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Over-expression of EphB4 by cancer cells provides survival advantage by interrupting death receptor-mediated apoptotic pathways
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Over-expression of EphB4 by cancer cells provides survival advantage by interrupting death receptor-mediated apoptotic pathways
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Content
OVER-EXPRESSION OF EPHB4 BY CANCER CELLS PROVIDES
SURVIVAL ADVANTAGE BY INTERRUPTING DEATH RECEPTOR-
MEDIATED APOPTOTIC PATHWAYS
by
Ram Kumar Subramanyan
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
December 2006
Copyright 2006 Ram Kumar Subramanyan
ii
Epigraph
In search of that Ultimate Truth that causes and governs us all…..
iii
Dedication
To my wonderful parents, Jaya and Subramanyan,
For this blessed life
For all the love and intellect
For those joys and opportunities, but most of all
For just being there and being you…..
To my lovely wife, Charanya,
For bringing to my blessed life
A fresh breath, a new meaning…….
iv
Acknowledgements
This research dissertation was supported in part by the Tobacco-related
disease research program, CA, through a generous fellowship 14DT-
0125 to study ‘EphB4-mediated inhibition of cell death’.
All of the work presented was done under the auspices of
Parkash S. Gill, MD, in his laboratory, supported by his grants and
donors. Over the course of this work, Parkash has become more than my
committee chair. Everything from laboratory supplies to research
ideology, from financial support to moral backing, from harsh research
criticisms to fun roadside lunches, this work is his. Fred A. Weaver, MD,
Professor of Surgery and Chief, Division of Vascular Surgery, created and
funded this research spot for me to pursue during my surgical residency.
This work was driven by the science that governs. That said, it was made
enjoyable by all the people that were part of this effort. From senior
colleagues that patiently taught me out of how little I knew, to peers and
junior colleagues who helped with innumerable parts of the grinding
research experiments, the journey down EphB4’s trail has been filled with
wonderful moments. My heartfelt gratitude is due to everyone whose path
crossed this trail.
v
Table of contents
Epigraph ii
Dedication iii
Acknowledgements iv
List of Figures vii
Abbreviations ix
Abstract xi
Chapter I: Introduction 1
Eph/Ephrin structure and function 2
Eph/Ephrins in tumors 4
Chapter II: Aims of proposal 8
Major aims 8
Specific aims 9
Chapter III: EphB4 is overexpressed in several epithelial
cancers
17
Table 3.1 EphB4 is expressed in tumor tissues of several
different epithelial cancers
18
EphB4 is overexpressed in colorectal carcinoma 18
EphB4 is overexpressed in prostate adenocarcinoma 21
EphB4 is overexpressed in squamous cell carcinoma of the
head and neck (HNSCC)
22
EphB4 is overexpressed in transitional cell carcinoma of the
bladder
25
EphB4 is expressed in carcinoma of the breast 26
EphB4 is expressed in pancreatic adenocarcinoma 27
EphB4 is overexpressed in ovarian and hepatocellular
carcinoma
28
Biologically active EphB4 is expressed on several different
cancer cell lines
29
Chapter IV: Expression of EphB4 is under control of different
oncogenes and tumor suppressor genes
32
EphB4 is upregulated by oncogenes 32
EphB4 is downregulated by tumor suppressor genes 36
vi
Chapter V: EphB4 provides survival signals to tumor cells in
vitro and in vivo
41
EphB4 knockdown inhibits cell viability in vitro 41
EphB4 knockdown inhibits cell viability in vivo 45
Chapter VI: EphB4 inhibits apoptotic pathways in tumor cells 48
EphB4 protects tumor cells from apoptosis in vitro and in
vivo
48
EphB4 predominantly interrupts the extrinsic apoptotic
pathway
51
Chapter VII: Extra-cellular domain of EphB4 inhibits TRAIL-
mediated cell death in tumor cells in a signaling independent
fashion
56
EphB4 knockdown sensitizes tumor cells to TRAIL-induced
cell death
56
Forced expression of EphB4 renders tumor cells resistant
to TRAIL
60
EphB4-eGFP mutant protein 62
The extracellular domain of EphB4 is sufficient to provide
