabolic activity. Therefore, the word “viability” in this context is used loosely to

refer to the collective effects of proliferation, cell death, and changes in cellular metabolic

rate.

30

Western blots

Total cellular protein was extracted using ice-cold EBC lysis buffer (120 mM NaCl, 50

mM Tris pH 8, 0.5% Nonidet P-40) containing Complete Mini Protease Inhibitor Tablets

(Roche) (1 tablet per 7-10 mL) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets

(Roche) (1 tablet per 7-10 mL). Lysates were run on a 12% SDS-PAGE gel under reducing

conditions, transferred onto PVDF membrane (Millipore), blocked in Odyssey Blocking

Buffer (LI-COR Biosciences), incubated overnight at 4°C with primary antibodies, and then

for 30-45 min at room temperature with anti-rabbit (680 nm) or anti-mouse (800 nm) infrared

dye-tagged secondary antibodies. Blots were scanned using the Odyssey Infrared Imaging

System (LI-COR Biosciences) at 700 and 800 nm (to detect anti-rabbit and anti-mouse

antibodies, respectively). To sequentially detect different proteins on the same blot, the

membrane was stripped with PVDF stripping buffer (LI-COR Biosciences), then re-blocked

and re-probed as before.

Background-adjusted densitometric values were taken using the Odyssey Infrared

Imaging System Application Software (LI-COR Biosciences). For the MAPK proteins (JNK,

p38, and ERK), densitometric results are presented as a ratio of phosphorylated MAPK over

total MAPK. For cleaved caspase-3 and cleaved PARP, results are normalized to vinculin

(the loading control). JNK (and phospho-JNK) appear as 2 distinct bands at 46 and 54 kDa;

densitometry was performed on each band separately. ERK (and phospho-ERK) appears as 2

distinct bands at 42 and 44 kDa; densitometry was performed on the collective intensity of

both bands (which are too close to densitometrically differentiate with precision). Cleaved

caspase-3 exists as 2 bands at 17 and 19 kDa, but densitometry was performed only on the 19

kDa band (the 17 kda band is weaker and does not show up consistently).

31

Primary mouse antibody for vinculin was purchased from Millipore. Primary rabbit

antibodies for cleaved caspase-3, cleaved PARP, Thr202/Try204-phosphorylated ERK1/2,

total ERK1/2, Thr183/Tyr185-phosphorylated JNK, total JNK, Thr180/Try182-

phosphorylated p38, and total p38 were purchased from Cell Signaling Technology. Anti-

rabbit and anti-mouse infrared dye-tagged secondary antibodies were purchased from LI-

COR Biosciences.

Statistical analysis

Statistical analyses were performed using two-tailed Student’s t-test, with unequal

variance. A p value of less than 0.05 was considered statistically significant. Data are

presented as mean +/− SEM (standard error of the mean) from experiments involving 3

independently passaged populations of cells.

32

CHAPTER 3: RESULTS

The results are organized into four general sections. The first section establishes the

effects of 4OHT on RMS cell lines (including caspase activation), consistent with a previous

finding in our lab [4]. The second and third sections explore the mechanisms of 4OHT,

focusing on the involvement of MAPKs and ERs. The final section addresses data that

suggest a link between MAPKs and ERs.

3.1 Effects of 4OHT on RMS cell lines

A previous study from our lab [4], the first to explore the ER pathway in RMS, has

shown that 4OHT leads to decreased cell viability and induces apoptotic signaling. The

treatments were carried out in 10% serum, and lasted at least 24 hr. In my experiments,

however, a shorter treatment time is preferred due to the extreme instability of the MAPK

inhibitors. Serum-starvation sensitized the cells to 4OHT, allowing the treatment time to be

reduced to 3 hr while retaining comparable levels of effect.

For most of my treatments, I used DMEM free of phenol red (a pH indicator that exhibits

weak estrogenic activity). This was a precautionary measure in case phenol red interferes

with the binding of 4OHT onto ERs, which would overcomplicate the mechanisms that we

are attempting to explore.

3.1.1 4OHT leads to loss of cell viability

Before exploring the mechanisms behind 4OHT, the overall effect of 4OHT in the

treatment regime was established: overnight serum-starvation followed by 3 hr 4OHT

33

treatment. Under these conditions, 10 µM 4OHT drastically reduced cell viability as detected

by the MTT assay (Figure 6), although 5 µM had little or no effect. In subsequent

mechanistic experiments, 10 µM 4OHT is used to establish the basal effect.

