THERAPEUTIC POTENTIAL OF EGFR DERIVED PEPTIDES IN BREAST CANCER by Hsin-Yuan Su

THERAPEUTIC POTENTIAL OF EGFR DERIVED PEPTIDES IN BREAST CANCER by Hsin-Yuan Su
THERAPEUTIC POTENTIAL OF EGFR DERIVED PEPTIDES IN BREAST CANCER
by
Hsin-Yuan Su
__________________________
A Dissertation Submitted to the Faculty of the
GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2013
2
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Hsin-Yuan Su
entitled Therapeutic Potential of EGFR Derived Peptides in Breast Cancer
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy.
_______________________________________________________________________
Date: 04/03/2013
Joyce Schroeder
_______________________________________________________________________
Date: 04/03/2013
Todd Camenisch
_______________________________________________________________________
Date: 04/03/2013
Jesse Martinez
_______________________________________________________________________
Date: 04/03/2013
Emmanuelle Meuillet
_______________________________________________________________________
Date:
Final approval and acceptance of this dissertation is contingent upon the candidate’s submission
of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend
that it be accepted as fulfilling the dissertation requirement.
________________________________________________ Date: 04/03/2013
Dissertation Director: Joyce Schroeder
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for an
advanced degree at the University of Arizona and is deposited in the University Library to be
made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided
that an accurate acknowledgement of the source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by the head
of the major department or the Dean of the Graduate College when in his or her judgment the
proposed use of the material is in the interests of scholarship. In all other instances, however,
permission must be obtained from the author.
SIGNED: Hsin-Yuan Su
4
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to the important individuals who
helped with my dissertation, my graduate career, and my life.
First, I would like to thank my mentor, Dr. Joyce Schroeder, for her guidance,
encouragement and support throughout my graduate career. I really appreciate Joyce for
what she has done for me. I would also like to acknowledge my committee members: Dr.
Camenisch, Dr. Martinez, Dr. Meuillet and Dr. Nagle, for their insightful comments and
helpful discussions for my project.
I would like to thank the previous and current lab members for their company,
assistance and discussions. I am especially grateful to Dr. Ben Bitler, who helped me a lot
for my first three years. I am also thankful to Dr. Matt Hart and Derrick Broka for
working together with me to finish the EJ1 project.
I would like to thank the Cancer Biology Program and all the CBIO students,
especially Dr. Kristy Lee who helped me a lot for the ROS part of my project. I would
also like to thank Anne Cione for her kindness to help me since the very first day I got
accepted into Cancer Biology Program
Last but not least, I would like to thank my family back in Taiwan and new family
here, the international friend family. I am grateful for their support in every way. In
particular, I would like to thank my wife, Hui-Hua Chang. Her support in my pursuit of a
PhD in the US has been constant. Without her being by my side, I would not be able to
make it this far.
5
TABLE OF CONTENTS
LIST OF FIGURES ....................................................................................................... 8
LIST OF TABLES ....................................................................................................... 11
LIST OF ABBREVIATIONS ...................................................................................... 12
ABSTRACT ................................................................................................................. 16
CHAPTER 1 – INTRODUCTION .............................................................................. 19
I.
II.
Breast Cancer.................................................................................................. 20
A.
Luminal A breast cancer ...................................................................... 21
B.
Luminal B breast cancer ...................................................................... 21
C.
HER2-enriched breast cancer .............................................................. 22
D.
Basal epithelial-like breast cancer ....................................................... 22
E.
Normal breast-like breast cancer ......................................................... 23
Treatment ...................................................................................................... 24
III. EGFR ........................................................................................................... 26
IV. Ligands of EGFR ......................................................................................... 29
V.
EGFR Internalization and Degradation ....................................................... 31
VI. EGFR in Breast Cancer................................................................................ 35
A.
Overexpression of EGFR ..................................................................... 35
B.
Overexpression of ligands.................................................................... 36
C.
Defective down-regulatory mechanism ............................................... 37
D.
Co-expression of other erbB family members ..................................... 38
VII. EGFR-Targeted Therapy ............................................................................. 39
A.
Anti-EGFR antibodies ......................................................................... 39
B.
Tyrosine kinase inhibitors .................................................................... 41
VIII. Resistance Mechanism to EGFR-Targeted Therapy.................................... 43
A.
Intrinsic resistance ............................................................................... 44
6
TABLE OF CONTENTS - Continued
B.
Acquired resistance .............................................................................. 46
IX. Kinase-Independent EGFR Function ........................................................... 47
X.
EGFR Juxtamembrane Domain ................................................................... 48
XI. Statement of the Problem ............................................................................. 52
CHAPTER 2 - A POTENTIAL DRUG DERIVED FROM EGFR NLS
SEQUENCE ................................................................................................................. 56
I.
Introduction .................................................................................................. 56
II.
Materials and Methods................................................................................. 60
III. Results .......................................................................................................... 62
A.
Design and synthesis of the ENLS peptides ........................................ 62
B.
ENLS peptide significantly inhibits activated EGFR to
translocate into the nucleus .................................................................. 63
C.
ENLS peptide does not affect cell viability of MDA-MB-468 cells ... 65
D.
ENLS peptide does not affect radiosensitivity of MDA-MB-468
cells ...................................................................................................... 67
E.
ENLS peptide sensitizes AG1478-resistant cells ................................. 68
IV. Discussion .................................................................................................... 70
CHAPTER 3 –A POTENTIAL DRUG DERIVED FROM JUXTAMEMBRANE
DOMAIN OF EGFR .................................................................................................... 74
I.
Introduction .................................................................................................. 74
II.
Materials and Methods................................................................................. 75
III. Results .......................................................................................................... 82
A.
EGFR juxtamembrane peptide reduces cell viability .......................... 82
B.
EJ1 inhibits EGFR activation through promoting inactive dimers ...... 88
C.
EJ1 affects cell survival through apoptosis/necrosis ........................... 92
7
TABLE OF CONTENTS - Continued
D.
EJ1 causes membrane dynamic change through affecting Ca2+/CaM
downstream MLCK signaling ............................................................ 104
E.
EJ1 causes mitochondrial disruption and reactive oxygen
species (ROS) generation ................................................................... 107
F.
EJ1 reduces tumor growth and metastasis in mouse models of
breast cancer....................................................................................... 114
IV. Discussion .................................................................................................. 117
CHAPTER 4 – EFFECTS OF MUC1 EXPRESSION ON ERBB3 ACTIVITY ...... 123
I.
Introduction ................................................................................................ 123
II.
Materials and Methods............................................................................... 125
III. Results ........................................................................................................ 127
A.
MUC1 expression affects NRG1β-induced ErbB3 degradation and
downstream signaling ........................................................................ 127
B.
MUC1 expression facilitates EGFR/ErbB3/MUC1 complex
formation ............................................................................................ 131
C.
Knockdown of MUC1 expression promotes both NRG1βdependent and NRG1β-independent cell survival and migration ...... 133
IV. Discussion .................................................................................................. 136
CHAPTER 5 – CONCLUDING REMARKS............................................................ 138
I.
Nuclear EGFR as a potential therapeutic target ......................................... 139
II.
Juxtamembrane domain of EGFR as a potential therapeutic target ........... 141
III. MUC1 sustains NRG1β-dependent ErbB3 activities................................. 144
IV. Conclusions ................................................................................................ 145
REFERENCES .......................................................................................................... 147
8
LIST OF FIGURES
Figure 1.1 Structural model of EGFR activation. ...................................................... 28
Figure 1.2 The erbB signaling network. .................................................................... 29
Figure 1.3 EGFR extracellular domain structure. ...................................................... 31
Figure 1.4 Model of EGFR trafficking. ..................................................................... 33
Figure 1.5 Schematic model of EGFR juxtamembrane domain. ............................... 51
Figure 1.6 Schematic model of nuclear translocation of EGFR. ............................... 52
Figure 2.1 Schematic working model of ENLS peptide. ........................................... 59
Figure 2.2 ENLS peptide design. ............................................................................... 63
Figure 2.3 ENLS peptide affects phosphorylated EGFR (pY845) to translocate
into the nucleus but not EGFR activation. ............................................... 65
Figure 2.4 ENLS peptide does not affect cell viability of MDA-MB-468 cells........ 66
Figure 2.5 ENLS peptide does not affect radiosensitivity of MDA-MB-468 cells. .. 68
Figure 2.6 ENLS peptide sensitizes AG1478-resistant MDA-MB-468 cells. ........... 70
Figure 3.1 Peptide design and nomenclature. ............................................................ 83
Figure 3.2 Juxtamembrane domain peptides reduce cell viability. ........................... 84
Figure 3.3 Dose-dependent response of EJ1 peptide in MDA-MB-468 cells. .......... 86
Figure 3.4 EJ1 peptide affects cell viability in different cell lines. ........................... 87
Figure 3.5 EGFR, ErbB2 and ErbB3 expression in different cell lines. .................... 88
Figure 3.6 EJ1 peptide inhibits EGFR activation. ..................................................... 89
Figure 3.7 EJ1 peptide promotes EGFR homodimer formation. ............................... 91
Figure 3.8 EJ1 peptide induces apoptosis and autophagy. ........................................ 94
Figure 3.9 Inhibition of autophagy causes more cell death upon EJ1 treatment. ...... 95
Figure 3.10 EJ1 peptide induces a minor fraction of cells to undergo apoptosis
after 6 hours of treatment. ...................................................................... 96
Figure 3.11 EJ1 peptide induces a minor fraction of cells to undergo apoptosis
after 24 hours of treatment. .................................................................... 97
9
LIST OF FIGURES - Continued
Figure 3.12 EJ1 causes membrane dynamic change and intracellular vesicle
formation. ............................................................................................. 100
Figure 3.13 EJ1 causes double-membrane-vacuole formation. ............................... 101
Figure 3.14 EJ1 treatment affects membrane integrity. ........................................... 102
Figure 3.15 EJ1 causes an increase of intracellular calcium concentration. ........... 103
Figure 3.16 EJ1 causes necrosis. ............................................................................. 104
Figure 3.17 EJ1 induces membrane blebbing through activation of Ca2+/CaM
and its downstream effector, MLCK. .................................................. 106
Figure 3.18 MLC phosphorylation inhibitors can rescue EJ1-induced cell death. .. 107
Figure 3.19 EJ1 causes mitochondrial swelling. ..................................................... 109
Figure 3.20 EJ1 treatment disrupts mitochondrial membrane potential. ................. 110
Figure 3.21 EJ1 causes accumulation of ROS. ........................................................ 111
Figure 3.22 Inhibition of ROS reduces EJ1-induced cell death. ............................. 112
Figure 3.23 The mechanisms of EJ1-induced cell death are MLCK-regulated
membrane blebbing and accumulation of ROS. .................................. 114
Figure 3.24 EJ1 reduces tumor growth in a MMTV-PyMT mouse model. ............ 116
Figure 3.25 EJ1 reduces lung metastasis in a MMTV-PyMT mouse model. .......... 117
Figure 4.1 MUC1 inhibits the degradation of NRG1β-stimulated ErbB3 and
sustains downstream signaling in BT20 cells. ....................................... 129
Figure 4.2 MUC1 inhibits the degradation of NRG1β-stimulated ErbB3 and
sustains downstream signaling in MDA-MB-231 cells. ........................ 130
Figure 4.3 MUC1 expression facilitates EGFR/ErbB3/MUC1 complex
formation. ............................................................................................... 132
Figure 4.4 Knockdown of MUC1 expression promotes both NRG1β-dependent
and NRG1β-independent cell survival in BT20 cells. ........................... 134
Figure 4.5 Knockdown of MUC1 expression promotes both NRG1β-dependent
and NRG1β-independent cell migration in BT20 cells.......................... 135
10
LIST OF FIGURES - Continued
Figure 5.1 Schematic model of EJ1-induced cell death. ......................................... 142
Figure 5.2 EJ1 peptide affects cell viability of AG1478-resistant cells. ................. 144
11
LIST OF TABLES
Table 1 Features of the gene expression profiling for defined molecular subtypes
of breast cancer. ........................................................................................... 24
12
LIST OF ABBREVIATIONS
ADAM
a disintegrin and metalloprotease
ADCC
antibody-dependent cellular cytotoxicity
AR
amphiregulin
ATP
adenosine triphosphate
BCRP
breast cancer resistance protein
bFGF
basic fibroblast growth factor
BTC
betacellulin
CaM
calmodulin
Cbl
Casitas B-lineage lymphoma
CoxII
cytochrome oxidase subunit II
DNA
deoxyribonucleic acid
DNA-PK
DNA-dependent protein kinase
EGF
epidermal growth factor
eGFP
enhanced green fluorescent protein
EGFR
epidermal growth factor receptor
Egr1
early growth response factor 1
EPR
epiregulin
Eps15
epidermal growth factor receptor substrate 15
ER
estrogen receptor
ER
endoplasmic reticulum
13
FBS
fetal bovine serum
FDA
Food and Drug Administration
HB-EGF
heparin-binding EGF
HGF
hepatocyte growth factor
HIF
hypoxia-inducible factor
HMGB1
high mobility group box 1
HNSCC
head and neck squamous cell carcinoma
Hsp
heat shock protein
IF
immunofluorescence
IHC
immunohistochemistry
IL-8
interleukin-8
INM
inner nuclear membrane
iNOS
inducible NO synthase
IP
immunoprecipitation
JAK
Janus kinase
MAPK
mitogen-activated protein kinase
MLC
myosin light chain
MLCK
myosin light chain kinase
mRNA
messenger RNA
MVB
multivesicular bodies
NAC
N-acetyl cysteine
NPC
nuclear pore complex
14
NRG
neuregulin
NSCLC
non small cell lung cancer
ONM
outer nuclear membrane
ORR
overall response rate
PCNA
proliferating cell nuclear antigen
pI
isoelectric point
PI3K
phophatidylinositol 3-kinase
PLC
phospholipase C
PNPase
polynucleotide phosphorylase
PTB
phosphotyrosine-binding
PR
progesterone receptor
ROS
reactive oxygen species
RT-PCR
reverse transcriptase-polymerase chain reaction
RTK
receptor tyrosine kinase
SGLT1
sodium/glucose cotransporter 1
SH2
Src homology 2
STAT
signal transducers and activators of transcription
TGF-α
transforming growth factor-α
TNFα
tumor necrosis factor α
TKI
tyrosine kinase inhibitor
UIM
ubiquitin interacting motif
VEGF
vascular endothelial growth factor
15
VEGFR
vascular endothelial growth factor receptor
16
ABSTRACT
The epidermal growth factor receptor (EGFR) belongs to the erbB family of receptor
tyrosine kinases which consists of four members (EGFR, ErbB2, ErbB3 and ErbB4).
Upon ligand binding, the EGFR is capable of dimerization with other erbB receptors and
propagates signals regulating a diverse array of cellular physiologies, including cell
growth, migration and survival. Dysregulation of the EGFR is important for development
and progression of different types of cancers, including breast cancer.
Breast cancer is the second leading cause of cancer death in women. EGFR
overexpression has been observed in about 15% of all breast cancers. Moreover, in triple
negative breast cancer (TNBC), which is a more aggressive type of breast cancer and
lacks effective therapies, up to 50% of tumors are found to overexpress EGFR. Targeted
therapy against EGFR has been used in TNBC. However, limited efficacy has been
observed in TNBC due to intrinsic and acquired resistant mechanisms. In order to
overcome this issue, we have developed two novel therapeutic peptides derived from the
nuclear localization signal (NLS) sequence and juxtamembrane domain of EGFR and
investigated their efficacy in regard to inhibiting EGFR translocation and activation in
17
TNBC.
EGFR has been found to translocate into the nucleus and nuclear EGFR can affect
gene transcription, cell proliferation, stress response and DNA repair through interacting
with different components in the nucleus. Importantly, these functions of nuclear EGFR
correlate with cancer prognosis and therapeutic resistance. We found that an EGFR
NLS-derived peptide (ENLS peptide) could inhibit activated EGFR (pY845) undergoing
nuclear translocation. We also showed that this ENLS peptide sensitized breast cancer
cells to AG1478 (EGFR tyrosine kinase inhibitor) treatment.
The juxtamembrane domain of EGFR regulates its trafficking to the nucleus and
mitochondria, interaction with calmodulin and calcium signaling, and participates in
dimerization and activation of EGFR. These non-traditional kinase related functions of
EGFR represent a novel target for EGFR therapy. We found that a mimetic peptide of the
juxtamembrane domain of EGFR (EJ1 peptide) could effectively inhibit EGFR activation
through promoting inactive dimer formation. It could also effectively kill cancer cells
through processes of apoptosis and necrosis. Mechanistically, this EJ1 peptide affects
membrane integrity thereby leading to calcium influx, disruption of mitochondrial
membrane potential and reactive oxygen species (ROS) accumulation. Importantly, EJ1
18
peptide appeared to be effective in inhibition of tumor growth and metastasis in a
transgenic mouse model of breast cancer and showed no observable toxicity.
ErbB3, another member of the erbB family, represents an important driver of the
parallel signaling pathway to EGFR as well as a key regulator of PI3K/AKT activity
which is important for therapeutic resistance. ErbB3 has been shown to interact with
MUC1. The interaction between MUC1 and EGFR promotes EGFR stability through
recycling of receptors. We found that MUC1 expression also affected ErbB3 activity and
stability through ErbB3/EGFR/MUC1 complex formation.