resistance to TRAIL
66
The extracellular domain of EphB4 provides resistance to
TRAIL in vivo
68
Chapter VIII: Conclusions and future directions 70
Bibliography 72
vii
List of figures
Figure 1.1 EphB4 and EphrinB2 structure 2
Figure 1.2 Apoptotic pathways in cells 6
Figure 3.1 EphB4 is overexpressed in colon cancer 20
Figure 3.2 Expression of EphB4 in prostate tumor specimens 21
Figure 3.3 EphB4 is overexpressed in HNSCC primary tissues and
metastases
23
Figure 3.4 EphB4 is overexpressed in transitional cell cancer of
the bladder
25
Figure 3.5 EphB4 is expressed in breast cancer 27
Figure 3.6 EphB4 is expressed in pancreatic adenocarcinoma 28
Figure 3.7 Functional EphB4 is expressed on the membrane of
colon cancer cells
30
Figure 4.1 EGFR signaling upregulates EphB4 expression 32
Figure 4.2 EGF upregulates EphB4 by signaling via JAK-STAT
and PI3K/Akt
34
Figure 4.3 HER2/neu signaling upregulates EphB4 expression 35
Figure 4.4 Akt signaling upregulates EphB4 expression 36
Figure 4.5 PTEN downregulates EphB4 expression 37
Figure 4.6 p53 downregulates EphB4 expression 38
Figure 4.7 APC downregulates EphB4 expression 39
Figure 4.8 Regulation of EphB4 expression in cancer cells 40
Figure 5.1 EphB4 provides survival signals to cancer cells in vitro 43
Figure 5.2 AS-mediated knockdown of EphB4 inhibits cancer cell
survival
44
viii
Figure 5.3 EphB4 provides survival signals to cancer cells in vivo 46
Figure 6.1 EphB4 protects cancer cells from apoptosis in vitro 49
Figure 6.2 EphB4 protects cancer cells from apoptosis in vivo 51
Figure 6.3 EphB4 interferes with apoptotic pathways in vitro 52
Figure 6.4 EphB4 interferes with apoptotic pathways in vivo 54
Figure 7.1 EphB4 inhibits death receptor pathway 58
Figure 7.2 EphB4 inhibits extrinsic apoptotic pathway 60
Figure 7.3 Forced expression of EphB4 renders TRAIL resistance
to tumor cells
61
Figure 7.4 EphB4-eGFP mutant protein characterization 63
Figure 7.5 EphB4-extracellular domain is sufficient to provide
resistance against TRAIL-induced apoptosis
67
Figure 7.6 Extracellular domain of EphB4 provides resistance to
TRAIL in vivo
69
ix
Abbreviations
AP – Alkaline Phosphatase
APC – Adenomatous Polyposis Coli
AS ODN – AntiSense OligoDeoxyNucleotides
Bcr-Abl – Breakpoint Cluster Region – Abelson gene
CXCR4- Chemokine Receptor 4, fusin
DAPI - 4',6-DiAmidino-2-PhenylIndole
DCIS – Ductal Carcinoma In-Situ
DR4/DR5 – Death Receptor 4/5
EGF/EGFR – Epidermal Growth Factor and its cognate Receptor
FACS – Fluorescence Associated Cell Sorting
FITC – Fluorescein IsoThioCyanate
FLIP – Flice Inhibitory Protein
GAPDH - Glyceraldehyde-3-Phosphate Dehydrogenase
GFP – Green Fluorescence Protein
GPCR – G-Protein Coupled Receptor
GPI – Glycosylphosphatidylinositol
H/E – Hematoxylin/Eosin staining
HUVEC – Human Umbilical Venous Endothelial Cells
IC50 – Inhibitory Concentration 50
IGF-1 – Insulin-like Growth Factor -1
x
IHC - ImmunoHistoChemistry
IP – ImmunoPrecipitation
JAK – Janus kinase
MAPK – Mitogen-Activated Protein Kinase
MTT - 3-(4,5-diMethylThiazol-2-yl)-2,5-diphenylTetrazolium bromide
NMDA - N-Methyl-D-Aspartate
ODN – OligoDeoxyNucleotides
PDGF – Platelet-Derived Growth Factor
PI – Propidium Iodide
PI3K – Phosphatidyl-Inositol 3 Kinase
PR – Progesterone Receptor
PTEN - Phosphatase and Tensin homolog
SAM – Sterile Alpha-Motif
siRNA – Small Interfering RiboNucleic Acid
STAT – Signal Transducers and Activators of Transcription
TNF-alpha – Tumor Necrosis Factor-alpha
TRAIL – Tumor necrosis factor Related Apoptosis Inducing Ligand
TUNEL – Terminal deoxynucleotidyl transferase-mediated dUTP Nick End
Labeling
WB – Western Blotting
xi
Abstract
An increasing body of evidence suggests overexpression of Eph receptor
tyrosine kinases in tumors. However, their function in tumor biology is
unknown. This thesis proposal evaluates the expression and biological
significance of EphB4 in eight different epithelial cancers. Consistently,
membranous expression of EphB4 was observed at much higher levels in
tumor tissue compared to adjacent normal tissue, with a trend towards
increased expression in higher grade, stage and proliferative front of tumors.