0%

20%

40%

60%

80%

100%

120%

RD RH4 RH30Cell line

Perc

ent c

ell v

iabi

lity

0 µM 4OHT 5 µM 4OHT 10 µM 4OHT

* ***

***

Figure 6: 4OHT leads to loss of viability in RMS cell lines. Cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight, and treated with 0, 5, or 10 µM 4OHT (and equal concentrations of vehicle) in serum-free medium. After 3 hr of treatment, cell viability was measured using MTT assay. Shown here are mean (+/− SEM) percent cell viability relative to the 0 µM treatment. * p < 0.05, *** p < 0.001, two-tailed unequal variance t-test.

34

3.1.2 4OHT induces apoptotic signaling

At the same treatment conditions (overnight serum-starvation followed by 3 hr 4OHT

treatment), 4OHT also led to increased cleavage of caspase-3 and poly(ADP-ribose)

polymerase (PARP), both of which are markers of apoptosis (Figure 7). Consistent with the

MTT results, 10 µM 4OHT had a significant effect, while 5 µM had little or no effect.

RD RH4

4OHT (µM) 0 5 10 0 5 10 0

cleaved casp-3

cleaved PARP

vinculin

A B

0.1

1

10

100

1000

RD RH4 RH30

Cell line

Rel

ativ

e un

its


*

*

0.1

1

10

100

1000

RD RCe

Rel

ativ

e un

its

0 µM 4OHT 5 µM 4

*

Figure 7: 4OHT induces apoptotic signaling in RMS cell lines. Cells were10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free (treated with prfDMEM containing 0, 5, or 10 µM 4OHT (and equal concentratihr of treatment, whole-cell protein lysates were harvested for detection of cleacleaved PARP (B) via western blot. Background-adjusted densitometric valuecleaved caspase-3, only the 19 kDa band was measured by densitometry). S(+/− SEM) values, normalized to both the loading control vinculin and to the 01 relative unit). Note the logarithmic scale (y-axis). * p < 0.05, *** p < 0.001, two-tailed unequal variance t-test.

35

RH3

5 10

17 kDa

19 kDa

H4 RH30ll line

OHT 10 µM 4OHT

****

plated in DMEM + prf) DMEM, and ons of vehicle). After 3 ved caspase-3 (A) ands were taken (for hown here are mean µM values (defined as

3.2 Involvement of MAPK in the effects of 4OHT

Having established that 10 µM 4OHT leads to loss of cell viability, as well as increased

apoptotic signaling, we next explored whether this may be linked to MAPK signaling.

3.2.1 4OHT-induced apoptotic signaling is associated with increased MAPK

phosphorylation

At the same conditions in which apoptotic signaling is observed (Figure 7), an increase in

the phosphorylated forms of JNK, p38, and ERK were detected, whereas total

(phosphorylation + non-phosphorylated) levels of these proteins remained approximately

constant (Figure 8).

3.2.2 MAPK inhibition protects against 4OHT-induced loss of viability

Having established a correlation between 4OHT-induced apoptotic signaling and MAPK

activation, we next explored whether there is a causal link. To this end, we pretreated RD

cells for 30 min with MAPK inhibitors, before initiating the 3 hr 10 µM 4OHT treatment

(with sustained inhibitor treatment throughout this 3 hr period). In cells treated with 4OHT

alone, an extensive loss of cell viability was detected, as expected (Figure 9). However, when

a JNK inhibitor (SP600125) or ERK inhibitor (PD98059, a direct inhibitor of MEK1/2,

which is responsible for ERK phosphorylation) was present, this effect of 4OHT was

partially blunted (Figure 9).

36

3.2.3 MAPK inhibition blocks 4OHT-induced apoptotic signaling

Consistent with the functional data shown in Figure 9, inhibition of JNK or ERK under

the same conditions also blunted the apoptotic signaling induced by 4OHT, suggesting a

causal link between MAPK activation and apoptotic signaling. When treated with 4OHT

alone, RD cells showed increased JNK phosphorylation and increased apoptotic markers

(cleaved caspase-3 and cleaved PARP), but pre- and co-treatment with the JNK inhibitor

SP600125 not only reduced phosphorylated JNK, but also reduced levels of the apoptotic

markers (Figure 10). A similar effect is observed using the p38 inhibitor SB203580 and the

ERK inhibitor PD98059 (Figure 10).