In conclusion, we demonstrated that two EGFR-derived peptides, working through
novel strategies, represent a new foundation of effective therapeutic agents to breast
cancer. ErbB3/EGFR/MUC1 complex formation under MUC1 expression also represents
a druggable target for ErbB3 activity and stability.
19
CHAPTER 1 – INTRODUCTION
Breast cancer represents the second leading cause of cancer death in women. With
the advances in early diagnosis and targeted therapies, the mortality and morbidity of
breast cancer patients have decreased significantly since 1990 (2.2% death rate decrease
per year between 1990-2007, [1]). However, the basal epithelial-like group of breast
cancer (clinically referred as triple negative breast cancer, ER(-), PR(-), and HER2(-))
still harbors a poor prognosis compared with other groups of breast cancers; one of the
possible reasons is the lack of specific targeted therapies. Interestingly, the basal
epithelial-like group of breast cancer was found to overexpress EGFR in more than 50%
of cases. In addition, aberrant localization of EGFR was found to be related to therapeutic
resistance. Thus, EGFR represents a new therapeutic target for this group of patients.
Therefore, we investigated two novel strategies to target both EGFR activity and
trafficking. We have provided evidence to support these two novel strategies as potential
therapeutics for breast cancer patients.
20
I.
Breast Cancer
Breast cancer is the most common form of cancer and the second leading cause of
cancer death in women. In 2013, it is estimated that there will be 232,340 new cases of
invasive breast cancer to be diagnosed and 40,030 breast cancer deaths to be seen among
women in the US [2]. The prognosis depends on the stage and classification of the
disease. According to the Surveillance, Epidemiology, and End Results (SEER) Summary
Stage system, breast cancer can be separated into three stages: local, regional and distant.
Local-stage tumors are cancers confined to the breast. Regional-stage tumors have spread
to surrounding tissue or nearby lymph nodes. Distant-stage cancers have metastasized to
distant organs. The five year survival rate is 99% for localized breast cancer, 84% for
regional disease, and 23% for distant-stage disease [3].
Traditionally, breast cancers have long been classified according to their
morphological characteristics, histological type, and grade (aggressiveness). Recently,
gene expression analysis using DNA microarray technology has identified additional
breast cancer subtypes that were not apparent with traditional histopathological methods.
Based on gene expression profiles, breast cancer can be classified into five main groups
[4, 5]. They are luminal A group, luminal B group, HER2-enriched group, basal
21
epithelial-like group, and normal breast-like group (Table 1). This classification has been
shown to be of clinical relevance for disease prognosis, incidence of relapse rate, and
sensitivity to therapies.
A.
Luminal A breast cancer
Most breast cancers originate from the luminal cells that line the mammary ducts.
Luminal A is the most common type of breast cancer and accounts for 50-60% of breast
cancers. This type of breast cancer tends to express both estrogen receptor (ER) and
progesterone receptor (PR) with low to zero HER2 and proliferation-associated gene
expression. Patients with luminal A breast cancer have an excellent prognosis with a
10-year survival rate of 70% and a 15-year relapse rate of 27.8% [6].
B.
Luminal B breast cancer
Luminal B makes up 10-20% of total breast cancers. This type of breast cancer also
expresses ER and PR. In contrast to luminal A, luminal B breast cancers are more likely
to express high levels of proliferation-related genes. As such, luminal B breast cancers
have a worse prognosis compared to luminal A, with a 10-year survival rate of 54.4% and
a 15-year relapse rate of 42.9% [6].
22
C.
HER2-enriched breast cancer
The HER2-enriched group is composed of tumors that are preferentially
ER-negative and express high levels of HER2 and genes located to the HER2 amplicon.
This group of cancer accounts for 10-15% of total breast cancers and has a poor
prognosis, even though anti-HER2 treatment has substantially improved survival rates. Its
10-year survival rate is 48.1% and 15-year relapse rate is 51.4% [6].
D.
Basal epithelial-like breast cancer
The basal epithelial-like group represents 10-20% of all breast cancers. This group
of cancers is characterized by a lack or low levels of expression of ER and PR, and by
frequent
absence
of
HER2
overexpression,
high
levels
of
expression
of
proliferation-related genes, and expression of genes usually found in basal and
myoepithelial cells of the breast, including cytokeratins 5/6 and 17, and the EGFR.
Clinically, this group and the normal breast-like group are classified as triple negative
breast cancers because they lack expression of ER, PR and HER2. Basal epithelial-like
breast cancers tend to have a poor prognosis with a 10-year survival rate of 52.6% and
15-year relapse rate of 43.1% [6].
23
E.
Normal breast-like breast cancer
Normal breast-like tumors express genes similar to those expressed by normal breast
cells. Only 5-10% of total breast cancers belong to this group. The normal breast-like
tumors are usually ER-, PR- and HER2- as well as cytokeratin 5- and EGFR-, which
distinguish them from basal epithelial-like tumors. Patients with this type of breast cancer
have an intermediate prognosis with a 10-year survival rate of 62.6% and 15-year relapse
rate of 35.1% [6].
24
Table 1. Features of the gene expression profiling for defined molecular subtypes
of breast cancer.
Molecular
Subtype
Frequency
ER/PR/HER2
EGFR
Genes of
Proliferation
Prognosis
ER+:91-100%
Luminal A
50-60%
PR+:70-74%
-
Low
-
High
+/-
High
Poor
+
High
Poor
Excellent
HER2+:8-11%
ER+:91-100%
Luminal B
10-20%
PR+:41-53%
HER2+:15-24%
Intermediate/
Poor
ER+:29-59%
HER2-enriched
10-15%
PR+:25-30%
HER2+:66-71%
ER+:0-19%
Basal epithelial-
10-20%
like
PR+:6-13%
HER2+:9-13%
ER+:44-100%
Normal breastlike
5-10%
PR+:22-63%
HER2+:0-13%
-
Low/
Intermediate
Intermediate
ER: estrogen receptor. PR: progesterone receptor. +: positive. -: negative. Modified from
[7]
II. Treatment
The treatment options for breast cancer are similar to those for other cancers,
including surgery, radiation therapy, chemotherapy and targeted therapy. Usually, a
multimodality approach is required for treatment, depending on the stage, the histological
and pathological features, and the molecular profiling subtypes of the disease. Surgery is
25
still the primary treatment for operable tumors and can be combined with pre-operative
chemotherapy for women who desire breast conservation surgery, even though
pre-operative chemotherapy does not affect disease outcome as compared to
post-operative chemotherapy [8]. Radiation therapy is usually employed after surgery.
The main goal of radiation therapy is to eradicate residual disease thereby reducing local
recurrence. In order to systemically control the disease and to reduce metastasis,
chemotherapy and targeted therapy can be used based on the stage and molecular features
of the disease. Chemotherapy usually involves drugs that less selectively kill
hyper-proliferative cells in the body whereas targeted therapy acts more selectively on
tumor cells.
Hormonal therapy is one of the targeted therapies for breast cancer. There are two
classes of hormonal agents for breast cancer: selective ER modulators, e.g. tamoxifen,
and aromatase inhibitors, e.g. letrozole. Patients with ER positive breast cancers such as
luminal A and luminal B groups of breast cancer mostly benefit from hormonal therapy.
For ER positive breast cancer, allocation to about five years of adjuvant tamoxifen
reduces the annual breast cancer death rate by 31% [9]. Letrozole in ER positive
postmenopausal women who received tomoxifen for five years have a significant
26
increase in disease free survival when compared to placebo-receiving women [10].
Besides ER, HER2 is another molecular target amplified in 20% of breast cancers
and plays an important role in tumorigenesis and progression in these tumors.
Trastuzumab (Herceptin® ) is the first antibody drug against HER2-overexpressing breast
cancer. Trastuzumab has been shown to prolong the overall survival of patients with
HER2-overexpressing metastatic breast cancer [11]. Adjuvant trastuzumab after
chemotherapy also reduced the one-year relapse rate by 46-52% [12, 13]. Triple negative
breast cancer patients have a poor prognosis which could be due to lack of ER and PR for
hormonal therapy, as well as lack of HER2 for anti-HER2 antibody therapy. Importantly,
triple negative breast cancers express EGFR, which also belongs to the erbB family of
receptor tyrosine kinases as HER2. Thus, anti-EGFR therapies could be an alternative
option for triple negative breast cancers.
III. EGFR
The epidermal growth factor receptor (EGFR/ErbB1/HER1) is a 170 kDa type I
transmembrane protein. It belongs to a receptor tyrosine kinase (RTK) subfamily that has
four members, including EGFR, ErbB2 (HER2/Neu), ErbB3 (HER3) and ErbB4 (HER4)
[14]. All of the four receptors have a similar structure which contains an extracellular
27
ligand-binding domain, a transmembrane domain, a juxtamembrane domain, a
cytoplasmic tyrosine kinase-containing domain and a carboxy-terminal tail containing
tyrosine phosphorylation sites. Of which, juxtamembrane domain is believed to regulate
various functional aspects of erbB receptor, including control of the tyrosine kinase
activity, and receptor trafficking, as well as mediating interaction with second messengers
such as calmodulin [15, 16]. ErbB2 does not bind to a known ligand but instead functions
as a preferred co-receptor for the other three receptors [17]. ErbB3 has a defective kinase
domain thus its activation depends on the dimerization with other erbB receptors [18].
After ligand binding, receptors can either form a homodimer or heterodimer.
Dimerization of receptors brings the intracellular tyrosine kinase domains in proximity
and forms an asymmetric dimer. The juxtamembrane segment from the receiver then
makes contact with the C-terminal lobe of the activator kinase domain [19], leading to
autophosphorylation of tyrosine residues on the C-terminal tail (Figure 1.1).
Phosphorylated tyrosine residues, in turn, allow for docking of second messenger
proteins, which contain Src homology 2 (SH2) and phosphotyrosine- binding (PTB)
domain motifs, and activating multiple downstream pathways involved in cell
proliferation, survival and motility (Figure 1.2). Major pathways associated with erbB
28
signaling include the Ras/mitogen-activated protein kinase (MAPK) pathway, the
phophatidylinositol 3-kinase (PI3K)/AKT pathway, the Janus kinase (JAK)/signal
transducers and activators of transcription (STAT) pathway, and the phospholipase Cγ
(PLCγ) pathway. A different dimer formation represents the mechanism by which
receptors within this family can regulate a diverse array of cell signals.
Figure 1.1. Structural model of EGFR activation.
Activation of EGFR by EGF results in a receptor dimerization which brings the
intracellular tyrosine kinase domain in proximity with an asymmetric fashion. The
juxtamembrane segment from the receiver (green line) makes contact with the C-terminal
lobe of the activator kinase domain, leading to autophosphorylation of tyrosine residues
on C-terminal tail (red dots). The illustration is adapted from [20].
29
Figure 1.2. The erbB signaling network.
Ligands and the ten dimeric receptor combinations comprise the input layer.
Numbers in each ligand block indicate the respective high-affinity erbB receptors.
Dimerization of receptors leads to activation of multiple downstream pathways, as shown
in the signal-processing layer, which in turn up-regulate different transcription factors in
the nucleus. At the end, different cellular responses are elicited as the output layer. This
illustration is adapted from [21].
IV. Ligands of EGFR
ErbB family members are activated by extracellular ligand binding. This EGF
family of ligands can be divided into three groups. The first group of ligands includes
EGF, transforming growth factor α (TGFα) and amphiregulin (AR), all three of which
bind specifically to EGFR. The second group of ligands includes betacellulin (BTC),
30
heparin-binding EGF (HB-EGF) and epiregulin (EPR), which bind both EGFR and
ErbB4. The third group is composed of neuregulins (NRGs) and can be further divided
into two subgroups based on their ability to bind ErbB3 and ErbB4 (NRG1 and NRG2) or
only ErbB4 (NRG3 and NRG4) (Figure 1.2, reviewed in [22]). These ligands are
synthesized as transmembrane precursors that can be cleaved by cell surface proteases
and released into the extracellular environment. The main proteases involved in cleavage
of the EGF family of ligands belong to the metalloproteinase family, in particular the
ADAM (a disintegrin and metalloprotease) family of metalloproteinases [23].
Following the release of ligands from the membrane, ligand binding to erbB
receptors induces a conformational change which in turn promotes dimerization [24]. For
example, in the absence of EGF, interactions between subdomains II and IV of the
extracellular domain of EGFR maintain the extracellular domain in an “off” status and
prevent dimerization. When EGF binds to domains I and III, it changes the conformation
of the extracellular domain of EGFR and turns it to an “on” status, which exposes domain
II and allows the protruding arm of this domain to participate in dimerization (Figure 1.3).
Dimerization of receptors then leads to activation of downstream pathways and regulates
cell proliferation, migration, differentiation and survival (Figure 1.2, reviewed in [22]).
31
Figure 1.3. EGFR extracellular domain structure.
The extracellular domain is composed of four subdomains designated I, II, III and IV.
The domains I, II and III form a ligand-binding pocket, where a ligand is docked between
the domains I and III. This illustration is adapted from [25].
V.
EGFR Internalization and Degradation
Since EGFR regulates various important cellular physiologies, it is not surprising
that EGFR activity is tightly regulated. Degradation of the activated receptors is an
32
important mechanism by which the cells can control the extent of signaling. Binding of
ligands leads to dimerization of the receptor and transphosphorylation of certain tyrosine
residues on the C-terminal domain (reviewed in [26]). One of these tyrosine residues
(pY1045) then acts as a docking site for the Cbl (Casitas B-lineage lymphoma) protein,
which along with E2 ligase, binds EGFR and facilitates its ubiquitination [27]. EGFR
ubiquitination promotes the recruitment of Eps15 (epidermal growth factor receptor
substrate 15) through its ubiquitin interacting motif (UIM) and mediates translocation of
EGFR to a clathrin-coated pit which initiates internalization [28]. A clathrin-coated
vesicle is then formed and subsequently the clathrin coat is removed to produce an early
endosome where sorting for receptor recycling or degradation occurs. The early
endosome then matures to form multivesicular bodies (MVB). EGFR destined for
degradation is then internalized into the MVB lumen, fused with lysosomes and degraded
[29] (Figure 1.4).
33
Figure 1.4. Model of EGFR trafficking.
Binding of EGF leads to dimerization of the receptors and transphosphorylation of
tyrosine 1045 (pY1045), then acts as a docking site for the Cbl protein, which binds and
facilitates the ubiquitination of EGFR. EGFR ubiquitination promotes the recruitment of
Eps15 and mediates translocation of EGFR to a clathrin-coated pit which initiates
internalization. A clathrin-coated vesicle is then formed and subsequently the clathrin
34
coat is removed to produce an early endosome. The early endosome then matures to form
MVB. EGFR destined for degradation is then internalized into the MVB lumen, fused
with lysosomes and degraded. P, phosphate; Ub, ubiquitin. This illustration is adapted
from [30].
Deregulation of the degradation pathway can lead to over-activation of EGFR
signaling. Both inhibition of endocytosis and promotion of recycling have been reported
in several circumstances [31-34]. They can be a result of different ligand binding,
different heterodimer formation or of interaction with other proteins. Binding of EGF or
TGF-α to EGFR has a similar affinity at pH 7.4. However, these two ligands have
substantially different pH sensitivities at lower pH levels, which is a characteristic of
endosomes. These different pH sensitivities are due to their difference in pI (isoelectric
point, pH 6.2 for TGF-α vs. pH 4.3 for EGF). TGF-α is dissociated from EGFR in acidic
conditions which leads to recycling of EGFR [31]. EGFR/ErbB2 heterodimer can also
alter its degradation. Overexpression of ErbB2 causes constitutive activation of ErbB2 as
well as of its interaction partner, EGFR. Endocytosis of EGFR is not affected by
overexpression of ErbB2. On the other hand, degradation of EGFR is strongly inhibited
due to a preferential recycling pathway instead of the lysosomal targeting pathway for
EGFR/ErbB2 heterodimers [32]. Aberrant protein-protein interaction also affects EGFR
35
trafficking. For example, Src overexpression has been observed in many different types
of cancers, and Src/EGFR interaction induces EGFR activation and redistributes EGFR
into recycling endosomes [33]. MUC1/EGFR interaction also potentiates EGFR
activation and inhibits EGFR degradation through promoting recycling of EGFR [34].
Over-activation of EGFR due to alteration in receptor trafficking can ultimately lead to
tumor formation and progression.
VI. EGFR in Breast Cancer
EGFR can be oncogenic by a variety of mechanisms, including over-expression or
mutation of receptors, over-expression of ligands, defective down-regulatory mechanisms,
and co-expression of other erbB family members (reviewed in [22]).
A.
Overexpression of EGFR
EGFR over-expression has been observed in about 15% of unselected breast cancers.
In triple negative breast cancer, up to 50% of tumors are found to overexpress EGFR [35].
This overexpression can be a result of several different events including gene
amplification, up-regulated EGFR transcription or translation. EGFR gene amplification
which results in increased protein expression has been found in approximately 6% of
36
breast cancers [36]. Another important inducer for EGFR overexpression is hypoxia,
which occurs during development of cancers and is correlated with cancer progression.
Hypoxia has been observed to modulate EGFR expression through transcriptional and
translational regulation. Hypoxia-induced early growth response factor 1 (Egr1) protein
up-regulates EGFR transcription by binding to EGFR promoter and in turn enhances
EGFR expression [37]. In addition, activation of hypoxia-inducible factor 2α (HIF2α)
protein leads to up-regulation of EGFR mRNA translation [38]. Although overexpression
of EGFR seems to be a frequent phenomenon in triple negative breast cancers, EGFR
mutation is not. Only about 11% of triple negative breast cancers were positive for EGFR
mutations in a recent analysis [39].