EphB4 expression was upregulated by oncogenes such as EGFR, HER-
2/neu and Akt, and downregulated by tumor suppressors such as PTEN, p53
and APC. EphB4 provides direct survival signals to tumor cells both in vitro
and in vivo by interrupting extrinsic apoptotic pathways. Expression of the
extracellular domain of EphB4 is sufficient for tumor cells to overcome
apoptotic signals from the TRAIL-death receptor pathway. These results
confirm that targeted overexpression of EphB4 on epithelial tumor cells
provides a survival advantage to tumors by interrupting death-receptor-
mediated apoptosis.
1
Chapter I: Introduction
The Eph family of receptors is comprised of fourteen different
structurally related receptors and constitutes the largest family of
receptor tyrosine kinases. Eph receptors bind membrane-associated
ligands called Ephrins and are subdivided into EphA and EphB
receptors based on their preferential binding to EphrinAs, proteins
linked to the cell membrane via GPI-linkages, or EphrinBs, also
transmembrane proteins. EphB4 and its cognate ligand EphrinB2 were
initially studied for their role in the central nervous system in regulating
spatial migration, axonal pathfinding, and segmentation during
embryonic development (Tickle et al, 1999). Recently, evidence from
knockout animals has shown a critical role for EphB4-EphrinB2
mediated signaling in vessel maturation, in that, knocking out either
protein results in a mirror image capillary arrest vascular phenotype that
causes early embryonic lethality (Gerety et al, 1999). In addition, EphB4
is specifically expressed on endothelial cells targeted to the venous
lineage, whereas EphrinB2 labels arterial endothelial cells.
1.1 Eph/Ephrin structure and function
Eph receptors have an extracellular domain composed of the ligand-
binding globular domain, a cysteine rich region followed by a pair of
fibronectin type III repeats. The cytoplasmic domain consists of a
juxtamembrane region containing two conserved tyrosine residues; a
protein tyrosine kinase domain; a sterile α-motif (SAM) and a PDZ-
domain binding motif (Figure 1.1). EphB4 is specific for the membrane-
bound ligand EphrinB2 (Sakano et al, 1996). EphrinB2 belongs to the
SAM domain
Globular domain
Receptor-binding
domain
Vein
Artery
PDZ binding motif
Cysteine-rich domain
Fibronectin III repeat
Juxta-membrane region
Kinase domain
PDZ binding motif
EphrinB2 EphB4
SAM domain
Globular domain
Receptor-binding
domain
Vein
Artery
PDZ binding motif
Cysteine-rich domain
Fibronectin III repeat
Juxta-membrane region
Kinase domain
PDZ binding motif
EphrinB2 EphB4
SAM domain
Globular domain
Receptor-binding
domain
Vein
Artery
PDZ binding motif
Cysteine-rich domain
Fibronectin III repeat
Juxta-membrane region
Kinase domain
PDZ binding motif
EphrinB2 EphB4
Figure 1.1 EphB4 and EphrinB2 structure. - Tyrosine motif
2
3
class of Eph ligands that have a transmembrane domain and
cytoplasmic region with five conserved tyrosine residues and a PDZ
domain (Torres et al, 1998). In contrast, the A class ligands have a
glycosylphosphatidylinositol membrane anchor. Eph receptors are
activated by binding of clustered, membrane attached Ephrins (Davis et
al, 1996) indicating that contact between cells expressing the receptors
and cells expressing the ligands is required for Eph activation. A
corollary of this is that soluble monomeric ligands would act as
inhibitors of Eph clustering and hence activation (Kertesz et al, 2006).
Upon ligand binding, Eph receptor dimers autophosphorylate the
juxtamembrane tyrosine residues to acquire full activation (Himanen et
al, 2003). Forward signaling via EphB4 regulates cell adhesion and
migration (Fuller et al, 2003). In addition to forward signaling through
the Eph receptor, reverse signaling can occur through the EphrinBs.