37

02468

101214

RD RH4 RH30

Cell line

Rel

ativ

e ph

osph

o-/to

tal J

NK

(46

kD)


*

*

RD RH4 RH30

10 5 0 0 5 1050 10

0

1

2

3

4

RD RH4 RH30

Cell line

Rel

ativ

e ph

osph

o-/to

tal p

38


*

**

02468

10

RD RH4 RH30

Cell line

Rel

ativ

e ph

osph

o-/to

tal E

RK


** ***

38

4OHT (µM)
54 kDa
phospho-JNK

46 kDa

totalJNK

vinculin

Figure 8: 4OHT leads to increased MAPK phosphorylation in RMS cell lines. Cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free(prf) DMEM, and treated with prfDMEM containing 0, 5, or 10 ìM 4OHT (and equal concentrations of vehicle). After 3 hr of treatment, whole-cell protein lysates were harvested for detection of the MAPK proteins JNK, p38, and ERK (and their phosphorylated forms). Shown are mean (+/− SEM) background-adjusted densitometric ratios between phosphorylated and total MAPKs, normalized to the 0 ìM value (defined as 1 relative unit). JNK appears as two distinct bands (46 kDa and 54 kDa) due to alternative splicing. * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed unequal variance t-test.

0%

20%40%

60%

80%100%

120%

vehicle 10 µM 4OHT 50 µM SP600125 10 µM 4OHT + 50µM SP600125

Treatments

Per

cent

cel

l via

bilit

y

0%20%40%60%80%

100%120%

vehicle 10 µM 4OHT 50 µM PD98059 10 µM 4OHT + 50µM PD98059

Treatments

Per

cent

cel

l via

bilit

y

Figure 9: MAPK inhibition protects against 4OHT-induced loss of viability. RD cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight, pre-treated for 30 min with vehicle or a MAPK inhibitor (SP600125 or PD98059), and treated for 3 hr with the four basic permutations: no drug (vehicle), 4OHT alone, MAPK inhibitor alone, or a combination of 4OHT + inhibitor. Following treatment, cell viability was measured using MTT assay. Shown here are mean (+/- SEM from 3 experiments) percent cell viability relative to that of vehicle treatment (defined as 100%).

39

02468

1012

veh 10 µM 4OHT 50 µM SP 10 µM 4OHT +50 µM SP

Treatments

Rel

ativ

e un

its

phospho-/total JNK (46 kDa)phospho-/total JNK (54 kDa)cleaved caspase-3cleaved PARP

* *

10 µM 4OHT50 µM SP

＋－

－

－

－

＋

＋

＋

cCasp-3

cPARP

46 kDaphospho-

JNK 54 kDa

total JNK

vinculin

0.1

1

10

100

Vehicle 10 µM 4OHT 20 µM SB 10 µM 4OHT +20 µM SB

Treatments

Rel

ativ

e un

its

phospho-/total p38cleaved caspase-3cleaved PARP

*

10 µM 4OHT20 µM SB

－－

＋－

－

＋

＋

＋

cCasp-3

cPARP

phospho-p38

total p38vinculin

10 µM 4OHT50 µM PD

＋－

－

－

－

＋

＋

＋

05

10152025303540

Vehicle 10 µM 4OHT 50 µM PD 10 µM 4OHT +50 µM PD

Treatments

Rel

ativ

e un

its

phospho-/total ERKcleaved caspase-3cleaved PARP

*

cCasp-3

cPARP

phospho-ERK

total ERK

vinculin

Figure 10: MAPK inhibition protects against 4OHT-induced apoptotic signaling. RD cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight, pretreated for 30 min with vehicle or MAPK inhibitor, and treated for 3 hr with the four basic permutations: vehicle, 4OHT, inhibitor, or 4OHT + inhibitor. Whole-cell protein lysates were harvested for western blot. Data shown here are background-adjusted mean (+/− SEM) densitometric values/ratios. For cleaved caspase-3 and cleaved PARP, values are normalized to both the loading control (vinculin) and the vehicle values (defined as 1 relative unit). For JNK, p38, and ERK, values shown are background-adjusted densitometric ratios between phosphorylated and total forms, normalized to vehicle ratios (defined as 1 relative unit). * p < 0.05, two-tailed unequal-variance t-test.

40

3.3 Involvement of estrogen receptors in the effects of 4OHT

Having established a likely causal role for MAPKs in activating apoptotic signaling

following 4OHT treatment, we now move on to another candidate target: estrogen receptors.

In this section, we explore whether estrogen receptors might also be involved in 4OHT-

mediated effects.