B.
Overexpression of ligands
The concept that EGFR ligands could be oncogenic was first demonstrated in a
mouse model engineered to overexpress TGF-α [40]. TGF-α overexpression in a
post-lactational mammary gland induces secretory mammary adenocarcinomas. The
underlying mechanism could be that ligand-dependent EGFR activation results in a
positive feedback autocrine loop, which further induces the expression and/or activation
of ligands. Other EGFR ligands, such as amphiregulin, have also been shown to promote
37
proliferation, invasion and migration of normal and neoplastic mammary epithelial cells
[41]. It is not surprising that up-regulation of ligands mRNA can be detected by reverse
transcriptase-polymerase
chain
reaction
(RT-PCR)
in
80-96%
and
by
immunohistochemistry (IHC) in 53-67% of breast cancers [42, 43].
C.
Defective down-regulatory mechanism
A defective down-regulatory mechanism is seen in activating mutations of EGFR,
such as point mutation L858R (substitution of leucine to arginine at position 858) or
EGFRvIII mutation. The L858R mutation results from an amino acid substitution at
position 858 within the EGFR activation loop. EGFR L858R mutation leads to defective
ubiquitination and impaired degradation of EGFR [44, 45]. EGFRvIII has an in-frame
deletion of the extracellular domain and is unable to bind ligands; however, the receptor
is constitutively active. The constitutive activity is because the degradation of EGFRvIII
is inhibited. Ubiquitination of EGFRvIII is less effective which leads to inefficient
internalization and impaired trafficking to lysosomes [46]. Other than activating
mutations in EGFR, aberrant protein-protein interaction can also cause defective
degradation of EGFR; EGFR/MUC1 interaction is one of the examples [34].
38
D.
Co-expression of other erbB family members
Lastly, cross-talk between EGFR and other erbBs serves as another mechanism for
oncogenic activation of EGFR. ErbB2 has no known ligand and ErbB3 harbors a
defective kinase domain which implies the necessity of heterodimerization of these
receptors to effectively transduce extracellular signals. Co-overexpression of multiple
erbB receptors has been found in different cancers, including oral, brain and breast
cancers [47-49]. The EGFR-ErbB2 heterodimer appears to be the most common and the
most potent inducer of cell transformation [50]. The enhanced EGFR-ErbB2 signaling
could be due to impaired endocytosis of ErbB2 and thus a sustained signaling from the
receptors [51]. In addition, EGFR-ErbB3 heterodimers have been shown to play an
important role in mediating resistance to EGFR-targeted therapy through their
predominant engagement with PI3K/AKT survival signaling [52, 53].
EGFR can gain its oncogenic activity through a variety of mechanisms; all of these
can be demonstrated in breast cancers which suggests targeting EGFR can be an excellent
therapy for those EGFR-dependent or EGFR-addicted breast cancers.
39
VII. EGFR-Targeted Therapy
The finding that EGFR is activated in a variety of cancers and plays critical roles in
tumor initiation and progression prompts the development of EGFR-targeted therapeutics.
Currently, there are two types of strategies focused on blocking EGFR signaling,
including anti-receptor antibodies and small molecule tyrosine kinase inhibitors (TKI).
A.
Anti-EGFR antibodies
Anti-EGFR antibodies bind the extracellular domain of the receptor. In one of the
very first studies, anti-EGFR monoclonal antibody treatment resulted in more than 75%
of growth reduction in a xenograft tumor model of athymic mice [54]. Interestingly,
neither the expression level of EGFR in tumor cells nor the capability of competing with
EGF for receptor binding of the monoclonal antibody is a determinant of
anti-proliferative activity in vivo, suggesting that some host animal responses may be
involved in the antitumor effect. As an example of anti-receptor antibodies, cetuximab is
a chimeric IgG1 anti-EGFR monoclonal antibody derived from the murine anti-EGFR
monoclonal antibody M225. Cetuximab was the first anti-EGFR antibody approved by
the FDA in February of 2004 for the treatment of colorectal cancer. It is also considered
for the treatment of EGFR-overexpressing breast cancer patients including the triple
40
negative cases that overexpress EGFR [55]. A phase II study on metastatic breast cancer
patients was recently done to evaluate the combination of cetuximab with irinotecan and
carboplatin, two standard chemotherapeutic agents used for breast cancer therapy. The
preliminary result showed an improved overall response rate (ORR) with the addition of
cetuximab over irinotecan and carboplatin alone (39% vs. 19%) [56]. Mechanistically,
cetuximab competitively binds to the accessible extracellular subdomain III of EGFR
with high affinity preventing EGFR ligand binding and promoting receptor dimerization,
endocytosis and degradation [57]. Cetuximab also induces cell-cycle arrest through
upregulation of p27kip1 [58]. In addition, cetuximab has been shown to inhibit
angiogenesis through reduction of angiogenic factors, such as interleukin-8 (IL-8),
vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF)
[59, 60]. Lastly, cetuximab also works by mediating antibody-dependent cellular
cytotoxicity (ADCC) [61, 62]. From different clinical trial results, cetuximab seems to be
a promising agent for the treatment of breast cancer, especially for the subgroup of
patients with aggressive triple-negative tumors that overexpress EGFR. Selection of
patients who will benefit from targeted therapy will be the key to improve the clinical
outcome of EGFR-targeting agents.
41
B.
Tyrosine kinase inhibitors
The second type of anti-EGFR therapeutics is small molecule TKIs. This type of
inhibitor specifically targets the ATP-binding pocket of EGFR and inhibits the EGFR
phosphorylation and downstream cascade. One of the advantages for small molecule
TKIs over anti-EGFR monoclonal antibodies is that they are orally bioavailable and well
tolerated. Gefitinib, a reversible inhibitor of EGFR tyrosine kinase, is the first anti-EGFR
TKI approved by the FDA in May of 2003. It was approved for the treatment of
refractory non small cell lung cancer (NSCLC) as an accelerated-track drug based on
tumor response rates in a phase III trial [63]. However, when survival data became
available for the trial, gefitinib failed to show benefit and was subsequently placed on
restricted-use status. Further investigation showed activating mutations in the EGFR
dictate responsiveness of NSCLC to gefitinib [64]. Meanwhile, a second EGFR-TKI,
erlotinib, which is a reversible inhibitor of wild type EGFR and EGFRvIII mutant,
completed phase III trials. In contrast to gefitinib, erlotinib showed a 2-month
improvement in median survival compared to placebo when used as monotherapy in
previously-treated NSCLC. Based on these data, erlotinib was approved by the FDA in
November of 2004 for advanced NSCLC [65]. Further investigation showed an increased
42
responsiveness to erlotinib of never-smokers and patients with EGFR mutation [66, 67].
Noteworthy is that somatic mutations in EGFR are found in 10% to 15% of Caucasian
and in 30% to 40% of Asian NSCLC patients [68-70]. EGFR mutations associated with
increased response to gefitinib and erlotinib are found to be predominantly of two types:
45% are deletions involving at least 12 nucleotides in exon 19, eliminating a conserved
LREA motif (corresponding to amino acid residues leucine, arginine, glutamic acid and
alanine located at 747-750), and 40% are single point mutations in exon 21 (L858R) [71].
Unfortunately, neither gefitinib nor erlotinib showed success in the treatment of breast
cancer. One possible reason seems to be the lack of activating mutations in breast cancer.
Among EGFR-TKIs, lapatinib is the only one to receive FDA approval for the
treatment of metastatic breast cancer. It is a dual inhibitor of the tyrosine kinase domain
of both EGFR and ErbB2. Lapatinib was found to be an active and well-tolerated oral
dual TKI for the treatment of ErbB2-overexpressing breast cancer [72]. It is also active in
trastuzumab refractory, ErbB2-overexpressing metastatic breast cancer patients [73].
Hence, lapatinib has potential as a successful targeted therapy for erbB2 positive breast
cancer patients. There are two more EGFR-TKI, canertinib and neratinib, currently under
clinical evaluation. Both of them are irreversible pan-erbB inhibitors forming covalent
43
binding to the ATP-binding site. Since they both nonspecifically inhibit all four erbB
receptors, they hold the potential for a broader range of action. For example, neratinib
also inhibits the growth of cultured cells that contain resistance-associated EGFR
mutations [74].
From the experience of these two types of anti-EGFR therapeutics, it is clear that
only a small portion of patients with certain types of EGFR mutations will be responsive
to these anti-EGFR drugs. Discouragingly, a majority of patients either do not respond to
the treatment at all (intrinsic resistance) or respond to the initial treatment but relapse
quickly and ultimately develop drug resistance in 6 to 12 months (acquired resistance).
VIII.
Resistance Mechanism to EGFR-Targeted Therapy
Resistance is a significant issue impairing the use of anti-EGFR therapies. As
mentioned above, resistance to EGFR-targeted therapy can be intrinsic or acquired in
nature. Intrinsic, or primary, resistance to EGFR inhibitors presents as rapidly progressing
disease despite treatment. Acquired, or secondary, resistance typically appears in
responding patients within 12 months of initiation of therapy. It is still controversial in
some cases regarding whether acquired resistance arises from the selection of resistant
44
subclones that are present before the initial therapy, or whether it develops de novo in
response to the treatment [75, 76]. So far, multiple mechanisms have been shown to
contribute to resistance to EGFR-targeted therapies, including EGFR mutations,
activating downstream signaling, alternative activating parallel signaling, and
translocalization of EGFR.
A.
Intrinsic resistance
Intrinsic resistance to EGFR-targeted therapy is likely the result of lack of
EGFR-dependency or presence of redundant pathways. The best examples of intrinsic
resistance
to
EGFR-targeted
therapy
are
KRAS
mutation
[77,
78]
and
Met/ErbB2/VEGF/VEGFR overexpression [79-83]. EGFR controls different downstream
signaling, including Ras/Raf/MAPK and PI3K/AKT pathways and regulates cell
proliferation and survival. A constitutively active downstream effector will render the
cancer cells independent of EGFR activity. One of the most frequent mutations is KRAS
mutation. The frequency of point mutations at codons 12 and 13 of the KRAS gene was
detected in a variety of cancers, including 75% of adenocarcinomas of the pancreas, 40%
of adenomas and carcinomas of the colon and rectum, 30% of carcinomas of the bile duct,
25% of carcinomas of the lung, and 5% in breast [84]. Thirty metastatic colorectal cancer
45
patients treated by cetuximab were screened for KRAS mutation. As a result, 43% of
them were found to have KRAS mutation and none of them responded to cetuximab [77].
A different study determining the association of KRAS mutation and sensitivity to EGFR
TKI in 60 lung adenocarcinomas also concluded that mutation in KRAS was associated
with a lack of sensitivity to both gefitinib and erlotinib [78]. Both studies suggest KRAS
mutations are associated with intrinsic resistance to EGFR-targeted agents and can be
used as a biomarker to select candidates for EGFR-targeted therapy.
PI3K/AKT pathway regulates cell survival and thus is important for cancer cell
maintenance. Activation of parallel pathways that can up-regulate PI3K/AKT activity
also leads to resistance to EGFR inhibitors. Different receptor tyrosine kinases have been
found to compensate down-regulation of PI3K/AKT activity in the treatment of EGFR
inhibitors. Well characterized examples include Met and its ligand, hepatocyte growth
factor (HGF), as well as ErbB2, VEGFR and its ligand, VEGF. Met was found to
cooperate with c-Src to phosphorylate EGFR in an EGFR kinase-independent manner
which leads to insensitivity to EGFR TKI in some breast cancer cell lines [79]. Another
study showed that the Met ligand, HGF, as well as tumor-stromal interactions can both
play important roles in resistance to EGFR TKI. Specifically, fibroblast-secreted HGF
46
activated Met and led to EGFR/Met crosstalk and resistance to EGFR TKI in triple
negative breast cancer [80]. In addition to Met signaling, the activation level of ErbB2
correlated with intrinsic resistance to gefitinib in head and neck squamous cell carcinoma
(HNSCC) and may have potential as a predictive biomarker and as a therapeutic target
for combination therapy in treatment of HNSCC with gefitinib [81]. VEGFR-1
expression also contributes to resistance to EGFR-targeted therapy in different human
cancer cells [82]. VEGF overexpression was found to correlate with resistance to
cetuximab in metastatic colorectal cancer and can be a useful predictive biomarker for
cetuximab treatment [83].
B.
Acquired resistance
Acquired resistance is also a significant hurdle in the clinical use of EGFR-targeted
agents. EGFR secondary mutation (EGFR T790M, substitution of threonine to
methionine at position 790 of EGFR) and Met amplification are the two best-studied
mechanisms among other acquired resistance mechanisms and account for ~60% to 70%
of all known causes of acquired resistance to gefitinib or erlotinib.
Uncovered in clinical trials of erlotinib in NSCLC, EGFR T790M mutation is a
common mechanism for acquired resistance. This T790M mutation results in an increase
47
in the ATP binding affinity and thus reduces the potency of any ATP-competitive kinase
inhibitor. The irreversible inhibitors, such as canertinib and neratinib, overcome this
resistance simply through covalent binding [85].
Met amplification is also identified as an acquired resistance mechanism to EGFR
TKI and accounts for ~20% of the cases [52]. Other mechanisms of acquired resistance
are also discovered, such as up-regulation of ErbB2 and ErbB3 activities [53, 86], and
changes of subcellular localization of EGFR to nucleus [87, 88] or cytosol [89].
IX. Kinase-Independent EGFR Function
Current strategies to target EGFR mainly focus on EGFR kinase activities. However,
a recent study showed that a kinase-dead mutant of EGFR was able to inhibit autophagic
cell death by maintaining intracellular glucose levels through interaction and stabilization
of the sodium/glucose cotransporter 1 (SGLT1) in cancer cells [90]. This finding
indicates an EGFR oncogenic function that is independent of its kinase activity. In
addition, a myriad of separate, non-traditional kinase related functions of the EGFR
receptor have also been demonstrated in the past two decades. These functions include
the ability to translocate to the nucleus and act as transcriptional co-factors, as well as
48
participate in DNA damage repair and replicative pathways, involvement in calcium
signaling, and ability to traffic to mitochondria, where EGFR can interact directly with
cytochrome oxidase subunit II (CoxII) to affect cellular ATP levels and apoptosis [91-94].
These
non-traditional
kinase
related
functions
of
EGFR
along
with
the
kinase-independent function of EGFR [90] suggest that in order to control
EGFR-dependent tumors, solely targeting kinase activity may not be enough.
X.
EGFR Juxtamembrane Domain
Curiously, the highly conserved juxtamembrane domain of the erbB receptors,
composed of amino acids 645-682 of EGFR and located just c-terminal of the
transmembrane domain, has been shown to be involved in all of these non-traditional
kinase related processes.
The N-terminal portion of the EGFR juxtamembrane domain contains a tripartite
sequence of three clusters of basic amino acids that promotes EGFR nuclear translocation
[95] (Figure 1.5). Following EGFR internalization, the receptor is trafficked to the
endoplasmic reticulum (ER) where it associates with Sec61β, a component of the Sec61
translocon, and is then retrotranslocated from the ER to the cytoplasm [96] (Figure 1.6).
49
In the cytoplasm, the EGFR then interacts with Importin α1/β1 through its nuclear
localization signal sequence (NLS, RRRHIVRKRTLRR) [97, 98] (Figure 1.5) and
translocates to the nucleus through the nuclear pore complex (NPC) (Figure 1.6). Prior
work from our lab and that of others has shown that once there, EGFR is able to interact
with the promoters of several genes, including Cyclin D, b-myb, Cox2, iNOS (inducible
NO synthase), and BCRP (breast cancer resistance protein) to up-regulate their
transcription, thereby effecting proliferation, stress response, and resistance to
chemotherapeutics [99-103]. In addition, nuclear localized EGFR can interact with
DNA-PKs (DNA-dependent protein kinases) and PCNA (proliferating cell nuclear
antigen) to enhance double strand DNA repair in response to ionizing radiation and to
promote DNA replication [104, 105]. Importantly, the presence of nuclear EGFR has
been shown to correlate with increased aggressiveness and a higher rate of recurrence in
several cancer types [106-108].
Along with its involvement in nuclear trafficking, this same juxtamamebrane region
is important for EGFR trafficking to the mitochondria as its deletion reduces
ligand-mediated EGFR/mitochondrial co-localization [109] (Figure 1.5). Additionally, the
influence of the transmembrane domain in this process has been demonstrated, as only
50
the combination of transmembrane domain amino acids, 622-644, and the juxtamembrane
amino acids, 645-666, was able to direct an eGFP (enhanced green fluorescent protein)
fusion protein to the mitochondria. Once inside the mitochondria, EGFR can interact
directly with and phosphorylate CoxII, causing a reduction in its activity and resulting in
a temporary decrease in cellular ATP levels [109].
In addition to its effects on EGFR subcellular localization, the juxtamembrane
domain of the receptor is integral to receptor dimerization. This domain is responsible for
both maintaining the inactive conformation of an EGFR monomer through electrostatic
interactions with the plasma membrane, as well as facilitating the interaction of two
EGFR receptors in an active dimer formation [110]. It has been proposed that during
ligand-mediated activation, the helical N-terminal portion (amino acids 645-663) of this
domain participates in the formation of a stabilizing anti-parallel dimer, while the
C-terminal portion (amino acids 664-682) of the domain from the “receiver” receptor
forms a clamp to engage the C-terminal lobe of the kinase domain from the “activator”
receptor in an asymmetric dimerization (Figure 1.1). The specific interactions of both
portions of the juxtamembrane domain are necessary for activation to occur [20].