Eph engagement of Ephrins results in rapid phosphorylation of the
conserved intracellular tyrosines (Holland et al, 1996) and somewhat
slower recruitment of PDZ binding proteins (Lu et al, 2001). Reverse
signaling regulates lateral cell migration, capillary network formation
and sprouting angiogenesis (Hamada et al, 2003).
Evidence suggests that the Eph/Ephrin interaction influences and is
influenced in turn by other signaling pathways. The endothelial-specific
receptor Tie-2 can directly phosphorylate Ephrin cytoplasmic domains
4
(Adams et al, 1999), while EphrinB1 is phosphorylated by the PDGF
receptor, and inhibits PDGF induced focus formation (Bruckner et al,
1997). The Ephrins have also been shown to couple to GPCRs, such
as the chemokine receptor CXCR4, via the PDZ linking proteins (Lu et
al, 2001) and a ternary complex involving the extracellular domains of
EphrinB1, EphB2 and the 7-transmembrane GPCR subunit of the
NMDA glutamate receptor has been demonstrated (Dalva et al, 2000).
1.2 Eph/Ephrins in tumors
The Eph receptors have generated considerable enthusiasm for their
role in tumor biology. EphA2 induces malignant transformation (Zelinski
et al, 2001) and is being evaluated as a potential target for therapy in
lung and ovarian cancer. Several recent reports have suggested an
important role for EphB4 expression in the progression of epithelial
cancers. EphB4 is specifically expressed in tumor cells at higher levels
than adjacent normal tissue (Berclaz et al, 2003, Lee et al, 2005, Wu et
al, 2004). The level of expression of EphB4 correlates with tumor stage,
grade and aggressiveness (Wu et al, 2004). EphB4 transcripts are
more abundant in the invasive front of the tumor (Berclaz et al, 2003,
Wu et al, 2004). Although organ-targeted overexpression of EphB4
does not initiate transformation, double transgenic mice expressing
EphB4 and neuT show early development of aggressive mammary
5
tumors and increased metastasis (Munarini et al, 2002). EphB4 on
tumor cells can engage EphrinB2 on tumor endothelial cells
augmenting tumor angiogenesis (Noren et al, 2004).
Preliminary data from our lab indicates that EphB4 is expressed in
several clinical cancer samples, and expression is restricted to tumor
cells. Importantly, expression of EphB4 is necessary for tumor cell
viability, migration and invasion. Blocking EphB4 expression results in
cell cycle arrest followed by caspase-mediated apoptosis in vitro.
Understanding the molecular mechanism that underlies EphB4-
mediated survival advantage is of significant scientific merit. One of the
mechanisms by which growth factor receptors influence cell survival is
interaction with apoptotic pathways. The hepatocyte growth factor
receptor tyrosine kinase, met, inhibits apoptosis by directly binding to
and sequestering Fas, thereby preventing Fas activation by its ligand
(Wang et al, 2002). The Bcr-Abl kinase augments Stat5 DNA-binding
activity, thereby transcriptionally increases Bcl-x
L
expression, and
prevents apoptosis in leukemia cells (Huang et al, 2002). Along similar
lines, our preliminary data shows that EphB4 affords survival advantage
to cancer cells by preventing the induction of apoptosis.
Apoptosis can be induced in cells by two distinct mechanisms – the
extrinsic pathway that originates in the cell membrane is initiated in
response to signaling from death-inducing receptors following their
activation by specific ligands such as the FAS-ligand, TNF- α, TNF
alpha-related apoptosis inducing ligand (TRAIL), etc. This results in
activation of the enzyme casapse-8 that leads to further activation of a
number of downstream caspases, inducing cell death (Figure 1.2). The
intrinsic pathway originates in the mitochondria in response to cell-
intrinsic signals. Activation of these signals is usually secondary to
radiation or complement-mediated injury, DNA damage, etc. and results
Figure 1.2 TRAIL and death receptor in apoptosis. TRAIL signaling pathway
and the induction of ‘extrinsic’ or membrane-originating apoptosis
6
7
in activation of caspase-9. This leads to activation of downstream
caspases and, ultimately, cell death. EphB4 knockdown selectively
activates caspase-8, but not caspase-9, thereby indicating its influence
on the membrane-originating, extrinsic caspase pathway. This could
result from two non-mutually exclusive effects – (a) inhibition of death
receptor signaling by interrupting ligand-receptor interaction or (b)
downstream signaling through death effector domain interactions. Our
preliminary data indicates that EphB4 knockdown sensitizes cells to
TRAIL-mediated cell death. TRAIL is a naturally occurring apoptosis-
inducing ligand that acts via the death receptor (DR4 and DR5)
pathways. The focus of this proposal is to study the interaction between
EphB4 and cell death induced by the death receptor pathways.