3.3.1 ER inhibition protects against 4OHT-induced loss of viability

Fulvestrant is a pure antagonist of ERα and ERβ in humans. We decided to test whether

inhibition of ERs using fulvestrant can block the effects of 4OHT. RMS cells were pretreated

for 30 min with fulvestrant, before being subjected to the standard 3 hr 4OHT treatment (with

continued fulvestrant co-treatment throughout this 3 hr period). When treated with 4OHT

alone, RMS cells showed a drastic reduction in viability as expected, but pre- and co-

treatment with fulvestrant partially abolished this effect (Figure 11). Note that treatment with

fulvestrant had no effect on cell viability, which at first may be surprising given that

fulvestrant is a potent ER inhibitor. However, recall that these experiments were done under

serum-free (and hence, estrogen-free) conditions. In other words, there is no estrogen binding

onto the ERs in the first place, so therefore ER inhibition by fulvestrant has no effect on the

cells.

The cytotoxic effects of 4OHT can be easily observed under a light microscope. RMS

cells rapidly lose their well-defined spindle shape and adopt a smaller rounded shape, and

eventually lose their adherence to the culture plate. The striking protective effects of

fulvestrant can also be observed under the microscope. Figure 12 shows images taken from a

differential interference contrast (DIC) microscope, with four panels corresponding to each

41

of the four treatments (vehicle, 4OHT alone, fulvestrant alone, and 4OHT + fulvestrant) in

RD cells. The vehicle panel shows the normal appearance of RD cells, a characteristic

sharply defined spindle shape. In the 4OHT alone panel, the cells are shrunk into a small

rounded shape, and have a darkened colour (indicative of floating cells). However, when

treated with a combination of 4OHT and fulvestrant, the cells are largely reversed back to

their normal appearance. No staining was done on these cells, so the specific biological

conclusions from Figure 12 alone are limited. However, the images are meant to complement

results from the MTT assay (Figure 11) and western blots (Figure 13), and to show the

protective effect of fulvestrant against 4OHT on a raw appearance level.

0%

20%

40%

60%

80%

100%

120%

RD RH30Cell line

Perc

ent c

ell v

iabi

lity

vehicle 10 µM 4OHT 5 µM Fulv 10 µM 4OHT + 5 µM Fulv

* **

Figure 11: The ER inhibitor fulvestrant protects against 4OHT-induced loss of viability. Cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free DMEM, pre-treated for 30 min with vehicle or fulvestrant, and treated for 3 hr with the four basic permutations: no drug (vehicle), 4OHT alone, fulvestrant alone, or a combination of 4OHT + fulvestrant. Cell viability was measured using MTT assay. Shown hereare mean (+/− SEM) percent cell viability relative to that of vehicle treatments (defined as 100%). * p < 0.05, ** p < 0.01, two-tailed unequal-variance t-test.

42

vehicle 10 µM 4OHT

10 µM 4OHT + 20 µM Fulv 20 µM Fulv

Figure 12: Visible changes in cell appearance associated with 4OHT-induced cell death can be partially blocked with the ER inhibitor fulvestrant. RD cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free DMEM, pretreated for 30 min with vehicle or fulvestrant, and treated for 3 hr with the four basic permutations: no drug (vehicle), 4OHT alone, fulvestrant alone, or a combination of 4OHT + fulvestrant. Differential interference contrast (DIC) microscopy pictures were taken after the 3 hr treatment.

3.3.2 ER inhibition blocks 4OHT-induced apoptotic signaling

Consistent with the functional data shown in Figure 11, inhibition of ERs using

fulvestrant also blunted the apoptotic signaling induced by 4OHT, suggesting that ERs may

be involved in mediating apoptotic signaling. When treated with 4OHT alone, RMS cells

showed increased apoptotic markers (cleaved caspase-3 and cleaved PARP) as expected, but

pre- and co-treatment with fulvestrant almost completely abolished both apoptotic markers

(Figure 13).