Dimerization of erbB receptors can be further modulated by calcium influx, another
51
function regulated by the juxtamembrane domain. Upon ligand binding, an induction of
cytosolic Ca2+ is quickly observed, and this Ca2+ influx is likely a Ras-mediated
phenomenon depending on interactions of the receptor kinase domain with PLCγ [111].
The resultant elevation of free Ca2+ levels in the cytoplasm induces activation of
calmodulin (CaM) and leads to the association of Ca2+/CaM and the juxtamembrane
domain (Figure 1.5). This interaction was shown to increase the rate at which the
juxtamembrane domain of EGFR dissociates from the plasma membrane and affects both
the trafficking and kinase activity of EGFR [16, 110, 112].
Figure 1.5. Schematic model of EGFR juxtamembrane domain.
Different EGFR domains are shown and juxtamembrane domain (JXM) is
emphasized. Sequences involved in different functions are highlighted.
52
Figure 1.6. Schematic model of nuclear translocation of EGFR.
Following EGFR internalization, the receptor is trafficked through Golgi to the ER
where it associates with Sec61 and retrotranslocated to the cytoplasm. In the cytoplasm,
the EGFR interacts with Importin β1 through its NLS sequence and translocates to the
nucleus through the NPC.
XI. Statement of the Problem
The EGFR and its family members, including ErbB2 and ErbB3, have been shown
53
to actively contribute to the pathogenesis and progression of breast cancer [113-116].
This evidence led to the development of different anti-EGFR and anti-ErbB2 agents, and
some of them have undergone clinical trials, including trastuzumab and lapatinib being
FDA-approved as monotherapy for breast cancer. However, clinical benefit obtained
from these drugs as monotherapy is usually limited. Even patients who have responded to
initial treatment are likely to develop resistance within one year. These observations
clearly indicate that intrinsic and acquired resistance to targeted therapy, especially
EGFR-targeted therapy, is a common phenomenon in breast cancer. Moreover, although
response of NSCLC patients to gefitinib/erlotinib is directly related to the occurrence of
specific activating mutations in the EGFR, breast cancer patients cannot benefit from this
observation because such EGFR mutations are rare in breast carcinomas. Importantly, the
triple-negative subtype of breast cancer lacks effective therapies due to loss of expression
of ER, PR and ErbB2. Among these tumors, 50% of them actually overexpress EGFR.
Targeting EGFR through a novel therapeutic strategy to improve responsiveness and
overcome resistance mechanisms in breast cancer is critical in order to improve the
outcome of triple negative breast cancer patients.
EGFR juxtamembrane domain contains important sequences for its activation,
54
dimerization, regulation of trafficking, and interaction with calmodulin. All of these
functions of EGFR are also important for its oncogenic activity as well as for drug
resistance. From the success of inhibitory peptide derived from MUC1 to interrupt
EGFR/MUC1 interaction and attenuate proliferation and migration in breast cancer cells
[117], it was hypothesized that targeting the nuclear localization domain or
juxtamembrane domain of EGFR will inhibit oncogenic activities of EGFR and
circumvent resistant mechanisms to conventional targeted therapy. ErbB3, another
member in the erbB family, is also overexpressed in breast cancers and plays important
roles in therapeutic resistance to EGFR targeted therapy. MUC1, a transmembrane mucin,
has been shown to interact with all four erbB receptors and inhibit EGFR degradation [34,
118]. It was hypothesized that ErbB3/MUC1 interaction will alter ErbB3 signaling
through affecting ErbB3 trafficking and/or degradation. To test these hypotheses, three
different approaches were undertaken to answer three specific questions. These questions
are the following:
1. Could the peptide derived from EGFR nuclear localization signal sequence
inhibit EGFR nuclear translocation and prevent nuclear EGFR-dependent
radiation resistance, chemoresistance or EGFR-targeted therapy resistance?
55
2. Could the peptide derived from EGFR juxtamembrane domain inhibit
EGFR oncogenic activity and suppress tumor growth?
3. Does MUC1 expression affect ErbB3 activities?
56
CHAPTER 2 - A POTENTIAL DRUG DERIVED FROM EGFR NLS SEQUENCE
Note: The work presented in this chapter has not been published. All experiments were
performed by Hsin-Yuan Su.
I.
Introduction
EGFR represents an important anti-cancer therapeutic target and many
EGFR-targeted therapies have been developed in hope to replace or work synergistically
with non-targeted chemotherapy and radiation therapy. Resistance to chemotherapy and
radiation therapy has also been linked to EGFR expression, activity, and its nuclear
translocation [119-121]. Nuclear translocation of EGFR starts at the plasma membrane
after ligand binding and receptor activation. Following EGFR internalization, the receptor
is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61β, a
component of the Sec61 translocon, and is then retrotranslocated from the ER to the
cytoplasm [96]. In the cytoplasm, the hydrophobic regions of EGFR interact with Hsp70
(heat shock protein 70kDa) to prevent protein aggregation. EGFR then interacts with
Importin
α1/β1
through
its
nuclear
localization
signal
sequence
(NLS,
57
RRRHIVRKRTLRR) [97, 98] and translocates to the nucleus through the nuclear pore
complex (NPC) (Figure 2.1).
Recently, another model of nuclear transport of EGFR was proposed by Hung’s
group, namely the integral trafficking from the ER to the nuclear envelope transport
(INTERNET) pathway [122]. In this model, membrane-associated EGFR interacts with
importin β all the way from the endocytic vesicle to the nucleus and travels from the
ER/ONM (outer nuclear membrane) to the INM (inner nuclear membrane) via the NPCs.
When utilizing this pathway, EGFR remains embedded in the membrane from the cell
surface to the nucleus envelope in the entire trafficking process. In both trafficking
models, EGFR/Importin α1/β1 interaction is critical for EGFR nuclear translocation.
Studies showed that both ionizing radiation and cisplatin were able to induce EGFR
nuclear translocation, and the nuclear EGFR was able to regulate repair of double-strand
DNA breaks through both activation of DNA-PK and inhibition of polynucleotide
phosphorylase (PNPase) activity. Therefore, it was hypothesized that targeting EGFR
could overcome chemoresistance or radiation resistance. However, targeting EGFR
through antibodies against the extracellular ligand binding domain or small molecules
against the intracellular kinase domain has demonstrated a limited efficacy in breast
58
cancer patients. In addition, nuclear EGFR was found to contribute to resistance to
cetuximab [87] and gefitinib [88]. Therefore, specifically targeting EGFR nuclear
translocation can be an alternative strategy to circumvent the nuclear EGFR-dependent
resistance.
Based on these findings and notions, it was hypothesized that inhibiting
EGFR/Importin α1/β1 interaction by an EGFR NLS mimetic peptide could prevent
nuclear translocation of EGFR and thereby prohibit the emergence of therapeutic
resistance (Figure 2.1).
59
Figure 2.1. Schematic working model of ENLS peptide.
Ionizing radiation and cisplatin can induce nuclear translocation of EGFR and this
nuclear EGFR is able to regulate DNA repair for double-strand breaks, which ultimately
leads to therapeutic resistance. ENLS peptide designed to prevent EGFR binding to
Importin β1 could potentially inhibit nuclear translocation of EGFR.
60
II. Materials and Methods
Cell culture MDA-MB-468 cells were obtained from American Type Culture Collection
and were grown in RPMI (Mediatech, Inc., Manassas, VA) and supplemented with 5%
fetal bovine serum (FBS, PAA, Piscataway, NJ) in 37°C incubator with 5% CO2.
Compounds and reagents EGFR 1005 antibody was obtained from Santa Cruz
Biotechnology, Inc. (Dallas, TX). p-EGFR (pY845) antibody was obtained from Cell
Signaling Technology, Inc. (Danvers, MA). β-actin and laminB1 antibodies were
purchased from Sigma-Aldrich (St. Louis, MO). AG1478 and MTT reagent (thiazolyl
blue tetrazolium bromide) were also purchased from Sigma-Aldrich (St. Louis, MO).
EGF was purchased from Invitrogen Life Technologies Inc. (Carlsbad, CA).
Peptide Synthesis ENLS and CP polypeptides were synthesized by GenScript (Scotch
Plains, NJ) and delivered lyophilized. The peptides were resuspended at a concentration
of 2mM in water and stored at 4°C in single-use aliquots.
Differential Detergent Fractionation The differential detergent fractionation protocol
61
was modified from [123]. Briefly, cells were collected in an eppendorf tube and
resuspended in digitonin lysis buffer. The cytosolic fraction was collected from the
supernatant after centrifugation at 400  g for 7.5 minutes. The pellet was then
resuspended in Triton X-100 lysis buffer and then subjected to centrifugation for 7.5
minutes at 5100  g. The supernatant was stored as the membrane fraction and the pellet
was further resuspended in Tween 40/ DOC lysis buffer. After brief sonication, nuclear
fraction was collected from the supernatant after centrifugation for 10 minutes at 6900 
g.
Western blotting Following treatments, cells were harvested and lysed in lysis buffer
consisting of 20mM Tris, pH 7.5, 150mM NaCl, 1% NP-40, and 5mM EDTA, pH 8.0,
along with protease and phosphatase inhibitors. Protein concentration was determined
using Bicinchoninic Acid assay (Thermo Fisher Scientific Inc., Waltham, MA). Lysates
were then separated using SDS-PAGE before being transferred to PVDF membranes
(EMD Millipore Inc., Billerica, MA) and immunoblotted using indicated antibodies.
Cell viability assay Cells were analyzed by MTT following manufacturer’s instructions
62
(Sigma) and analyzed using a U-Quant Spectrophotometer (Bio-TEK Instruments, Inc.).
III. Results
A.
Design and synthesis of the ENLS peptides
EGFR NLS sequence was shown to contain three clusters of basic amino acids and
all three clusters of basic amino acids were required for efficient nuclear translocation of
EGFR [98]. The NLS sequence is recognized by a group of proteins called Importins.
EGFR NLS sequence has been shown to interact with Importin α1/β1 and this
protein-protein interaction is critical for nuclear transport of EGFR [97]. Therefore, a
13-amino acid peptide was synthesized in tandem with a protein transduction domain
(PTD4, [124]) to help peptides penetrate cells to be used as a decoy to interrupt
EGFR/Importin α1/β1 interaction. Basic amino acids (R or K) at each of the three clusters
were replaced with acidic amino acids (D) and this peptide was used as the control
peptide (CP). As shown in Figure 2.2, these peptides are hereafter referred to as ENLS
(EGFR Nuclear Localization Signal) or CP (Control Peptide) peptides.
63
Figure 2.2. ENLS peptide design.
Peptides were synthesized according to EGFR NLS sequence. The control peptide
was EGFR NLS sequence with 3 amino acid mutations at each of the three clusters of
basic amino acids. Mutated amino acids are shown in red. In order to allow peptides get
into cells, both peptides were synthsized in tandem with a protein transduction domain
(PTD4, [124]).
B.
ENLS peptide significantly inhibits activated EGFR to translocate into the nucleus
In order to test whether the ENLS peptide could affect nuclear translocation of
EGFR, a time course experiment was done first to determine the nuclear translocation of
EGFR after EGF stimulation (data not shown). As phosphorylated EGFR at tyrosine 845
(pY845) had been used to demonstrate nuclear localization of activated EGFR [125], this
same phospho-EGFR was used in our experiments. In a triple- negative breast cancer cell
line, MDA-MB-468, phosphorylated EGFR (pY845) was detected in the nucleus at 30
minutes after EGF stimulation. The same time point was used to determine whether
ENLS peptide affected nulcear translocation of phosphorylated EGFR. As shown in the
left panel of Figure 2.3, EGFR activation was not affected either by water or CP
64
treatment. In addition, it was not affected by ENLS peptide up to 20μM. The decrease of
phospho-EGFR (pY845) in 50μM of ENLS treatment could be due to a nonspecific
cytotoxic effect of the peptide. In nuclear fraction, phosphorylated EGFR levels were the
same for water and CP treated samples. However, the phosphorylated EGFR level was
significantly less in the ENLS peptide treated group. A concentration of 10μM of ENLS
was able to decrease the phophorylated EGFR level in the nuclear fraction (Figure 2.3,
right panel). Since EGFR/importin α1/β1 interaction is important for EGFR nuclear
translocation, interruption of EGFR/importin β1 interaction was then determined by
immunoprecipitation (IP). Both anti-EGFR and anti-importin β1 antibodies were first
used for immunoprecipitation to detect EGFR/importin β1 interaction from cell lysates
collected 15 minutes after EGF-stimulation. However, no obvious interaction between the
two could be detected. As a consequence, a direct evidence showing that decreased
phospho-EGFR (pY845) in the nucleus was due to interruption of EGFR/importin β1
interaction by ENLS peptide could not be provided. Nevertheless, data presented in
Figure 2.3 still indicated that the ENLS peptide could inhibit nulcear translocation of
phosphorylated EGFR (pY845) without affecting EGFR activation.
65
Total protein lysates
Nuclear fraction
Figure 2.3. ENLS peptide affects phosphorylated EGFR (pY845) to translocate
into the nucleus but not EGFR activation.
MDA-MB-468 cells were serum-starved overnight and pretreated with different
concentration of control peptide or ENLS peptide for 30 minutes then followed by
100ng/ml of EGF for 10 min on ice. Cells were then incubated with water (vehicle),
different concentration of CP or ENLS peptide in serum free medium for 30 minutes at
37°C and lysed. Total protein lysates and nuclear fraction of proteins were separated by
SDS–PAGE and immunoblotted with antibodies as indicated.
C.
ENLS peptide does not affect cell viability of MDA-MB-468 cells
As mentioned earlier, nuclear EGFR can also affect cell cycle progression through
transactivational regulation of Cyclin D1 exrpession [126]. We next investigated whether
inhibiting nuclear translocation of activated EGFR could affect Cyclin D1-regulated cell
proliferation. MDA-MB-468 cells were used because overexpression of EGFR as well as
nuclear localization of EGFR were both found in this cell line. After three days of
treatment with either the control peptide or the ENLS peptide, viable cells were
66
quantified by MTT assay. Surprisingly, as shown in Figure 2.4, the ENLS peptide did not
affect cell viability compared to the control peptide. This result indicated that inhibiting
nuclear EGFR with 20μM ENLS peptides was not enough to affect cell viability in
MDA-MB-468 cells.
Figure 2.4. ENLS peptide does not affect cell viability of MDA-MB-468 cells.
MDA-MB-468 cells were seeded in a 24-well plate (1× 104 cells/well) and treated
with 20μM control peptide (CP) or 20μM ENLS peptide (ENLS) for 3 days. A MTT
assay was done to quantify viable cells at day 3. CP-treated cells at day 3 is set as 100%.
Values are the means of 3 independent determinations ± SD.
67
D.
ENLS peptide does not affect radiosensitivity of MDA-MB-468 cells
Nuclear EGFR also regulates DNA double-strand break repair through activation of
DNA-PK and PCNA, and therefore has been linked to chemoresistance to
DNA-damaging agents, such as cisplatin, and radiation resistance. We next determined
whether ENLS peptide affected radiosensitivity in MDA-MB-468 cells by inhibiting
nuclear translocation of activated EGFR. Four Gy ionizing radiation (IR) has been
demonstrated to increase EGFR translocation into the nucleus of MDA-MB-468 cells
[121]. Thus, MDA-MB-468 cells were pre-treated with water, 20μM CP or ENLS for 1
hour then irradiated with 4Gy IR and allowed to grow for 3 days. Viable cells were then
quantified by MTT assay. As shown in Figure 2.5, radiation caused a ~50% decrease in
viable cells across three different treatments. However, no differences between each
treatment could be detected. This result indicated that ENLS peptide was not able to
affect radiosensitivity in MDA-MB-468 cells, even though it was able to prevent
phospho-EGFR (pY845) translocation to the nucleus.
68
Figure 2.5. ENLS peptide does not affect radiosensitivity of MDA-MB-468 cells.
MDA-MB-468 cells were seeded in a 24-well plate (1× 104 cells/well) and
pre-treated with vehicle (water), 20μM control peptide (CP) or ENLS peptide (ENLS) 1
hour before radiation (4Gy). A MTT assay was done to quantify viable cells at day 3.
Cells of vehicle/no radiation treated group at day 3 are set as 100%. Result is shown as
one representative of two independent experiments. Error bars, SD.
E.
ENLS peptide sensitizes AG1478-resistant cells
Lastly, nuclear EGFR is also important for acquired resistance to EGFR-targeted
therapy, including both monoclonal antibodies and tyrosine kinase inhibitors. Huang et al.
demonstrated a possible mechanism that nuclear EGFR-mediated breast cancer resistant
protein (BCRP/ABCG2) expression might contribute at least in part to the acquired
69
resistance to gefitinib [88]. In this study, the authors found an increase of nuclear EGFR
in the gefitinib resistant-MDA-MB-468 cells. Thus, we tested the effects of the ENLS
peptide in this TKI-resistant cell line model and investigated whether inhibition of
nuclear translocation of phospho-EGFR affected drug sensitivity of an established
resistant cell line. We first established AG1478-resistant clones of MDA-MB-468 by
culturing and selecting them with increasing concentrations of AG1478 from 0.1μM to
10μM over a period of three months. Cells that grew in the presence of 10μM AG1478
were then treated with vehicle, CP or ENLS, in the presence or absence of 10μM AG1478,
and cell survival was measured after 3 days. As shown in Figure 2.6, removal of AG1478
could still promote cell growth in these AG1478-resistant cells. Without AG1478, there
were no differences in CP and ENLS treated cells, similar to the results observed in
Figure 2.4. With AG1478, interestingly, a statistically significant difference was detected
between CP- and ENLS-treated cells. ENLS peptide by itself did not affect cell viability
of MDA-MB-468 cells; in this case it could turn AG1478-insensitive MDA-MB-468 cells
into AG1478-sensitive cells (Figure 2.6). This indicated that inhibition of nuclear EGFR
could possibly overcome resistance to AG1478. However, the increased sensitivity only
caused ~10% cell death when CP and ENLS treatments were compared.