8
CHAPTER II: Aims of the proposal
Frequently, receptor tyrosine kinases that provide survival advantage to
cancer cells are overexpressed in malignancy. Targeting these
receptors for focused biological therapy is a distinct feasibility –
trastuzumab, the HER2/neu receptor targeting antibody, to quote an
example (Slamon et al, 1999). Validation of tumor-specific
overexpression of EphB4 and documentation of a survival advantage
consequent to this overexpression will thus help identify a novel target
for potential therapy in cancer. Along these lines, the central
hypothesis of this proposal is that –
Targeted overexpression of EphB4 on epithelial tumor cells provides a
survival advantage to tumors by interrupting death-receptor-mediated
apoptosis.
We propose to validate this hypothesis by addressing the following
aims:
2.1 Major Aims
Aim 1: To characterize the expression of EphB4 in different tumors
Aim 2: To examine the biological role of EphB4 in tumor cell survival
9
Aim 3: To examine the functional interaction between EphB4 and the
death receptor pathway
2.2 Specific Aims
2.2.1 Aim 1: To characterize the expression of EphB4 in different
tumors
Rationale: EphB4 is specifically expressed by tumor cells at higher
levels relative to normal cells. Overexpression is driven by signaling via
oncogenes and suppressed by tumor suppressor genes.
Specific Aim 1a: To study the expression of EphB4 protein in human
tumor tissue compared to adjacent normal tissue (quantify by
immunoblotting and confirm tissue distribution by
immunohistochemistry)
Experiment 1: EphB4 expression on several types of tumor
specimens will be studied. Tumor specimens and adjacent
normal tissue harvested at the time of surgery will be snap
frozen in liquid nitrogen and sectioned. Subsequently, tissue
arrays containing an array of matched normal, tumor and
metastatic tissue (if any) will be constructed for comparative
staining. Immunohistochemical (IHC) staining using monoclonal
antibodies will be used to evaluate intensity of staining of tumor
10
tissue relative to normal tissue and define tissue-specific
expression of EphB4.
Experiment 2: Western Blotting of proteins extracted from tumor
tissue and matched control tissue will be used to quantitate
EphB4 expression.
Experiment 3: In order to establish in vitro models to study the
biological significance of EphB4 expression, we will evaluate
EphB4 expression in tumor cell lines as above.
Specific Aim 1b: Study the regulation of EphB4 expression in tumor cell
lines by select oncogenes and tumor suppressor proteins (by
knockdown, overexpression, or kinase inhibition)
Experiment 1: I propose to study the regulation of EphB4 by the
following candidate oncogenes –
EGFR – By complimentary stimulation and inhibition experiments
– EGFR will be stimulated in EGFR expressing cells using EGF
and expression of EphB4 evaluated. EGFR signaling will be
inhibited using specific kinase inhibitors and EphB4 levels
studied. To further elucidate the second messengers through
which EGFR signals, kinase inhibitors to specific second
11
messengers (PI3K, Akt, MAPK, etc.) will be used to study their
effect on EphB4 levels.
HER-2/neu - By complimentary stimulation and inhibition
experiments – We will study the effect of forced expression of
HER-2/neu on EphB4 levels in fibroblasts. Complimentarily,
HER-2/neu downregulation by degrading antibodies will be used
to study the effect of inhibition of HER-2/neu on EphB4 levels.
Akt – The effect of Akt on EphB4 expression will be evaluated by
inhibiting Akt signaling using specific-kinase inhibitor in cells that
express active Akt. In cells lacking EphB4 expression, forced
expression of wild-type or kinase-dead Akt will be used to study
its effect on EphB4 expression.
Experiment 2: I then propose to study the regulation of EphB4 by
the following candidate tumor suppressor genes –
p53 – Targeted knockdown of p53 in cell lines that harbor the
wild-type protein will be used to study the regulation of EphB4 by
p53. Re-introduction of wild-type p53 in these cells or forced
expression in cells with mutant p53 will be used as
complimentary strategy.