43

RH30 RD RH4

− −

+−

− +

+ +

−−

+−

−+

++

−−

+−

− +

+ +

10 µM 4OHT

20 µM Fulv

cCasp-3

cPARP

vinculin

0

10

20

30

40

50

60

RD RH4 RH30

Cell line

Rel

ativ

e un

its

vehicle10 µM 4OHT20 µM Fulv10 µM 4OHT + 20 µM Fulv

0

10

20

30

40

50

60

70

RD RH4 RH30

Cell line

Rela

tive

units


* **

** **

A B

Figure 13: The ER inhibitor fulvestrant protects against 4OHT-induced apoptotic signaling. Cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free (prf)DMEM, pretreated for 30 min with vehicle or fulvestrant, and treated for 3 hr with the four basic permutations: no drug (vehicle), 4OHT alone, fulvestrant alone, or a combination of 4OHT + fulvestrant. Whole-cell protein lysates were harvested for detection of cleaved caspase-3 (A) and cleaved PARP (B) via western blot. Shown here are mean (+/− SEM) background-adjusted densitometric values, normalized to both the vehicle values (defined as 1 relative unit) and to the loading control vinculin. * p < 0.05, ** p < 0.01, two-tailed unequal-variance t-test.

44

3.4 Linking the MAPKs with ERs

Having shown evidence that both ER and MAPK may be involved in the apoptotic

signaling induced by 4OHT, I next asked whether these two targets are part of the same

pathway, or represent independent mechanisms.

So far, I have shown that in RMS cells treated with 4OHT, there is an increase in JNK

phosphorylation. However, when the cells were pre- and co-treated with fulvestrant, a

reduced induction of JNK phosphorylation was observed (Figure 14). Fulvestrant is not a

JNK inhibitor, yet it was somehow able to inhibit 4OHT-induced JNK phosphorylation. This

suggests that 4OHT-induced JNK phosphorylation is dependent on ERs, thus inhibition of

ERs by fulvestrant also led to downstream inhibition of JNK.

A similar effect was observed on ERK and p38, though to a smaller extent (data not

shown).

45

RD RH4 RH30 --

+ -

-+

++

--

+-

-+

++

--

+-

- +

+ +

10 µM 4OHT

20 µM Fulv46 kDa

phospho-JNK 54 kDa

total JNK

vinculin

0

1

2

3

4

5

6

7

8

RD RH4 RH30

Cell line

phos

pho-

/tota

l JNK

(46

kDa)


0

0.5

1

1.5

2

2.5

3

3.5

4

RD RH4 RH30

Cell line

phos

pho-

/tota

l JNK

(54

kDa)


**

**

**

**

**

Figure 14: The ER inhibitor fulvestrant blocks 4OHT-induced JNK phosphorylation but does not affect basal phospho-JNK levels. Cells were plated in DMEM + 10% FBS, recovered for 6 hours, serum-starved overnight in phenol red-free (prf) DMEM, pretreated for 30 min with vehicle or fulvestrant, and treated for 3 hr with the four basic permutations: no drug (vehicle), 4OHT alone, fulvestrant alone, or a combination of 4OHT + fulvestrant. Whole-cell protein lysates were harvested for detection of phosphorylated and total JNK via western blot. Shown here are mean (+/− SEM) background-adjusted densitometric ratios between phosphorylated and total JNK, normalized to vehicle ratios (defined as 1 unit). JNK appears as two distinct bands (46 kDa and 54 kDa) due to alternative slicing. Densitometric ratios are plotted individually for each JNK variant (46 kDa on the left, 54 kDa on the right). ** p < 0.01, two-tailed unequal-variance t-test.

46

CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS

Although a great wealth of research exists on the mechanisms of TAM and 4OHT, our

understanding of their mechanisms consists of a mass of disconnected leads, with many gaps

to be filled, and some contradictions to be clarified.

In this study, we explored the mechanisms of 4OHT in RMS, following a previous study

[4] in our lab that demonstrated the anti-RMS effects of 4OHT. In particular, we looked at

the involvement of ERs, MAPKs, and the apoptotic markers caspase-3 and PARP. All of

these have been individually implicated in the mechanisms of TAM or 4OHT in various

studies, though few studies show the connections between these targets in the context of

4OHT activity.

4.1 Involvement of caspases

We showed a strong induction of apoptotic signaling following 4OHT treatment, as

indicated by the upregulation of cleaved caspase-3 and cleaved PARP. Caspases are cysteine

proteases that initiate and execute apoptosis in many systems, generally divided into two

classes: the initiators and effectors. Both forms exist in their inactive pro-enzyme forms in a

non-apoptotic cell. Upon induction of apoptosis, initiator caspases are auto-activated by

multimeric complexes (such as the apoptosome and DISC) [199]. The activated initiator

caspases cleave downstream effector caspases, which in turn execute apoptosis by cleaving a

wide range of cellular targets, leading to cell death. Caspase-3 is an effector caspase found in

mammals, and one of its targets is poly(ADP-ribose) polymerase (PARP) [200].