70
*
Figure 2.6. ENLS peptide sensitizes AG1478-resistant MDA-MB-468 cells.
AG1478-resistant cells were selected and grown with an escalated AG1478
concentration to 10μM. These AG1478-resistant cells were seeded in a 24-well plate (1×
104 cells/well) and treated with water (Vehicle), 20μM control peptide (CP) or ENLS
peptide (ENLS) in the presence or absence of 10μM AG1478 for 3 days. A MTT assay
was done to quantify viable cells at day 3. CP/AG1478-treated cells at day 3 are set as
100%. Result is shown as one representative of three independent experiments. *, P <
0.05, Student’s t-test. Error bars, SD.
IV. Discussion
In cancer cells, EGFR has been shown to translocate into the nucleus where it can
act as a transcriptional co-activator to regulate target gene expression, including Cyclin D,
b-myb, COX2, iNOS, and BCRP [88, 127, 128]. Nuclear EGFR is also able to regulate
71
DNA double-strand break repair through both activation of DNA-PK and inhibition of
PNPase, and increased DNA repair has been linked to therapeutic resistance [119, 121,
129]. In this study, we set out to determine whether a peptide derived from EGFR NLS
sequence could inhibit nuclear translocation of EGFR and overcome nuclear EGFRdependent therapeutic resistance. We found that the ENLS peptide could effectively
inhibit phospho-EGFR (pY845) to translocate into the nucleus without affecting EGFR
activation (Figure 2.3). However, by itself, 20μM ENLS peptide was neither able to affect
cell viability of MDA-MB-468 cells (Figure 2.4) nor able to change radiosensitivity of
MDA-MB-468 cells (Figure 2.5). Even though ENLS peptide demonstrated an ability to
sensitize AG1478-resistant cells to AG1478, the effect was observed in only 10% of the
cells (Figure 2.6).
In Figure 2.3, we found that EGF stimulation only increased phospho-EGFR in the
nucleus but not total EGFR. This finding was consistent with what Lin et al. reported
[126]. Therefore, ENLS peptide could only affect translocation of phospho-EGFR. The
fact that EGFR activation was not affected by ENLS peptide further supported that only
trafficking of EGFR was inhibited. Interaction between EGFR and Importin β1 was first
demonstrated in A431 cells and the interaction was only slightly increased after EGF
72
stimulation [97]. An optimization for this immunoprecipitation experiment is needed to
be able to directly determine the mechanisms of action of ENLS peptide.
The lack of obvious biological output shown in Figure 2.4 and Figure 2.5 could be
due to the high expression level of EGFR in MDA-MB-468 cells. The high expression
level of EGFR could drive a strong signal through canonical cytosolic signaling pathways
which could mask or easily compensate for the nuclear effect of EGFR. The stability of
the peptide could also be an issue affecting the biological effect of this peptide.
Modification of peptides with N-terminal acetylation and C-terminal amide/PEGylation
might increase stability of peptides inside cells [130-132]. Replacement with D-amino
acids could also increase peptide stability [132]. Besides being used as a strategy for
improving stability, peptide modification may be rationally designed to achieve better
efficacy. For example, Dittmann et al. used a phospho-peptide (Ac-RKRpTLRRLK) to
inhibit radiation-induced nuclear shuttling of EGFR, based on the finding that
phosphorylation of threonine 654 within NLS sequence of EGFR was critical for nuclear
translocation of EGFR [133]. The efficacy of modified peptides warrants further
investigation.
Nuclear EGFR contributes to resistance to cetuximab [87] and gefitinib [88]. We
73
found that inhibition of nuclear phospho-EGFR could sensitize AG1478-resistant
MDA-MB-468 cells to AG1478 again. However, this sensitization only affected 10% of
the cells. This finding indicated that there were other possible mechanisms involved in
AG1478-resistance, at least in this case.
So far, we provide evidence to support the possibility of overcoming therapeutic
resistance with a peptide derived from EGFR NLS sequence to inhibit nuclear
translocation of EGFR. A further investigation is definitely warranted to explore this as a
combination therapy in the future.
74
CHAPTER 3 –A POTENTIAL DRUG DERIVED FROM JUXTAMEMBRANE
DOMAIN OF EGFR
Note: The work presented in this chapter has already been combined with more data from
Matt Hart and submitted to Molecular Therapy. Experiments in Figure 3.2, Figure 3.2,
Figure 3.4a, Figure 3.8, Figure 3.14, Figure 3.17, Figure 3.21, Figure 3.22, Figure 3.23
and Figure 3.24 were performed by Derrick Broka. Experiments in Figure 3.4b, Figure
3.4c and Figure 3.18 were performed by Matt Hart.
I.
Introduction
EGFR belongs to the family of receptor tyrosine kinases based on its function as
both “receptor” and “tyrosine kinase”. Upon ligand binding, a conformational change
leads to transactivation of the receptor dimers and propagates signals downstream of the
pathway, ultimately controlling cell proliferation, migration and survival (reviewed in
[22]). Aberrant activation of EGFR results in tumor formation and progression. First
attempts to target EGFR were based mainly on the most important characteristics of
EGFR, which are the “receptor” and the “tyrosine kinase”. Yet, both the anti-EGFR
antibody against “receptor” and TKI against “tyrosine kinase” showed somewhat
unsuccessful results as treatments for breast cancer patients. A different approach may be
75
needed as an EGFR-targeted therapy.
Interestingly, the juxtamembrane domain of EGFR controls various non-traditional
kinase related functions of the receptor, including trafficking to the nucleus/mitochondria,
involvement in calcium signaling and regulation/stabilization of activation/inactivation
structure of EGFR [15, 20, 94, 134, 135]. Disruption of this important domain of EGFR
would affect multiple aspects of EGFR biology, all of which contribute to tumorigenesis.
Therefore, It was hypothesized that targeting the juxtamembrane domain of EGFR by
peptide inhibitors could inhibit oncogenic activities of EGFR.
II. Materials and Methods
Cell culture All cell lines were obtained from American Type Culture Collection (ATCC).
MDA-MB-468, MDA-MB-231, T47D breast cancer cell lines were grown in RPMI
(Mediatech, Inc., Manassas, VA) and supplemented with 10% (5% for 468 cells) FBS
(PAA, Piscataway, NJ). BT20 cells were grown in Modified Eagles Medium (MEM,
ATCC, Manassas, VA) supplemented with 10% FBS. MCF10A cells were grown in
DMEM F12 (Invitrogen Life Technologies Inc., Carlsbad, CA), supplemented with 5%
Horse Serum, 20ng/ml EGF, 0.5μg/ml Hydrocortisone, 100ng/ml Cholera-Toxin,
76
10μg/ml Insulin, and 1% Penicillin/Streptomycin (Mediatech, Inc., Manassas, VA) as
described in [136]. All lines were grown under 5% CO2.
Compounds and reagents EGFR 1005 antibody and ML-7 were obtained from Santa
Cruz Biotechnology, Inc. (Dallas, TX). EGFR Ab-13 was obtained from NeoMarkers
(Fremont, CA) and the following antibodies were obtained from Cell Signaling
Technology, Inc. (Danvers, MA): p-EGFR (pY845), LC3B, PARP, p-AKT (pS473), AKT,
p42/44 MAPK (ERK1/2), HMGB1, p-p38 (pT180/Y182), and p38. dp-ERK and β-actin
antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Fluo-4 calcium assay kit
and Vybrant apoptosis assay kit were purchased from Invitrogen Life Technologies Inc.
(Carlsbad, CA). 3-MA was obtained from Calbiochem (Billerica, MA). Carbonyl cyanide
3-chlorophenyl- hydrazone (CCCP) was purchased from Sigma-Aldrich (St. Louis, MO).
Y-27632 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). W-13
hydrochloride was purchased from Tocris Bioscience (Minneapolis, MN).
Western blotting Following treatments, cells were harvested and lysed in lysis buffer
consisting of 20mM Tris, pH 7.5, 150mM NaCl, 1% NP-40, and 5mM EDTA, pH 8.0,
77
along with protease and phosphatase inhibitors. Protein concentration was determined
using Bicinchoninic Acid assay (Thermo Fisher Scientific Inc., Waltham, MA). Lysates
were then separated using SDS-PAGE before being transferred to PVDF membranes
(EMD Millipore Inc., Billerica, MA) and immunoblotted using indicated antibodies.
Crosslinking/dimerization assay MDA-MB-468 cells were plated in 100mm dish with
3106 cells. Cells were pre-treated with water (vehicle), 20μM control peptide (CP) or
20μM EJ1 (EJ1) in serum-free medium for 10 minutes on ice and then stimulated with
100ng/ml EGF and respective treatments for another 10 minutes on ice and then washed
with PBS twice. Proteins were crosslinked by 3μM DMS (Thermo Fisher Scientific Inc.,
Waltham, MA) for 30 minutes on ice and then the cells were lysed.
Annexin V/propidium iodide staining (Apoptosis assay) MDA-MB-468 cells were
treated with 20μM CP or EJ1 in complete medium for indicated times. Following
treatment, cells were then trypsinized and collected in eppendorf tubes and stained with
Vybrant apoptosis assay kit (Invitrogen Life Technologies Inc., Carlsbad, CA) following
the manufacturer’s protocol to assess the percentage of non-stained, Annexin V only, PI
78
only, or Annexin V plus PI double-stained cells.
Cells were then sorted by a FACScan
flow cytometer (BD Biosciences, San Jose, CA) and analyzed by Cellquest Pro 4.0
software.
Mitochondrial morphology change MDA-MB-468 cells were treated in complete media
with 200nM MitoTracker Red CMXRos (Molecular Probes Life Technologies Inc.,
Carlsbad, CA) along with 5μg/ml Hoechst 33342 (Invitrogen Life Technologies Inc.,
Carlsbad, CA) nuclear stain for 15 minutes. Media was then removed and fresh media
containing 20μM CP or EJ1 were added. Images were taken on an Olympus IX71
microscope and deconvolved using softWoRx 4.0 image analysis software (Applied
Precision, Issaquah, WA) at the Imaging Shared Service in the Arizona Cancer Center
(AZCC). Images were brightened using Adobe Photoshop.
Cell growth/viability assay Cells were plated in 96 well plates with 2103 cells or in 24
well plates with 1104 cells. On the following day (day 0) treatment (water, CP or EJ1)
began and was changed every other day if not specified. On the final day of treatment,
media were removed and cells were incubated in 0.5-1mg/ml MTT reagent
79
(Sigma-Aldrich, St. Louis, MO) for 2-3 hours at 37°C. Following this incubation, media
and MTT were removed and formazan crystals were dissolved in 100μl DMSO/well for
96 well plates or 600μl DMSO/well for 24 well plates. Absorbance was read at 540nm
using a U-Quant Spectrophotometer (Bio-TEK Instruments Inc., Winooski, VT).
Calcium assay Following treatment in 96 well plates, Fluo-4 calcium assay kit was used
to stain cells following manufacture’s protocol. Fluorescence intensity was measured by
fluorescent plate reader using excitation/emission wavelengths set at 494 nm and 516 nm
respectively.
HMGB1 release assay Medium from treated cells was harvested, spun at 800g for 5
minutes and supernatant was filtered (0.45 mm filter) (EMD Millipore Inc., Billerica,
MA). Proteins were precipitated with 0.02% DOC (deoxycholate)
and 10% TCA
(trichloroacetic acid) and lysed in 2x SDS buffer for immunoblots as referenced from
[137].
Measurement of intracellular ROS formation The generation of intracellular ROS was
80
determined
using
a
fluorescein-labeled
dye,
2’,7’-dichlorofluorescin
diacetate
(DCFH-DA) (Invitrogen Life Technologies Inc., Carlsbad, CA). The non-fluorescent dye
permeates cells easily and is hydrolyzed to 2’,7’-dichlorofluorescin (DCF) upon
interaction with intracellular ROS. Cells were first labeled with 20μM DCFH-DA for 30
min at 37°C. Then, cells were treated as indicated and washed twice with ice-cold PBS
and harvested by trypsin. Then, the cells were immediately analyzed by a FACScan flow
cytometer at the Flow Cytometry Shared Service at the AZCC and excitation/emission
wavelengths were set at 488 nm and 525 nm respectively [138].
Mitochondrial membrane potential Cells were stained with 2 μM 5,5’,6,6’tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolcarbocyanine
iodide
(JC-1)
(Molecular
Probes Life Technologies Inc., Carlsbad, CA) in serum-free media for 15 minutes and
washed twice with PBS after staining. Cells were then treated as indicated. Fluorescence
was determined with a plate reader at wavelengths of 514 nm (excitation) and 529 and
590 nm (emission). The ratio of green and red fluorescence signals serves as a parameter
for the mitochondrial membrane potential independent of the mitochondrial mass.
81
MMTV-PyMT mouse experiments Female MMTV-PyMT mice were palpated weekly
until tumors >5.0 mm in diameter. At this point, animals were placed on study, and
injected daily for 21 days or until total tumor burden reached >10% of initial body weight,
an individual tumor was over 2 cm in average diameter, or a tumor ulcerated through the
skin. Tumors were measured on average every 5 days and size was calculated using the
formula:
(x*x)*(y/2), where x and y are separate horizontal and vertical measurements
(i.e. length and width), respectively.
The number of metastases to the lungs was assessed in control (6 mice) and EJ1 (7
mice) treated mice. Lungs from these mice were fixed, sectioned (10µm thickness) and
stained with Haematoxylin and Eosin. Metastatic foci of 5 individual sections spanning
200µm/mouse lung were then counted, and each section was included as a separate “n”
(i.e. control n=30 and EJ1 n=35).
Statistical Analysis All statistics were performed in Excel (Microsoft). Test implemented
was two-tailed Student’s t-test.
82
III. Results
A.
EGFR juxtamembrane peptide reduces cell viability
The juxtamembrane domain of EGFR contains sequences responsible for receptor
dimerization, calmodulin binding, nuclear localization, and mitochondrial localization
(Figure 1.5). Therefore, we set out to determine if blocking the function of the
juxtamembrane domain of EGFR would result in an effective, EGFR-dependent cancer
therapeutic. To do this, we created cell-penetrating peptides to act as dominant-negative
“decoys”, thereby inhibiting endogenous juxtamembrane domain interactions. Peptides
specific for juxtamembrane subdomains were synthesized downstream of the Protein
Transduction Domain-4 (PTD4, [139]) (Figure 3.1). Next, the effect of peptide treatment
on cell viability was analyzed on the breast cancer cell line MDA-MB-468 by MTT
analysis after three days of treatment (Figure 3.2).
We found that the amino acid region
between hEGFR643-663 (EJ1, >90% reduction) demonstrated optimal reduction in viability,
and partial reduction was also obtained with sub-sequences within EJ1, including EJ2
(hEGFR643-655, ~30% reduction), EJ3 (hEGFR649-663, ~60% reduction) and EJ5
(hEGFR653-663, ~40% reduction) (Figure 3.2).
83
Figure 3.1. Peptide design and nomenclature.
The amino acid number of EGFR is shown in the left column, which corresponds to
the specific amino acids shown in the middle column (sequence). Peptides were
designated EJ1-14, as indicated in the right column. Using EJ1 as the parental sequence,
changes in EJ2-14 are denoted in the second column from the right.
84
***
***
***
***
***
Figure 3.2. Juxtamembrane domain peptides reduce cell viability.
MDA-MB-468 cells were treated daily with 20μM of different peptides or left
untouched for three days, and cell viability was determined by MTT assay. ***, p < 0.001,
Student’s t-test. Error bars, SD.
Interestingly, scrambling of the amino acids of EJ1 resulted in no loss of efficacy
(EJ8), indicating charge may be important. To test the role of charge of the peptide, one
of the basic amino acids (R or K) in each of the three basic clusters of EJ1 was
substituted with an acidic amino acid (D; EJ13 hereafter referred to as control peptide,
CP) and this completely ablated the effects on viability (Figure 3.2). Note that
replacement of the eight arginines and lysines with alanines resulted in an insoluble
peptide (EJ9). Substituting those same basic amino acids with polar amino acids (Q; EJ14)
85
instead only marginally blocked the anti-proliferative effects of EJ1 (Figure 3.2).
Together, these results strongly implicate charged residues in the function of EJ1.
To determine if either the minimal nuclear localization sequence (EJ4), or the
minimal basolateral domain (EJ6) was responsible for the anti-proliferative effects of EJ1,
peptides of these subdomains were created. No anti-proliferative effect was observed for
either peptide, implicating the calmodulin and dimerization domains as essential for cell
death (Figure 3.2). After determining the optimal peptide concentration in MDA-MB-468
cells (Figure 3.3, IC50=6.140.79μM), EJ1 was tested for its ability to affect cell viability
in additional breast cancer cell lines including MDA-MB-231 (Figure 3.4a), and T47D
(Figure 3.4b), and the immortalized breast epithelial cell line MCF10A (Figure 3.4c). Due
to limited availability of the peptides, we only determined IC50 for MDA-MB-468 cells.