12
APC – To study the effect of APC on EphB4 expression, forced
expression of wild-type APC in cells that harbor APC mutations
will be used. We will evaluate EphB4 expression in intestinal
mucosa of APC
min
mice and compare it with levels in intestinal
mucosa of matched wild-type mice.
PTEN – Similarly, forced expression of PTEN in cells that have
mutated PTEN will be used to study regulation of EphB4 by
PTEN.
2.2.2 Aim 2: To examine the biological role of EphB4 in tumor cell
survival
Rationale: EphB4 overexpression in tumor cells provides a survival
advantage by interrupting apoptotic pathways.
Specific Aim 2a: Study the effect of EphB4 knockdown on tumor cell
survival in vitro (MTT assay for dose dependence) and tumor growth
and proliferation in vivo
The general approach will be to ablate EphB4 expression and
study its consequence on tumor cell survival. Both siRNAs and
AS-Oligodeoxynucleotides (ODNs) that specifically ablate EphB4
expression have been identified and tested. EphB4-negative cell
lines will serve as controls to determine whether the AS-ODN or
13
siRNA cause pleiotropic changes by non-specific mechanisms.
We will also use mutated siRNA and scrambled ODNs as
controls. In this aim, we will expand our scope to investigate
several EphB4-positive tumor cell lines to determine whether
they exhibit a requirement of EphB4 expression for their survival.
We will use murine tumor xenografts to study the effect of
EphB4-specific antisense ODNs on tumor growth. As above,
scrambled ODN and EphB4-negative cell lines will be used as
negative control.
Specific Aim 2b: Study the effect of EphB4 knockdown on tumor cell
apoptosis and apoptosis pathways (intrinsic and extrinsic pathways)
Experiment 1: To study the mechanism by which EphB4
provides survival cues to cancer cells, we will study apoptosis in
cells following knockdown of EphB4. We will study the following
features of cells –
o Cell cycle changes – looking for accumulation of cells in
sub-G0 population
o Accumulation of cytoplasmic nucleosomes
o Annexin positivity in absence of PI staining
o TUNEL staining of tissue sections.
14
Experiment 2: To study the apoptotic pathways influenced by
EphB4, we will study
o Colorimetric assay for activation of caspase-8 or caspase-
9 to ascertain which of the apoptotic pathways (extrinsic
vs. intrinsic) is selectively induced upon EphB4
knockdown.
o Study the changes in levels of pro-apoptotic (Caspase-8,
Bid) and anti-apoptotic proteins (Bcl, Bcl-X
L
, Mcl-1, FLIP)
upon EphB4 knockdown by Western blotting.
2.2.3 Aim 3: To examine the functional interaction between EphB4 and
the death receptor pathway
Rationale: EphB4 interrupts TRAIL-induced apoptosis by inhibiting
TRAIL-mediated signaling via death receptor pathway.
Specific Aim 3a: Study the effect of EphB4 knockdown on tumor cell
sensitivity to TRAIL-induced apoptosis
The general approach to studying the effect of EphB4
knockdown on TRAIL-induced apoptosis will be to knockdown
EphB4 expression to various levels in cells and evaluate if loss
of EphB4 expression alters tumor cell sensitivity to TRAIL-
15
induced apoptosis. The approach will be to look for a synergistic
effect of EphB4 knockdown and TRAIL on tumor cell death.
Specific Aim 3b: Study the effect of EphB4 overexpression in tumor
cells sensitive to TRAIL
Complimentarily, we will select a cell line that does not express
EphB4 (or expresses at low levels) and is sensitive to TRAIL-
induced death. EphB4 will be overexpressed in these cells and
transfected cells sorted to enrich the resultant cell population
with cells of interest. EphB4 expression by these cells will be
confirmed by Western blotting and FACS analysis to ensure
membrane expression.
TRAIL-sensitivity of EphB4 transfected and mock transfected
cells will be compared to parent cells to ascertain if
overexpression of EphB4 renders TRAIL-resistance to tumor
cells.