47

Apoptosis can be initiated by two general pathways, either intrinsically triggered from

signals within the cell (leading to release of apoptotic factors from the mitochondria and

formation of the apoptosome), or extrinsically triggered from extracellular ligands binding

onto death receptors on the plasma membrane (leading to formation of DISC) [199].

Although our experiments did not distinguish between the two (since caspases-3 is activated

by both the intrinsic and extrinsic pathways), there is evidence that TAM alongside

simultaneous activation of the extrinsic pathway leads to synergistic apoptotic effects,

suggesting that TAM complements the extrinsic pathway, and therefore acts intrinsically

[201]. This is consistent with many studies showing mitochondria-mediated apoptosis after

TAM treatment [141, 155-157]. Therefore, we have reason to suspect that the apoptotic

signaling we observed here is likely triggered via the intrinsic pathway mediated by the

mitochondria. However, there is a recent report suggesting that estrogen itself could induce

apoptosis in estrogen-sensitive cells via both the intrinsic and extrinsic pathways [202].

Further experiments will need to be done to confirm whether the 4OHT-induced pathway is

intrinsic or extrinsic (or both), possibly by monitoring the translocation of apoptotic

mitochondrial factors such as cytochrome c and AIF, or by observing the sensitivity of RMS

cells to 4OHT while blocking the extrinsic pathway. It will also be informative to determine

whether extrinsically activated apoptosis can combine synergistically with 4OHT in our

experimental system.

In addition to apoptosis, there may be other modes of cell death not accounted for in our

assays and experimental conditions. Apoptosis itself is classified as Type I cell death [203].

Type II cell death involves autophagy, characterized by the formation of vacuoles that digest

the host cell from the inside [203, 204]. Type III cell death, necrosis, loosely refers to forms

48

of seemingly “uncontrolled” cell death leading to chaotic release of inflammatory cellular

contents [203, 204]. Oncosis is a form of cell death resulting from the collapse of membrane

ion gradients, leading to cell and organelle swelling as well as membrane permeation, with

eventual release of inflammatory cellular contents [204].

4.2 Involvement of MAPKs

In addition to apoptotic signaling, we observed a simultaneous activation of MAPKs,

especially JNK. To distinguish whether the MAPKs are acting to mediate apoptotic signaling,

or whether they are part of a protective response to cell death, we used inhibitors to block

MAPK phosphorylation. This led to partial protection against 4OHT, suggesting that MAPKs

are involved in mediating apoptotic signaling.

Both JNK and p38 have been commonly associated with apoptosis, including TAM- or

4OHT-induced apoptosis [127, 138]. While ERK is traditionally associated with proliferation,

many studies link ERK to apoptosis in a wide variety of systems [205-207]. More work will

be needed to confirm our results, using alternative methods of MAPK inhibition such as

siRNA knockdown or dominant-negative constructs. Once MAPK involvement is confirmed,

we may then further explore their downstream targets induced by 4OHT. So far, our

preliminary data (not shown) demonstrates an increase in ATF2 phosphorylation, but no

effect on c-Jun phosphorylation, both of which are classical JNK targets. Conversely, we

may trace the pathway in the other direction using inhibitors of upstream targets along the

MAPK pathways. Immediately upstream of the MAPKs are the MAP2Ks, including

MKK4/7 (which targets JNK), MKK3/6 (which targets p38), and MEK1/2 (which targets

ERK). Further upstream, at the MAP3K level, there are a much greater number of potential

49

targets, with multiple members from many kinase families, including the mixed lineage

kinases (MLK) and apoptotic signaling kinases (ASK). A screening approach may be needed

to trace the various targets involved at this level of the pathway.

4.3 Involvement of ERs

Finally, we have shown that ER inhibition using fulvestrant partially blocks both 4OHT-

induced apoptotic signaling, as well as 4OHT-induced phosphorylation of JNK (and to a

lesser extent, p38 and ERK). Therefore, we propose that ERs act upstream of the MAPKs,

which in turn lead to apoptotic signaling. A previous study in RMS showed that 17β-estradiol

(E2) was also able to partially block the effects of 4OHT, consistent with the idea that ERs

may be involved [4].