In analyzing the effects of EJ1 in these lines, we found that its effects range from a
minimum of 27% reduction in MCF10A cell viability (Figure 3.4c), to a maximum of
75% reduction in that of T47D cells over a three day treatment period (Figure 3.4b).
Analysis of the erbB expression profile (including EGFR, ErbB2 and ErbB3) in these cell
lines demonstrated expression of at least two of the three erbB receptors in each of the
lines (Figure 3.5).
86
***
***
***
***
Figure 3.3. Dose-dependent response of EJ1 peptide in MDA-MB-468 cells.
MDA-MB-468 cells were treated daily with a different concentration of EJ1 peptide
or left untouched (UNTX) for three days and cell viability was determined by MTT assay.
The UNTX group of cells was set as 100%. ***, p < 0.001, Student’s t-test. Error bars,
SD.
87
a
b
***
***
c
Figure 3.4. EJ1 peptide affects cell viability in different cell lines.
MDA-MB-231 (a), T47D (b), and MCF10A (c) cells were treated daily with 20μM
of EJ1 peptide or left untouched (UNTX) for three days, and cell viability was
determined by MTT assay. ***, p < 0.001, Student’s t-test. Error bars, SD.
88
Figure 3.5. EGFR, ErbB2 and ErbB3 expression in different cell lines.
EGFR, ErbB2 and ErbB3 expression were determined in breast cancer cell lines
(MDA-MB-231, MDA-MB-468, T47D, and BT20), an immortalized breast epithelial cell
line (MCF10A), and a pancreatic cell line (BxPC3). Forty micrograms of cell lysates
from each cell line were loaded on SDS-PAGE and immunoblotted with antibodies as
indicated.
B.
EJ1 inhibits EGFR activation through promoting inactive dimers
To determine whether EJ1 was affecting EGFR activity, we first treated
MDA-MB-468 with EJ1, CP or a vehicle in the presence or absence of EGF to activate
EGFR (Figure 3.6). We found that EJ1 significantly suppressed EGF-induced
phosphorylation of EGFR (pY845). This suppression also affected downstream signaling
partners, resulting in a reduction of p-AKT, dp-ERK (Figure 3.6 and data not shown).
89
Interestingly, treatment with EJ1 also resulted in a loss of total protein for AKT, p38, and
p42/44 MAPK (Figure 3.6 and data not shown). In addition, an increase of the activated
stress response kinase, p38, was observed upon EJ1 treatment.
Figure 3.6. EJ1 peptide inhibits EGFR activation.
MDA-MB-468 cells were serum-starved overnight and treated with 100ng/ml EGF
for 10 minutes on ice and then incubated with water (V), 20μM CP (CP) or EJ1 (EJ1) in
serum-free medium for the indicated times at 37°C and lysed. Protein lysates were
separated by SDS–PAGE and immunoblotted with antibodies as indicated.
90
As the EJ1 peptide mimics the dimerization domain of EGFR, we next evaluated the
ability of EJ1 to block dimerization. To evaluate the effects of EJ1 on EGFR homodimers,
MDA-MB-468 cells were treated with EGF and EJ1 or controls in the presence of a
non-cleavable cross-linker. Surprisingly, we found that EJ1 induced the formation of
EGFR dimers (Figure 3.7). Together, these results indicate that EJ1 inhibits EGFR
activation and promotes the dimerization of inactive EGFR receptors.
91
Figure 3.7. EJ1 peptide promotes EGFR homodimer formation.
MDA-MB-468 cells were serum-starved overnight and pre-treated with water
(Vehicle), 20μM CP (CP) or EJ1 (EJ1) in serum-free medium for 10 minutes on ice. Cells
were then stimulated with 100ng/ml of EGF and respective treatment for another 10
minutes on ice and then washed with PBS twice. Proteins were crosslinked by 3μM DMS
for 30 minutes on ice and then lysed. Protein lysates were separated by SDS–PAGE and
immunoblotted with antibodies as indicated. Arrow indicates EGFR homodimers.
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C.
EJ1 affects cell survival through apoptosis/necrosis
In order to understand how EJ1 peptide affected cell viability, MDA-MB-468 cells
were evaluated for cleaved PARP (apoptosis marker) and LC3B (autophagy marker [140])
expression (Figure 3.8). An induction of the cleavage of PARP as well as a conversion of
LC3B-I (upper band) to LC3B-II (lower band) were both observed upon EJ1 treatment
compared to controls. Autophagy has been linked with cell death as well as with the cell
survival mechanism of cancer cells (reviewed in [141]). To determine whether
EJ1-induced autophagy was a mechanism of cell death, we measured cell viability upon a
co-treatment of EJ1 and 3-MA, an autophagy inhibitor via the inhibition of type III PI3K
[142] (Figure 3.9). We found that the inhibition of autophagy results in more dead cells
upon EJ1 treatment, which indicated autophagy in this case was actually a pro-survival
signal for cells. To further test if apoptosis was the only mechanism of cell death caused
by EJ1, we performed a flow cytometry to quantitatively determine propidium iodide (PI)
staining and annexin V binding in EJ1-treated cells. As shown in Figure 3.10 and Figure
3.11, to our surprise, we observed only 6% and 2% of cells stained with annexin V alone
after 6 hours and 24 hours of EJ1 treatment, respectively. For annexin V/PI
double-stained cells, we also observed only 10% and 9% of cells after 6 hours and 24
93
hours of EJ1 treatment respectively. Of note, there were 4% and 2% of cells stained with
annexin V alone and there were 6% and 5% of cells double-stained with annexin V/PI
after 6 hours and 24 hours of CP treatment, respectively. Both the percentage of cells
undergoing apoptosis and the differences between EJ1 and CP treatment indicated that
apoptosis might not be the only mechanism of EJ1-induced cell death.
94
Figure 3.8. EJ1 peptide induces apoptosis and autophagy.
MDA-MB-468 cells were serum-starved overnight and treated with 100ng/ml EGF
for 10 minutes on ice, then washed with PBS to remove unbound ligands. Cells were then
incubated with water (Vehicle), 20μM CP or 20μM EJ1 in serum-free medium at 37°C for
6 hours and lysed. Protein lysates were separated by SDS–PAGE and immunoblotted
with antibodies as indicated.
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Figure 3.9. Inhibition of autophagy causes more cell death upon EJ1 treatment.
MDA-MB-468 cells were cultured in a 96-well plate (2 × 103 cells/well) and treated
with either water (Vehicle), 20μM EJ1, 1mM 3-MA or EJ1 in combination with 3-MA in
complete medium for seven days. A MTT assay was done to quantify viable cells at day 3.
Y-axis is expressed as percentage change of each treatment to untreated cells (UNTX). *,
P < 0.05, Student’s t-test. Error bars, SD.
96
Figure 3.10. EJ1 peptide induces a minor fraction of cells to undergo apoptosis
after 6 hours of treatment.
(a) MDA-MB-468 cells were treated with 20μM CP or EJ1 in complete medium for
6 hours at 37°C and then stained with propidium iodide (PI) and annexin V-FITC. Cells
were then sorted by a FACScan flow cytometer (BD Biosciences) and analyzed by
Cellquest Pro 4.0 software. The result is shown as flow cytometry dot plots with annexin
V staining in x axis and PI in y axis. (b) The result is shown as histogram to represent
percentage of cells in each quadrant.
97
Figure 3.11. EJ1 peptide induces a minor fraction of cells to undergo apoptosis
after 24 hours of treatment.
(a) MDA-MB-468 cells were treated with 20μM CP or EJ1 in complete medium for
24 hours at 37°C and then stained with propidium iodide (PI) and annexin V-FITC. Cells
were then sorted by a FACScan flow cytometer (BD Biosciences) and analyzed by
Cellquest Pro 4.0 software. The result is shown as flow cytometry dot plots with annexin
V staining in x axis and PI in y axis. (b) The result is shown as histogram to represent
percentage of cells in each quadrant.
98
To further explore the mechanism of cell death induced by the EJ1 peptide, cell
morphology was examined following EJ1 treatment. We found that by 15 minutes, the
EJ1 peptide induced the formation of large membrane protrusions or blebs (Figure 3.12f,
arrows) and by 60 minutes of treatment, cells had formed large intracellular vacuoles
(Figure 3.12g, arrowheads). After 16 hours, most EJ1-treated cells had died (Figure
3.12h). To investigate the vacuoles shown in Figure 3.12g, transmission electron
microscopy (TEM) was done (Figure 3.13). MDA-MB-468 cells were treated with 20μM
EJ1 and evaluated at several timepoints by TEM (Figure 3.13a-d). By 30 minutes,
double-membrane structures (Figure 3.13c’, arrowheads) filled with organelle debris
(Figure 3.13c’, filled arrows) and electron dense deposits (Figure 3.13c’, open arrows)
were observed in EJ1-treated cells. Electron-dense deposits in the swollen mitochondria
usually suggest an increase of Ca2+ influx [143] or even a calcium overload [144]. A
possible mechanism of massive Ca2+ influx is loss of membrane integrity. To test this
possibility, plasma membrane integrity was determined by a calcein-AM leakage assay.
In live cells, nonfluorescent calcein-AM can cross the membrane and be converted to a
green-fluorescent calcein (after acetoxymethyl ester hydrolysis by intracellular esterases),
becoming membrane impermeable. If the plasma membrane integrity is compromised,
99
the calcein dye will be released from the cells and can then be detected in the media.
Therefore, MDA-MB-468 cells were treated with calcein-AM for 30 minutes, followed
by treatment with vehicle, CP, EJ1, or Triton X-100 as a positive control for 15 minutes
(Figure 3.14). EJ1 treatment resulted in a significant increase of calcein signal in the
media, and the same results were seen with T47D, BT20 and MDA-MB-231 cells (data
not shown), indicating the plasma membrane integrity was breached by EJ1. To further
understand whether the observed membrane damage caused an increase of intracellular
calcium concentration, which in turn resulted in swollen mitochondria with
electron-dense deposits, a calcium assay was done to measure intracellular calcium
concentration after EJ1 treatment (Figure 3.15). Not surprisingly, a significant increase of
intracellular calcium concentration was observed after EJ1 treatment. These results
indicate that the EJ1 peptide affects plasma membrane integrity, leading in turn to a rapid
increase of intracellular calcium concentration. This calcium overload may further cause
mitochondrial swelling and intracellular vacuoles formation [145].
Loss of plasma membrane integrity is one of the main characteristics of necrosis. In
order to understand if EJ1-induced cell death was necrosis-related, culture media from
EJ1-treated MDA-MB-468 cells were collected and evaluated for the release of the
100
nuclear protein HMGB1 (high mobility group box 1), an indicator of necrosis [137]
(Figure 3.16). We found detectable HMGB1 in EJ1- but not control-treated cell media.
Interestingly, we also found AKT and p38 presented in EJ1-treated cell media (Figure
3.16), which was consistent with our previous findings (Figure 3.6) that EJ1 resulted in
loss of cellular AKT and p38. Taken together, these data indicate that EJ1 causes cell
death through both apoptosis and necrosis.
Figure 3.12. EJ1 causes membrane dynamic change and intracellular vesicle
formation.
MDA-MB-468 cells were treated with either 20μM CP (a-d) or 20μM EJ1 (e-h) in
complete medium at 37°C for 0 minute (a and e), 15 minutes (b and f), 60 minutes (c and
g) or 16 hours (d and h). Images represent the bright-field images. Arrows indicate
membrane blebbing. Arrowheads indicate intracellular vacuoles.
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Figure 3.13. EJ1 causes double-membrane-vacuole formation.
MDA-MB-468 cells were treated with 20μM EJ1 in complete medium at 37°C for 0
minute (a), 5 minutes (b), 30 minutes (c and c’) or 2 hours (d). Cells were then prepared
for TEM. Magnification is represented as scale bars indicated. Filled arrows indicate
organelle debris. Open arrows indicate electron-dense deposits. Arrowheads indicate
double-membrane structures.
102
Figure 3.14. EJ1 treatment affects membrane integrity.
MDA-MB-468 cells were cultured in a 24-well plate (5 × 104 cells/well) and loaded
with 1μM Calcein-AM in complete medium at 37°C for 30 minutes. Cells were then
treated with 1% Triton X-100, water (Vehicle), 20μM CP or EJ1 in PBS with 5% FBS at
37°C for 15 minutes. Media were then carefully transferred to a new plate and fluorescent
signal (Ex/Em 490/515 nm) was measured by a plate reader. Y-axis is expressed as
percentage of fluorescent intensity for each treatment compared to Triton X-100 treated
cells. **, P < 0.01, Student’s t-test. Values are the means of three independent
determinations ± SD.
103
Figure 3.15. EJ1 causes an increase of intracellular calcium concentration.
MDA-MB-468 cells were seeded in 96 well plates and treated with water (Vehicle),
20μM CP or EJ1 and measurement of intracellular calcium concentration was done
following manufacturer’s protocol as described in Materials and Methods section.
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Figure 3.16. EJ1 causes necrosis.
MDA-MB-468 cells were treated with water (Vehicle), 20μM CP, 20μM EJ1, 2μM
ionomycin and 50μM CCCP as necrosis inducers (necrosis), 10 ng/ml human tumor
necrosis factor α and 35μM cycloheximide as apoptosis inducers (apoptosis), or 100nM
rapamycin as an autophagy inducer (autophagy) for indicated times. Following treatments,
media were collected and processed as described in [137]. Proteins from media were then
separated in SDS-PAGE and immunoblotted with antibodies as indicated.
D.
EJ1 causes membrane dynamic change through affecting Ca2+/CaM downstream
MLCK signaling
EJ1 induces a rapid membrane dynamic change (Figure 3.12f) and a rapid increase
of intracellular calcium concentration (Figure 3.15). Some speculation occurred regarding
whether these two events were correlated. Binding of Ca2+ to CaM leads to activation of
Ca2+/CaM-regulated downstream signaling, which controls many different cellular events
105
such as membrane dynamics, cell survival, mitochondrial function, motility, and
exocytosis [146-148]. Myosin light chain kinase (MLCK) activity is responsible for
membrane dynamic regulation [149]. Once MLCK is activated by Ca2+/CaM it can then
phosphorylate myosin light chain (MLC) and regulate actinomyosin reorganization
during membrane blebbing. To investigate whether EJ1-induced membrane blebbing was
via the MLCK pathway, MDA-MB-468 cells were treated with vehicle, CP, or EJ1 alone
(Figure 3.17a-c) or EJ1 in combination with the calmodulin inhibitor W-13 or the MLC
phosphorylation inhibitors ML-7 and Y-27632 (Figure 3.17d-f). We found that both the
calmodulin inhibitor W-13 and the MLC phosphorylation inhibitors ML-7 and Y-27632
completely inhibited EJ1-induced membrane blebbing. To determine if these effects on
membrane blebbing were related to cell survival, the inhibitors were used in conjunction
with EJ1 in a MTT assay. To perform this MTT, cells were evaluated after only one day
of treatment due to long term cytotoxicity of ML-7. After one day of treatment, both
Y-27632 and ML-7 were able to significantly reduce the effects of EJ1 on cell viability
(Figure 3.18). Taken together, EJ1 affects membrane integrity which leads to calcium
influx and activation of MLCK pathway which is integral to the EJ1 mediated reduction
in cell survival.
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Figure 3.17. EJ1 induces membrane blebbing through activation of Ca2+/CaM and
its downstream effector, MLCK.
MDA-MB-468 cells were pre-treated with 50μM W-13 (d), 10μM ML-7 (e) or
10μM Y-27632 (f) in complete medium at 37°C for 30 minutes, then treated with either
water (vehicle) alone (a), 20μM CP alone (b), or 20μM EJ1 alone (c) or in combination
with W-13 (d), ML-7 (e) and Y-27632 (f) in complete medium at 37°C for 15 minutes.
Images represent the bright field images. Arrows indicates membrane blebbing.
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Figure 3.18. MLC phosphorylation inhibitors can rescue EJ1-induced cell death.
MDA-MB-468 cells were cultured in a 96-well plate (2 × 103 cells/well) and treated
with either water, 20μM EJ1 alone, 10μM Y-27632 alone, 10μM ML-7 alone or EJ1 in
combination with Y-27632 or ML-7 in complete medium for one day. A MTT assay was
done to quantify viable cells after one day. Y-axis is expressed as percentage change of
each treatment to untreated cells (UNTX). **, P < 0.01, Student’s t-test. Error bars, SD.
E.
EJ1 causes mitochondrial disruption and reactive oxygen species (ROS) generation
During the evaluation of intracellular vesicles created by EJ1 treatment (Figure 3.12
and Figure 3.13), what observed appeared to be damaged mitochondria (Figure 3.13c’,
filled arrows). To further explore the effects of EJ1 treatment on mitochondria,
MDA-MB-468 cells were labeled with Mitotracker, treated with either EJ1 or CP and
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then imaged (Figure 3.19a-d). Mitochondria appeared enlarged and rounded within five
minutes of EJ1 treatment (Figure 3.19d’, arrowheads). To determine if the mitochondrial
membrane was damaged during this process, cells were treated with JC-1 dye, a reporter
of mitochondrial membrane potential. MDA-MB-468 cells were labeled with JC-1 for 15
minutes and then treated for two hours with CP, EJ1 or carbonyl cyanide
3-chlorophenylhydrazone (CCCP), a compound disrupting mitochondrial integrity, as a
positive control for mitochondrial damage. A significant loss of mitochondrial membrane
potential was observed with EJ1 treatment (Figure 3.20). Overall, these results imply that
EJ1-induced loss of membrane integrity and calcium influx may cause mitochondrial
swelling and loss of mitochondrial membrane potential.