Specific Aim 3c: Study the domain-specific interaction between EphB4
and the TRAIL pathway (using EphB4 truncated and mutant variants)
Both EphB4 and TRAIL are membrane proteins. The major
question that begs to be answered is if interaction occurs at the
cell membrane or via second messenger molecules inside the
16
cytoplasm. Furthermore, is signaling via EphB4 a necessary
event for inhibition of apoptosis? The general approach to
ascertain whether the cytoplasmic tail and signaling is required
to inhibit apoptosis will be to generate a mutant EphB4 in which
the cytoplasmic domain is replaced with eGFP. The mutant
protein is targeted to the cell membrane, but is signaling-
incompetent (Noren et al, 2004). Cells carrying this mutant
protein will then be analyzed for their sensitivity to TRAIL-
induced death.
17
CHAPTER III: EphB4 is overexpressed in several
epithelial cancers
Growing evidence points to the expression of several Eph receptors in
different cancer types. Several recent reports have suggested an
important role for EphB4 expression, in particular, in the progression of
epithelial cancers. EphB4 is specifically expressed in tumor cells at
higher levels than adjacent normal tissue (Berclaz et al, 2003, Lee et al,
2005, Wu et al, 2004). The level of expression of EphB4 correlates with
tumor stage, grade and aggressiveness (Wu et al , 2004). EphB4
transcripts are more abundant in the invasive front of the tumor (Berclaz
et al, 2003, Wu et al, 2004). Although organ-targeted overexpression of
EphB4 does not initiate transformation, double transgenic mice
expressing EphB4 and neuT show early development of aggressive
mammary tumors and increased metastasis (Munarini et al, 2002). We
therefore sought to characterize the expression of EphB4 in several
epithelial tumor types and compare levels to those found in matched
adjacent normal mucosa. We used both Western blotting and
immunohistochemistry to study protein expression – while Western
blotting allows for more quantitative analyses, tissue staining allows
study of localization of expression in various compartments of tumor.
Table 3.1 represents a list of tumors types in which we have evaluated
EphB4 expression.
Table 3.1 EphB4 is expressed in tumor tissues of several different
epithelial cancers
Cancer type
No. of tumors
analyzed
No. expressing
EphB4 (%)
Colon adenocarcinoma 110 110 (100)
Prostate adenocarcinoma 62 41 (66)
Squamous cell carcinoma
(Head and Neck)
37 37 (100)
Bladder adenocarcinoma 15 14 (93)
Breast adenocarcinoma 12 7 (58)
Ovarian adenocarcinoma 10 7 (70)
Hepatocellular carcinoma 10 10 (100)
Pancreatic adenocarcinoma 4 4 (100)
3.1 EphB4 is overexpressed in colorectal carcinoma
We analyzed the expression of EphB4, EphB2, and EphrinB2 in 110
fresh frozen samples of colon tumors and metastatic deposits and
adjacent normal mucosa by immunofluorescence (IHC, Figure 3.1A)
18
19
and Western blotting (WB, Figure 3.1B) (manuscript under review). In
general, there was excellent correlation between WB and IHC. All tumor
samples expressed EphB4 on both WB and IHC. On IHC, tumors
expressed high levels of EphB4, whereas minimal to no expression was
observed in adjacent normal colonic mucosa. On WB, EphB4
expression was increased a median 2.4-fold in tumor samples
compared to adjacent normal mucosa, with a greater than 4-fold
increase in expression in 49 (45%) tumors. EphB2 on the other hand
was expressed in 74 (61%) tumors, whereas consistent and high levels
of expression were observed in normal colonic mucosa. EphB2
expression was significantly down-regulated in 48 (44%) tumors and
increased in 28 (25%) tumors. Frequently, EphB2 was downregulated
compared to normal mucosa in tumors in which EphB4 was
correspondingly upregulated (Figure 3.1A). EphrinB2 was expressed in
79 tumors (72%).
In addition, EphB4 expression was seen in vasculature in normal
colonic tissue and in tumor tissue, as confirmed by CD31/EphB4 co-
staining (Figure 3.1C). Even when there was significant increase in
tumor cell expression of EphB4 compared to normal colonic mucosa,
there appeared to be no difference in EphB4 staining intensity in normal
versus tumor vasculature. Also, we noted a consistent trend towards
increased expression of EphB4 in areas of tumor that were of higher
grade (Figure 3.1D)
Normal colon Colon cancer
EphB4 EphB2
Normal colon Colon cancer
EphB4 EphB2
A B
20
C
Tumor – EphB4/Nuclei
D
Figure 3.1 EphB4 is overexpressed in colon cancer. Fresh frozen samples of
colon cancer and adjacent normal colonic mucosa were sectioned and stained to
detect expression of EphB4 and EphB2 (A). Protein lysates from fresh frozen
colon cancer specimens and adjacent normal mucosa were serially probed for
expression of EphB4, EphB2, EphrinB2 and β-actin (B). N-normal colonic
mucosa, T-tumor. Normal colonic mucosal sections were triple stained for EphB4
(monoclonal antibody, green), CD31 (polyclonal antibody, red) and DAPI (blue) to
label vessels (C). For comparison, EphB4 staining in matched tumor section with
blood vessel is shown in bottom right panel. Some tumor sections comprised of
different grades within a single section (D). EphB4 expression appeared to be
higher in areas of high-grade tumor.