One immediate argument against this interpretation of our data is the possibility that

fulvestrant might be nonspecifically inhibiting JNK phosphorylation. However, this is

unlikely as fulvestrant is completely unrelated to any kinase inhibitors, and to our knowledge

there are no reports of fulvestrant inhibiting JNK phosphorylation. Furthermore, closer

examination of our data also argues against this possibility. As shown in Figure 14,

fulvestrant only affected 4OHT-induced phospho-JNK levels, and had no effect on basal

levels of phospho-JNK, in contrast to the true JNK inhibitor SP600125 (Figure 10), which

reduced both basal and 4OHT-induced phospho-JNK. This helps to rule out the possibility

that fulvestrant might be nonspecifically targeting JNK.

Alternative methods of inhibition (including siRNA knockout, dominant-negative

constructs, or other inhibitors) should be used to confirm this conclusion. Conversely, it

50

would also be informative to overexpress the ERs and observe whether this sensitizes the

cells to 4OHT.

Although we did not attempt to distinguish between ERα and ERβ in our experiments

(fulvestrant inhibited both), a previous study in our lab by Greenberg et al. [4] has shown that

RMS primary tumours and cell lines express ERβ but not ERα, at both the mRNA and

protein levels. This suggests that ERβ is likely the isoform that is mediating MAPK and

caspase activation in our experiments, with little or no contribution from ERα. To confirm

this idea, isoform-specific inhibition of ERβ will need to be achieved, possibly using siRNA

to downregulate ERβ mRNA, or ERβ-specific small-molecule inhibitors. We have attempted

siRNA knockdown of ERβ, but with only mild effects at the mRNA level. A stable inducible

shRNA system may be needed to achieve more robust and durable knockdown.

According to Greenberg et al., the various RMS cell lines (RD, RH4, and RH30) express

different levels of ERβ. We observed no correlation between ERβ expression and 4OHT

sensitivity. However, to confidently assess the role of ERβ without confounding factors from

other differences in the cell lines, we would need to vary ERβ expression across an otherwise

isogenic set of cell lines. This can be achieved by stable shRNA knockdown and

overexpression of ERβ to generate variant RMS cell lines with altered ERβ expression.

Mode of Action at the ER

Upon binding of 4OHT to the ER, one can postulate two general modes of action. First,

4OHT may occupy the ER, blocking it from endogenous ER ligands, leading to downstream

consequences of this blockade. In the second scenario, 4OHT not only binds to the ER, but

also continues on with a genomic or nongenomic pathway of its own. There is overwhelming

51

evidence for both possibilities, and both are likely to occur under different systems and

conditions [92, 93, 131, 134]. However, in our experimental system we can eliminate the first

possibility, due to two reasons.

First, all our experiments were carried out in serum-free conditions. Therefore, we have

removed estrogens, as well as other components of the serum that might bind onto the ERs.

In most of our experiments, we also removed phenol red (an artificial component of the

medium that weakly mimics estrogen and can bind onto the ERs). Since we have removed all

the ER ligands as much as possible, it is unlikely that 4OHT is merely acting to blockade the

ER (because there are no ligands left to block in the first place).

Second, in our experiments using fulvestrant, a potent inhibitor of the ERs, no effect was

observed on cell viability or on apoptotic signaling. In other words, simple blockade of the

ERs cannot account for the effects observed following 4OHT treatment.

Therefore, 4OHT is most likely binding onto the ER and initiating a pathway of its own

that leads to JNK activation and apoptotic signaling. 17β-estradiol alone cannot recapitulate

the effects of 4OHT under the same conditions (data not shown), supporting our notion that

4OHT may be distorting the normal estrogen pathway to achieve the observed anti-RMS

effects. The timescale of our treatments (3 hr) do not allow us to further narrow down the

possibility of genomic versus nongenomic signaling, as both events can occur within this

relatively large time window. To make this distinction, treatment times on the scale of

minutes may be needed, as nongenomic effects tend to occur much more rapidly.

52

Linking ERs with MAPKs

MAPK activation has traditionally been considered as part of an ER-independent

mechanism of TAM and 4OHT [138, 208, 209]. However, our study provides evidence for

ER-dependent MAPK activation. Despite the prevailing view that TAM- or 4OHT-induced

MAPK activation is ER-independent, there are still some studies that support our conclusions.

Zhang et al. provided strong evidence that causally linked p38 to ERα as part of a

mechanism for 4OHT [127]. ERα-negative HeLa cells are 4OHT-resistant and do not induce

p38 activation after 4OHT treatment. However, stable transfection of ERα not only sensitizes

the cells to 4OHT, but also leads to 4OH-induced p38 activation. When these ERα-

transfected HeLa cells are selected for resistance to 4OHT, p38 activation vanishes as well.