109
Figure 3.19. EJ1 causes mitochondrial swelling.
MDA-MB-468 cells were incubated with 200nM MitoTracker Red CMXRos and 5
μg/ml Hoechst 33342 nuclear stain, followed by either 20μM CP (a-b’) or 20μM EJ1
(c-d’), and imaged at 0 minute (a, a’ and c, c’) or at five minutes (b, b’ and d, d’).
Arrowhead indicates enlarged mitochondria.
110
Figure 3.20. EJ1 treatment disrupts mitochondrial membrane potential.
MDA-MB-468 cells were serum-starved overnight and first stained with 1μM JC-1
in serum-free medium for 15 min at 37°C and then incubated with 20μM CP, 20μM EJ1
or 50μM CCCP in serum-free medium for 2 hr at 37°C. Following the treatments, cells
were trypsinized and collected. After being washed twice with PBS, the cell pellets were
re-suspended in PBS and transfered to a 96-well plate. Fluorescent signals were then
detected at 514/529 nm and 514/590 nm by a plate reader. Results were calculated as the
ratio of the 514/590 nm to 514/529 nm and normalized to the CP-treated samples. *, P <
0.05, Student’s t-test. Values are the means of 3 independent determinations ± SD.
Loss of mitochondrial membrane potential is frequently linked to an accumulation
of ROS within the cells (reviewed in [150]). To measure intracellular ROS levels,
DCFH-DA, which becomes fluorescent DCFH in the presence of ROS, was used to
measure ROS levels in MDA-MB-468 cells. Cells were treated with either CP, N-acetyl
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cysteine (NAC, a ROS scavenger which reduces intracellular ROS levels), EJ1 or NAC +
EJ1 (Figure 3.21). While EJ1 treatment increased intracellular ROS levels as indicated by
DCFH fluorescence, co-treatment with NAC significantly reduced EJ1-induced ROS
levels. It was then to be determined whether decreasing ROS would prevent EJ1-induced
cell death. We found that NAC could significantly inhibit EJ1-induced cell death (Figure
3.22). These data demonstrate that EJ1 causes cell death at least in part through loss of
mitochondrial function and ROS accumulation.
Figure 3.21. EJ1 causes accumulation of ROS.
MDA-MB-468 cells were stained with 10μM DCFH in complete medium for 30
minutes at 37°C and then incubated with water (Vehicle), 20μM CP, 0.5mM NAC, 20μM
112
EJ1 or EJ1 in combination with NAC in complete medium for 1 hr at 37°C. Cells were
then sorted by a FACScan flow cytometer (BD Biosciences) and analyzed by Cellquest
Pro 4.0 software. The results are expressed as the percentage of green fluorescent cells. *,
P < 0.05, Student’s t-test. Values are the means of three independent determinations ± SD.
Figure 3.22. Inhibition of ROS reduces EJ1-induced cell death.
MDA-MB-468 cells were cultured in a 96-well plate (2 × 103 cells/well) and treated
with either 0.5mM NAC alone, 20μM EJ1 alone, or NAC in combination with EJ1 in
complete medium for 3 days. A MTT assay was done to quantify viable cells at day 3.
Y-axis is expressed as a percentage change of each treatment to untreated cells (UNTX).
***, P < 0.001, Student’s t-test. Error bars, SD.
113
So far, the effect of EJ1 was shown to be partially inhibited by both inhibitors of
MLC phosphorylation (ML-7 and Y-27632) and an inhibitor of ROS (NAC) (Figure 3.18
and Figure 3.22). To determine if a combination of these two classes of drugs could
effectively block EJ1-induced cell death, cells were treated with the inhibitors (alone or
in combination) in the presence or absence of EJ1 (Figure 3.23). Virtually all of the cell
death induction from EJ1 was eliminated by blocking both MLC phosphorylation and
ROS accumulation, indicating that these two pathways account for a majority of the
mechanism by which EJ1 kills cells.
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Figure 3.23. The mechanisms of EJ1-induced cell death are MLCK-regulated
membrane blebbing and accumulation of ROS.
MDA-MB-468 cells were treated with either water (Vehicle), 20μM CP, 20μM EJ1
alone, or in combination with Y-27632 (Y), ML-7 (M) and NAC (N) and MTT assays
were performed to quantify viable cells after one day. ***, p < 0.001, NS, no statistical
significance, Student’s t-test. Error bars, SD.
F.
EJ1 reduces tumor growth and metastasis in mouse models of breast cancer
We next set out to determine whether EJ1 would function as an anti-tumor therapy
in vivo. MMTV-pyMT murine model of breast cancer was used, which develops
synchronous, multifocal mammary tumors in all ten mammary glands with a multistep
115
progression that resembles human disease [151, 152]. To determine dosage, animals were
injected daily (intraperitoneal, i.p.) with 5, 10, 20 or 40 mg/kg body weight EJ1 or
vehicle (PBS). Twenty mg/kg body weight appeared to provide the optimal tumor
regression (data not shown), and this dose was then given to tumor-bearing
MMTV-pyMT mice for either EJ1 (n=6) or CP (n=3). Three additional mice were given
PBS (n=3). We found that both the average tumor size (~37.1% reduction in average
tumor size) and the growth rate of tumors were significantly reduced by treatment with
EJ1 compared to CP or PBS (12.1 mm3/day in control-treated versus 7.8 mm3/day in
EJ1-treated; Figure 3.24 and data not shown). Many EJ1-treated tumors were also
necrotic in appearance compared to controls (Figure 3.24, insets). Importantly, no toxicity
from this dose of EJ1 was observed (weight loss, grooming, behavior, or gross changes to
organs upon necropsy).
One thing to be noted is that MMTV-pyMT mice display highly penetrant secondary
metastases to the lungs [153]. Since EGFR expression and activity have been correlated
with tumor metastasis in different types of cancers [154-157], lung metastases were
evaluated to determine whether the EJ1 peptide also affected metastasis in this mouse
model of breast cancer. We found that on average, the lungs of EJ1-treated mice had
116
significantly less metastatic foci than did comparable CP-treated mice as assessed by
bright field microscopic analysis and H&E assessment of tissue architecture (Figure 3.25
and insets).
Figure 3.24. EJ1 reduces tumor growth in a MMTV-PyMT mouse model.
Tumor-bearing MMTV-pyMT mice were injected with 20 mg/kg body weight (i.p.)
EJ1 (filled squares) or control peptides (open squares) for 21 days and tumors were
measured every five days. **, p < 0.01, Student’s t-test. Error bars, SE. Representative
images of control- and EJ1-treated tumors are shown.
117
+ controls
+ EJ1
Figure 3.25. EJ1 reduces lung metastasis in a MMTV-PyMT mouse model.
Tumors metastasized to the lungs were evaluated by embedding the lungs and
enumerating microscopic metastasis by H&E. Horizontal line represents average number
of lung metastasis foci in each mouse. **, p < 0.01, Student’s t-test.
IV. Discussion
In recent years, the essential regulatory role of the juxtamembrane domain of the
EGFR has been investigated and hypothesized (reviewed in [158]). In the present study,
we set out to determine whether this domain could be targeted by an EGFR-dependent
anti-cancer therapeutic. We found that a peptide composed of a 21 amino acid portion of
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the juxtamembrane domain (EJ1, FMRRRHIVRKRTLRRLLQERE) could effectively
kill breast cancer cell lines. A similar peptide (TE-64562, RRRHIVRKRTLRRLLQER)
was used to demonstrate inhibition of EGFR signaling and anti-cancer activity by another
group [159]. We discovered that this peptide (EJ1) promotes the formation of inactive
EGFR dimers, resulting in the activation of MLCK signaling, a downstream effector of
Ca2+/calmodulin regulated signaling. The results of this selective signaling were
membrane blebbing and cell death. In addition, EJ1 can affect mitochondrial membrane
potential involving the generation of ROS and induction of apoptosis/necrosis. Finally,
these effects appear to be tumor-specific, as injection of EJ1 into an immune-competent
transgenic mouse model of breast cancer resulted in an inhibition of tumor growth and
metastasis without any gross toxicity.
Previous studies have shown that the helical transmembrane domain and
anti-parallel dimeric interaction of the helical juxtamembrane domain A (amino acids
645-663) are both likely important for functional dimerization of the EGFR, as well as
for EGFR activation [20, 158, 160-163]. Two studies using an EGFR transmembrane
fragment or ErbB2 transmembrane peptide both demonstrated an inhibition of receptor
dimerization and activation [164, 165]. We have demonstrated that treatment of breast
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cancer cells with EJ1 results in a dramatic reduction in the EGF-mediated
phosphorylation of EGFR through the promotion of dimer formation. Our data suggest
that EJ1 could affect EGFR activity by altering the structural interaction of endogenous
EGFR dimers. The ability of an EGFR inhibitor to promote the formation of inactive
dimers is not unprecedented. Several groups have demonstrated that the EGFR kinase
inhibitors AG1478, AG1517 and ZD1839 promote receptor dimerization, while at the
same time impairing kinase activity [166-168].
MLCK-mediated phosphorylation of the myosin light chain is one way by which
actin cytoskeleton reorganization is regulated, and it is known to be involved in
membrane blebbing [149, 169]. Membrane blebbing induced by EJ1 peptide indicates
that myosin light chain phosphorylation is upregulated (Figure 3.12). Complete inhibition
of membrane blebbing by pre-treatment of calmodulin inhibitor (W-13), MLCK inhibitor
(ML-7) or ROCK-1 inhibitor (Y27632), which also phosphorylates the myosin light
chain, further confirms that EJ1 induced membrane blebbing is controlled by
phosphorylation of the myosin light chain through both MLCK and ROCK-1 (Figure
3.17). In addition, phosphorylation of the myosin light chain has been correlated with
both cell survival and cell death under different conditions [170-173]. Under the
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circumstances of EJ1 treatment, MLCK and ROCK-1 inhibitors partially rescue
EJ1-induced cell death, indicating induction of phosphorylation of MLC by EJ1 treatment
is a regulator of membrane dynamics as well as of cell death.
We found that EJ1 treated cells were filled with vacuoles and TEM showed those
vacuoles were double membrane structures (Figure 3.13c’, arrowheads). It was
speculated that these vacuoles were swelled mitochondria and organelle debris inside the
vacuoles were cisternae remnants (Figure 3.12c’ filled arrows). How could the EJ1
peptide cause mitochondrial swelling? One explanation is mitochondrial permeability
transition (MPT). The opening of high conductance permeability transition pores in the
mitochondrial inner membrane causes MPT and that leads to mitochondrial
depolarization and uncoupling of oxidative phosphorylation, which will eventually result
in depletion of ATP and cell death [174]. Loss of mitochondrial membrane potential is
one of the measurable events of MPT. As shown in Figure 3.20, a significant loss of
mitochondrial membrane potential was seen in EJ1 treated cells.
There are multiple compounds that can act on permeability transition pore complex
to induce MPT. Other agents that increase cytosolic Ca2+ or stimulate ROS generation
can also trigger MPT [175]. Interestingly, we found that EJ1 treatment caused an
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immediately increase of intracellular calcium concentration (Figure 3.15) and
accumulation of ROS (Figure 3.21). This implies that the EJ1 peptide causes calcium
influx and overloads which leads to MPT and loss of mitochondrial membrane potential
as well as to cell death. MPT induced cell death can be apoptosis or necrosis depending
on ATP levels of the cells [176]. We found both types of cell death occurring in cells with
EJ1 treatment (Figure 3.8, Figure 3.10, Figure 3.11, and Figure 3.16).
Mitochondria control cell growth by regulating energy production and also guard
cell death by modulating the translocation of pro-apoptotic proteins from the
mitochondrial intermembrane space to the cytosol. Furthermore, mitochondria also play a
major part in multiple forms of non-apoptotic cell death and, in particular, in necrosis. As
mitochondria are key regulators of cell death, it is not surprising that many of the
mitochondrial functions are often altered in cancer, and mitochondrial targeted therapy
could be a promising strategy to kill cancer cells. Our results indicate that the EJ1 peptide
induces mitochondrial swelling (Figure 3.19) and loss of mitochondrial membrane
potential (Figure 3.20). This implies that the EJ1 peptide could be a potential
mitochondrial-targeted therapeutic which warrants further investigation.
In conclusion, EJ1 represents a novel EGFR therapeutic, targeting multiple activities
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of the EGFR receptor in a tumor-specific manner. In addition, EJ1 targets cells in an
EGFR-independent manner via its ability to disrupt membrane integrity in a
tumor-specific manner. Together, these effects result in a highly tumor-specific
anti-cancer therapeutic that may have significant clinical utility.
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CHAPTER 4 – EFFECTS OF MUC1 EXPRESSION ON ERBB3 ACTIVITY
Note: The work presented in this chapter has not been published. All experiments were
performed by Hsin-Yuan Su.
I.
Introduction
MUC1, a transmembrane mucin, is heavily glycosylated and normally found on the
apical surface of glandular epithelia [177]. The expression of MUC1 is aberrantly
regulated in more than 90% of breast carcinomas and metastases and its expression has
been linked to cell transformation in vitro and in vivo [178-180]. One mechanism by
which MUC1 can drive transformation is through cancer-dependent protein-protein
interaction. This has been shown in a WAP-TGFα transgenic mouse model, where the
expression
of
MUC1
dramatically
affects
EGFR-dependent
mammary
gland
transformation and cancer progression [181]. In addition, the interaction between MUC1
and EGFR inhibits ligand-induced ubiquitination and degradation of EGFR in human
breast cancer cell lines [34]. This indicates that expression of MUC1 may promote
oncogenic transformation through the inhibition of EGFR degradation. Additionally,
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MUC1 physically associates with all four erbB receptors and the interaction between
MUC1 and the receptor tyrosine kinase EGFR results in an increase in MAP kinase
activation [182]. The deregulation of erbB signaling frequently occurs during breast
cancer initiation and progression, making it important to understand the effects of MUC1
expression on other erbB receptors.
The erbB receptors belong to a receptor tyrosine kinase superfamily. Reviewed in
[183], there are four members in this family: EGFR, erbB2/neu, erbB3 and erbB4. All
members share a similar structure which contains an extracellular ligand-binding domain,
a single membrane-spanning domain, a juxtamembrane domain, a cytoplasmic
tyrosine-kinase-containing domain, and a carboxy-terminal tail. Ligands for erbB family
receptors comprise more than 11 different members, and upon binding to erbB receptors
they induce the formation of receptor homo- or heterodimers. Dimerization leads to
activation of the receptor and initiation of different downstream signaling pathways, such
as MAPK, PI3K/AKT, JAK/STAT and PLCγ. These different signals can promote cell
proliferation, migration and survival, resulting in breast cancer development and
metastasis [183]. EGFR has been shown to be either overexpressed or overactivated in
different types of human cancers, including breast cancer. The development of
125
EGFR-targeting antibodies or tyrosine kinase inhibitors has earned great success in vitro
and in vivo, reviewed in [184, 185]. However, these agents show very limited clinical
anti-tumor activity in breast cancer [186]. Recent studies have shown that EGFR
therapeutic resistance can be linked to ErbB3 expression [53].
ErbB3 receptors are highly expressed in more than 30% of invasive breast cancers
and are correlated to poor prognosis by many studies [187, 188]. Within the c-terminal
regulatory domain of ErbB3, there are six binding sites for the p85 regulatory subunit of
PI3K. Accordingly, the ErbB3 receptor is considered as a key regulator of PI3K activity
[189-191]. The PI3K/AKT pathway mediates survival signals that are required for tumor
maintenance as well as therapeutic resistance (reviewed in [192]). MUC1 can
dramatically alter EGFR-dependent breast cancer progression, and can also interact with
ErbB3 [182]. Therefore, it was hypothesized that MUC1 expression could alter ErbB3
signaling through affecting ErbB3 trafficking and/or degradation.
II. Materials and Methods
Western Blotting and Cell Viability Assay are the same as described in chapter 2.
126
Transfection with siRNA Transfection with siRNA was performed as in [87]. The MUC1
siRNA targets the extracellular domain (5’-AAGACTGATGCCAGTAGC- ACT-3’)
[160]. Additionally, the control siRNA has no known mammalian gene targets
(5’-AATTCTCCGAACGTGTCACGT-3’) (Qiagen Sciences Inc., German- town, MD).
The transfections were performed with Lipofectamine 2000 (Invitrogen Life
Technologies Inc., Carlsbad, CA) following the manufacturer’s instructions.
Immunoprecipitation BT20 cells were transfected with either MUC1 siRNA or control
siRNA. After 48 hours, cells were serum starved overnight and then treated with/without
5ng/ml of NRG1β for 10 minutes on ice. Protein lysates were collected and
immunoprecipitation with ErbB3 antibodies was performed as described by Pochampalli
et al., 2007 [34]. Immunoprecipitants were separated by SDS-PAGE and immunoblotted
with EGFR, ErbB3 and MUC1 antibodies.