Prifti et al. demonstrated ER-dependent activation of JNK by E2 and synthetic estrogens

[210]. T47D breast cancer cells treated with estrogen leads to increased JNK phosphorylation,

and pretreatment with fulvestrant completely abolishes JNK phosphorylation. As a control,

they showed that EGF-induced JNK phosphorylation (which is ER-independent) is not

affected by fulvestrant.

Various groups have also established a nongenomic ER pathway leading to MAPK

activation [211-213]. For example, Migliaccio et al. showed that E2 leads to activation of

ERK and upregulation of GTP-ras (another member of the ERK pathway) in MCF-7 cells

[212]. This E2-induced ERK pathway can be inhibited using fulvestrant. ERα-negative Cos-7

cells do not activate ERK in response to E2, but transfection of human ERα leads to E2-

responsive ERK activation. Cheskis et al. described a novel scaffold protein they called

modulator of nongenomic activity of estrogen receptor (MNAR), which is required for the

interaction between ERα and Src (a tyrosine kinase that can lead into the ERK pathway by

53

activating Raf), thus providing a mechanism for ER signaling to feed into the ERK pathway

[213].

Wong et al. originally identified MNAR by incubating a GST-tagged ERβ with MCF-7

whole-cell extracts in the presence or absence of E2 [214]. E2-dependent protein binding

partners were pulled down, separated by SDS-PAGE, silver-stained, and identified by mass

spectrometry. If the effects of 4OHT in RMS cells are mediated through a nongenomic

pathway, we might be able to use a similar approach to find 4OHT-dependent protein

binding partners that might shed light on the molecular events following binding of 4OHT to

the ER.

54

MAPK4OHT ER caspase

?

E2 ER

cytoplasm

nucleus

?

4OHT

?

?4OHT 4OHT

4OHT

?

4OHT 4OHT

nongenomic

genomic

Figure 15: Model of 4OHT activity in RMS cells. 4OHT-induced cell death in RMS cells involves binding of 4OHT to the ER, phosphorylation of MAPKs, and activation of apoptotic markers caspase-3 and PARP. These appear to be linked in a causal sequence, from ER to MAPK to apoptotic signaling. Furthermore, blockage of the ER alone is not sufficient to induce cell death. Therefore, the antiestrogen model does not fit our observations, leading us to propose a “pseudo-estrogen” model, in which 4OHT acts upon the ER to distort the pathway away from normal estrogen signaling, presumably leading to activation of MAPK and apoptotic signaling. It is conceivable for 4OHT to hijack the genomic pathway (leading to transactivation of target genes by the 4OHT-bound ER) or the nongenomic pathway (feeding into cytoplasmic signaling pathways, eventually also leading to transcriptional effects). Many blanks are yet to be filled in this still-simplistic model, and many intermediates are yet to be identified (candidates include MNAR and Src) to form a more complete picture.

55

4.4 Concluding remarks

Our study aims to define the mechanisms of 4OHT-induced cell death in RMS cell lines,

focusing on three groups of targets: ERs, MAPKs, and caspases. Furthermore, we show that

these targets may be causally linked in a sequence from ERs to MAPKs to caspases.

The immediate next steps are to confirm these conclusions using alternative inhibition

methods. Once we are confident that our basic pathway is valid, we can then begin to expand

it from each of the targets. From the caspases, we may explore whether the upstream

apoptotic triggers are intrinsic or extrinsic. From the MAPKs, we can monitor the

phosphorylation status of upstream and downstream targets. From the ERs, we may attempt

to narrow down the downstream events to a genomic or nongenomic pathway, and to pull

down 4OHT-dependent protein interaction partners.

Furthermore, 4OHT can be tested on normal cells to identify effects and mechanisms that

select against RMS cells (or tumour cells in general). New selective mechanisms might be

identified by comparing the changes in expression profile (in response to 4OHT treatment)

between normal versus RMS cells. Finally, primary RMS cells and murine xenografts may

offer more realistic systems to model the effects and mechanisms of 4OHT that are relevant

in a clinical setting.

A greater understanding of the pathways behind 4OHT-induced cell death may lead to

new therapeutic targets. Furthermore, it would allow us to design combinatorial treatments to

enhance the effects of TAM or 4OHT: drugs that employ a complementary mechanism to

4OHT may yield synergistic effects. Finally, greater understanding of the mechanisms

behind 4OHT or TAM will better equip us to understand the mechanisms of resistance to

these drugs.

56

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