Wound Healing 2D Migration Assay BT20 cells were transiently transfected with either
MUC1 siRNA (MUC1) or control siRNA (control). After 24 hours, cells were trypsinized
and re-plated in 24 well plates at 2104 cells per well to get a confluent monolayer. Then
127
the cells were serum-starved overnight. A p200 pipet tip was used to create a scratch in
the cell monolayer. Cells were then washed with PBS and refreshed with serum-free (SF)
medium containing different concentrations of NRG1β (NRG) as indicated. Pictures were
then taken at different time points and the distance between the closest margins was
measured.
III. Results
A.
MUC1 expression affects NRG1β-induced ErbB3 degradation and downstream
signaling
ErbB receptors, including EGFR and ErbB3, are typically endocytosed upon ligand
binding, trafficked to the lysosome and degraded [193]. MUC1 is known to prevent the
ligand-induced degradation of EGFR [34]. Therefore, we investigated whether it has a
similar effect on ErbB3. BT20 breast cancer cells were used to determine the effect of
MUC1 expression on ErbB3 stability. After transient transfection of either control siRNA
or MUC1 siRNA to knockdown MUC1 expression, NRG1β (neuregulin-1β, an ErbB3
ligand) was used to induce receptor internalization. We found that MUC1 expression
inhibited ligand-induced ErbB3 down-regulation at one- and two-hour time points, when
128
ErbB3 receptors began to be degraded in this cell line [194, 195]. Importantly, this effect
was only seen after ligand stimulation, indicating that this is a ligand-mediated event
(Figure 4.1, top 2 panels). A similar experiment was done in MDA-MB-231 cells in
which pCMV-MUC1 plasmid was transfected into cells to overexpress MUC1. Similarly,
MUC1
expression
inhibited
ligand-induced
ErbB3
down-regulation
of
phosphorylated (pY1289 [196]) and total protein levels (Figure 4.2, top 2 panels).
both
129
Figure 4.1. MUC1 inhibits the degradation of NRG1β-stimulated ErbB3 and
sustains downstream signaling in BT20 cells.
BT20 cells were transfected with either MUC1 siRNA or control siRNA, and treated
with 5ng/ml NRG1β for ten minutes on ice. Following treatments, endocytosis was
allowed for the indicated times before lysis. Protein levels were determined as indicated.
130
Figure 4.2. MUC1 inhibits the degradation of NRG1β-stimulated ErbB3 and
sustains downstream signaling in MDA-MB-231 cells.
MDA-MB-231 cells stably expressing empty vector (pcDNA3) or MUC1
(pCMV-MUC1) were treated with 5ng/ml NRG1β for ten minutes on ice. Following
treatments, endocytosis was allowed for the indicated times before lysis. Protein levels
were determined as indicated.
Activation of erbB receptors leads to different downstream signal transduction
events [183]. For ErbB3, PI3K/AKT is the prominent effector pathway regulated by the
receptor activity [197]. Thus, AKT activation was determined. As a result, p-AKT (pS473)
131
level was sustained by MUC1 expression in both BT20 and MDA-MB-231 cells (Figure
4.1 and Figure 4.2). MAP kinase signaling pathway, another important pathway
downstream of ErbB3, was also investigated. Similarly, the status of ERK (extracellular
signal-regulated kinase) phosphorylation was sustained by MUC1 expression in both cell
lines (Figure 4.1 and Figure 4.2). These results demonstrate that MUC1 expression has a
profound effect on NRG1β-induced ErbB3 degradation, and on downstream PI3K/AKT
and MAP kinase signaling pathways.
B.
MUC1 expression facilitates EGFR/ErbB3/MUC1 complex formation
ErbB receptors form homo- or heterodimers after ligand binding, and different
dimerization leads to different combinations of downstream signaling. BT20 cells were
used to determine whether MUC1 expression affects dimer formation. After transient
transfection of either control siRNA or MUC1 siRNA to knockdown MUC1 expression,
cells were treated with NRG1β to induce receptor dimerization. Complex formation was
determined by immunoprecipitation with the ErbB3 antibody. EGFR formed a complex
with ErbB3 only in the presence of MUC1 and NRG1β stimulation. In addition, MUC1
formed a complex with EGFR and ErbB3 only in the presence of NRG1β stimulation
132
(Figure 4.3). These results demonstrate that MUC1 expression facilitates an
EGFR/ErbB3/MUC1 complex formation.
Figure 4.3. MUC1 expression facilitates EGFR/ErbB3/MUC1 complex formation.
BT20 cells were transiently transfected with either MUC1 siRNA or control siRNA
to knockdown MUC1 expression. Cells were then treated with 5ng/ml NRG1β to induce
receptor dimerization. We then determined complex formation by immunoprecipitation
133
with the ErbB3 antibody and protein levels were determined as indicated.
C.
Knockdown of MUC1 expression promotes both NRG1β-dependent and
NRG1β-independent cell survival and migration
Both the PI3K/AKT and the MAP kinase pathways are important regulators for cell
proliferation, migration and survival. Since MUC1 expression affected both PI3K/AKT
and MAPK signaling activities, cell survival and migration were next used to determine
the effect of MUC1 expression. Cell survival was determined in BT20 cells with/without
MUC1. As shown in Figure 4.4, contrary to our expectations, the survival rates were
30%~50% higher in cells without MUC1 than in cells expressing MUC1, with/without
NRG1β at 5 or 10ng/ml. Next, wound healing 2D migration assay was used to investigate
whether MUC1 expression affects cell motility. Again, cells without MUC1 unexpectedly
moved faster than cells expressing MUC1, regardless of the presence of NRG1β (Figure
4.5).
134
Figure 4.4.
Knockdown of MUC1 expression promotes both NRG1β-dependent
and NRG1β-independent cell survival in BT20 cells.
BT20 cells were transiently transfected with either MUC1 siRNA or control siRNA.
After being serum-starved overnight (day 0), cells were then subjected to different
treatments (as indicated) for 48 hours (day 2). Relative numbers of viable cells were
determined by MTT assays on day 0 and day 2 of treatment, and cell survival was
assessed by the ratio of day 2 to day 0. CM, complete medium; SF, serum-free medium.
Values are the means of three independent determinations ± SD.
135
Figure 4.5.
Knockdown of MUC1 expression promotes both NRG1β-dependent
and NRG1β-independent cell migration in BT20 cells.
BT20 cells were transiently transfected with either MUC1 siRNA (MUC1) or
control siRNA (control). After the cells were serum-starved overnight, a p200 pipet tip
was used to create a scratch in the cell monolayer. Cells were then washed with PBS and
refreshed with serum-free (SF) media containing different concentrations of NRG1β
(NRG) as indicated. After indicated times, migration distance was measured between the
closest margins of the scratch. Values are the means of three independent determinations
± SD.
In summary, we found that MUC1 expression affected ErbB3 stability and
downstream signaling. This effect could be the result of EGFR/ErbB3/MUC1 complex
formation. However, we were not able to demonstrate a direct translation of these
136
molecular changes into biological effects, such as cell survival and 2D migration.
IV. Discussion
ErbB3 is an important partner for transducing PI3K/AKT signals and is one of the
key players involved in resistance to EGFR inhibitors [53]. We found that MUC1
expression sustained ErbB3 activity as well as downstream PI3K/AKT and MAPK
activities (Figure 4.1 and Figure 4.2). We also demonstrated that NRG1β-dependent
EGFR/ErbB3/MUC1 complex formation might be the cause of sustained signaling
(Figure 4.3). The complex formation might interfere with endocytosis or promote
receptor recycling, which need to be determined. Nevertheless, both possible events
could result in a sustained signaling, which would lead to cell proliferation, migration and
survival.
To our surprise, we did not observe a positive correlation of sustained signaling with
cell survival or migration (Figure 4.4 and Figure 4.5). The reasons for these observations
are obscure. Normally, serum starvation for two days would result in apoptosis in BT20
cells and that was what control cells showed (Figure 4.4, third column from the left)
[198]. However, the lack of apoptosis in MUC1 knockdown cells (Figure 4.4, fourth
column from the left) indicated that there might be other signaling pathways involved.
137
When the p-AKT or dp-ERK level was compared between cells with/without MUC1 in a
serum-free condition, a generally higher level of p-AKT or dp-ERK was observed in
MUC1-expressing cells (Figure 4.1 and Figure 4.2), which was also in disagreement with
the MTT result (Figure 4.4). MUC1 can promote cancer cell survival by modulating
apoptosis and autophagy pathways [199, 200]. The only situation where MUC1 promotes
cell death is in activated T cells [201]. Since there was only one cell line tested, what we
observed could be a cell line specific phenomenon.
Importantly, MUC1 expression sustains NRG1β-dependent ErbB3 activation
through an EGFR/ErbB3/MUC1 complex formation. This complex formation might
represent a potential therapeutic target as to inhibit ErbB3 activities.
138
CHAPTER 5 – CONCLUDING REMARKS
It is predicted that more than 230,000 new cases of invasive breast cancer will be
diagnosed and more than 40,000 cases of breast cancer-related death will be seen just in
2013 in the US. Early diagnosis of the diseases has already improved prognosis
significantly. Molecular profiling of breast cancers has also increased specificities of
targeted therapies. Anti-hormonal therapy and anti-HER2 therapy has significantly
improved prognosis of breast cancer patients. However, in a subgroup of breast cancer,
basal epithelial-like or clinically referred as triple negative breast cancer, which lacks
expression of estrogen receptor, progesterone receptor and HER2 receptor, the
anti-hormonal and anti-HER2 therapies are ineffective. Molecular profiling demonstrates
that EGFR and cytokeratin 5/6 are usually overexpressed in this type of breast cancer.
EGFR expression and activity are tightly regulated in normal cells. Consequently,
dysregulation of EGFR leads to tumor formation and progression. Anti-EGFR drugs
therefore are considered as a promising therapy specifically for EGFR-dependent cancers.
In triple negative breast cancer, more than 50% of cases overexpress EGFR. Thus,
EGFR-targeted therapies were introduced for the treatment of patients with this type of
139
cancer. Unfortunately, anti-EGFR therapies only achieve a limited efficacy in triple
negative breast cancer patients. The reasons are attributed to intrinsic and acquired
resistance to EGFR-targeted therapies. Although secondary mutation to EGFR is not
frequently seen in breast cancer, compensatory activation of parallel signaling pathways,
such as ErbB2, ErbB3 and Met, are often seen in EGFR-targeted refractory breast cancers
[52, 79]. These parallel pathways regulate an important survival pathway, PI3K/AKT
pathway, to escape cytotoxicity of anti-EGFR drugs. Nuclear localization of EGFR also
contributes to therapeutic resistance through up-regulation of DNA repair activity
[119-121] or expression of drug efflux proteins (ABCG2) [88]. In order to improve
therapeutic efficacy of treatments in patients with triple negative breast cancer, we
developed a three-part study to target nuclear translocation of EGFR, to inhibit EGFR
activity through a novel approach, and to understand mechanisms involved in ErbB3
over-activation.
I.
Nuclear EGFR as a potential therapeutic target
Nuclear EGFR is involved in many aspects of therapeutic resistance. We
demonstrate in this dissertation that it is possible to inhibit nuclear translocation of
140
activated EGFR (pY845) by a peptide derived from the EGFR NLS sequence.
Additionally, nuclear translocation of ErbB2 and ErbB3 has been demonstrated and both
are involved in tumor progression and therapeutic resistance [202-204]. It is noteworthy
that the predicted NLS sequences are highly conserved among EGFR, ErbB2 and ErbB3
[98]. It is therefore important to determine if the ENLS peptide also inhibits nuclear
translocation of ErbB2 and ErbB3. Alternatively, peptides based on sequence homology
among the three NLS sequences could be synthesized and investigated. Also, due to the
nature of the peptide half life, chemical molecules with similar structures could be used
instead for improved stability.
The limited biological effects after ENLS peptide treatment could simply indicate
that both cytosolic and nuclear activities of EGFR are important. Cytosolic activity of
EGFR is usually dominant and regulated by kinase activation to control cell proliferation,
motility and survival. When kinase activity is inhibited by either monoclonal antibodies
or TKIs, the nuclear activity of EGFR can become dominant to regulate transcription,
stress response, and DNA repair. Therefore, much work should be done to determine the
efficacy of the ENLS peptide in combination with EGFR-targeted therapy or other
tyrosine kinase inhibitors.
141
II. Juxtamembrane domain of EGFR as a potential therapeutic target
Juxtamembrane domain of EGFR plays important roles in receptor dimerization,
activation, trafficking and interaction with calmodulin. However, none of these
non-traditional kinase related functions are directly affected by monoclonal antibodies
and TKIs. We demonstrate that a peptide derived from juxtamembrane domain of EGFR
can effectively inhibit EGFR activation through promoting inactive dimer formation. It
can also effectively kill cancer cells through processes of apoptosis and necrosis.
Mechanistically, this peptide affects membrane integrity and leads to calcium influx,
disruption of mitochondrial membrane potential, and ROS accumulation (Figure 5.1).
Clearly, this peptide possesses EGFR-dependent and EGFR-independent properties.
The EGFR-independent property could provide a solution to overcome resistance to
EGFR-targeted therapy. In a preliminary experiment, we investigated whether the EJ1
peptide is still effective in AG1478-resistant cells. As shown in Figure 5.2, the EJ1
peptide still affects cell viability of AG1478-resistant cells, although the efficacy is not as
good as that observed in parental cells. Further studies need to be done to confirm that
this effect is actually through EGFR-independent mechanisms. Importantly, the effect of
142
EJ1 peptide appears to be cancer-specific in vitro (Figure 3.4). In addition, the EJ1
peptide shows no observable toxicity in vivo as determined by gross appearance, activity
and body weights of treated mice.
Figure 5.1. Schematic model of EJ1-induced cell death.
Left side of the figure represents the condition before EJ1 peptide treatment.
Activation of EGFR induces juxtamembrane domain mediated translocation of the
receptor to the nucleus and mitochondria. Right side of the figure represents possible
mechanisms by which EJ1 peptide induces cell death. EJ1 inhibits activation of EGFR
through promotion of inactive dimer formation. EJ1 peptide affects membrane integrity
and leads to calcium influx, activation of MLC-regulated membrane blebbing, disruption
of mitochondrial membrane potential, and ROS accumulation which eventually lead to
apoptotic and necrotic
cell death.
143
To improve its efficacy and stability, modification of the peptides and screening for
chemical mimetics are possible options. Combination therapy also warrants further
investigation. The EGFR-independent function of EJ1 could be attributed as a calcium
and ROS-dependent function. Even though the definitive mechanism to explain how EJ1
works through calcium and ROS is still lacking, targeting the calcium signaling pathway
and ROS as cancer therapy is not unprecedented [205-207]. Simultaneously targeting
multiple pathways could provide a better therapeutic efficacy and minimize the likelihood
of developing resistance. Therefore, the EJ1 peptide might provide hints for effective
combination therapy, such as EGFR inhibitors in combination with ROS inducers.
144
Figure 5.2. EJ1 peptide affects cell viability of AG1478-resistant cells.
AG1478-resistant cells were selected and grown as described earlier. These
AG1478-resistant cells were treated with 20μM control peptide (CP) or 20μM EJ1
peptide (EJ1) in the presence of 10μM AG1478 for 3 days. A MTT assay was done to
quantify cell viability at day 3. The reading for CP/AG1478-treated cells at day 3 was set
as 100%. ***, p < 0.001, Student’s t-test. Error bars, SD.
III. MUC1 sustains NRG1β-dependent ErbB3 activities
Crosstalk between different members of erbB receptors through heterodimerization
results in a wide array of downstream signaling events. Therefore, it is almost impossible
to completely downregulate signaling pathways by solely inhibiting one erbB receptor
member. ErbB3 is a kinase-defective receptor whose activation highly depends on its
145
dimer partner. ErbB3 has been shown to contribute to resistance to EGFR-targeted
therapy through membrane expression of ErbB3 and shifting phosphorylationdephosphorylation equilibrium [53]. MUC1, on the other hand, has been shown to
interact with four erbB receptors and was specifically demonstrated to promote EGFR
recycling [34]. In the present study, we investigated whether MUC1 expression also
affected ErbB3 activity and stability. We show that the MUC1 expression sustains
NRG1β-induced ErbB3 activation and downstream PI3K/AKT as well as MAPK
activities.
We
also
demonstrate
that
MUC1
promotes
NRG1β-dependent
EGFR/ErbB3/MUC1 interaction in BT20 breast cancer cells. This result might provide a
hint for the development of ErbB3-targeted therapy. Interruption of MUC1/ErbB3
interaction could be a potential therapeutic strategy to inhibit ErbB3 activity.
IV. Conclusions
The work presented in this dissertation provides evidence supporting several novel
strategies for targeting EGFR activity and localization as effective therapies. Although
more data need to be provided to consolidate our conclusion, this “proof-of-principle”
approach has offered a new avenue to further develop cancer-specific inhibitors targeting
146
protein-protein interactions to act against tumor-addicted proteins. Our work shows that
not only can the activity of receptors be targeted, but also localization of active receptors
can be manipulated. These results offer unique perspectives on the development of
potential novel therapeutics targeting the EGFR. By using this structure-function peptide
approach, we can determine whether a certain protein-protein interaction is targetable.
Once verified, modification of peptides and screening for chemical molecules with a
similar structure can be done to improve peptide stability and efficacy. Because
therapeutic resistance is an inevitable result for every single drug, the combination of
drugs working through different mechanisms might be the improved way to impede
emergence of resistance. Our work also provides insights for effective combination
therapy in the future.
147
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