3.2 EphB4 is overexpressed in prostate adenocarcinoma
To determine whether EphB4 is expressed in clinical prostate samples,
tumor tissues and adjacent normal tissues from prostate cancer
surgical specimens were examined (Xia et al, 2005). The histologic
distribution of EphB4 in the prostate specimens was determined by
immunohistochemistry. EphB4 protein expression is confined to the
neoplastic epithelium.
A B
Figure 3.2 Expression of EphB4 in prostate tumor specimens. Immunohistochemical
staining on clinical prostate cancer samples (A): a, b, and e – prostate cancer
showing EphB4 in tumor cells; c, isotype control IgG was used in place of primary
antibody to probe a serial section of (a); d, lack of EphB4 staining in benign prostate
hyperplasia; f, high-power view of (e) showing membrane staining of EphB4. Gene
expression for EphB1, EphB2, and EphB4 was studied by DNA microarray in 72
prostate cancer tissues (B). EphB4 was expressed in 64 (89%) cases.
Both membrane and cytoplasmic staining were seen (Fig. 3.2Aa, b, e,
and f), whereas, expression was absent in normal glands (Fig. 3.2Ad).
In a prostate tissue microarray generated from archival tissue, 41 of 62
21
22
(66%) prostate cancers examined were positive, whereas 3 of 20 (15%)
normal prostate tissues showed expression at low intensity (p < 0.01).
We also examined gene expression in 72 prostate cancer patients
using DNA microarrays. EphB4 was expressed in the majority of the
cases (64 of 72 or 89% with normalized expression above 50 units) with
signal intensity higher than that seen for EphB1 and EphB2 (Fig. 3.2B).
3.3 EphB4 is overexpressed in squamous cell carcinoma of the
head and neck (HNSCC)
Thirty-seven prospectively collected tumor tissues, comprising of 37
primary tumor samples, 37 matched normal adjacent tissue and 14
tumor-bearing lymph nodes, were evaluated (Masood et al, 2006).
Immunofluorescence was performed on fresh frozen tissue sections
using EphB4-specific monoclonal antibodies directed against the extra-
cellular domain of EphB4. All tumor sections expressed EphB4 in tumor
cells only and a higher intensity of staining was observed in advanced
stage disease. Figure 3.3A shows representative serial sections stained
for EphB4 and with H/E. In the left and middle panels, EphB4 staining is
discernable only in tumor tissue but not in adjacent stroma, as
confirmed by H/E staining. The right panel shows lack of EphB4
staining in normal mucosa. Figure 3.3B confirms membrane expression
of EphB4 in tumor cells. As expected, vasculature, presumably of
23
B
A
D
C
Figure 3.3 EphB4 is overexpressed in HNSCC primary tissues and metastases. A) Top
panel: Immunofluorescence of representative fresh frozen sections of tumors (left and
middle panels) or adjacent normal tissue (right panel) stained with EphB4-specific
monoclonal antibody and visualized with FITC (green color). Sections were counter-
stained with DAPI (blue) to identify cell nuclei. Bottom panel: Hematoxylin and Eosin
(H&E) staining of the next serial section. Arrowhead in middle panel shows a vessel
staining positive for EphB4. B) Representative high power photomicrographs of tumor
sections stained for EphB4 to document tumor cell membrane-specific expression. C)
Western blotting of representative matched tumor lysates consisting of tumor-bearing
lymph node (LN), primary tumor (T), and uninvolved adjacent normal tissue (N). D)
Quantification of EphB4 protein and DNA amplification in stage-stratified matched normal,
tumor or involved lymph nodes corrected for levels of β-actin and GAPDH is shown.
24
venous lineage, also expressed EphB4 (arrowhead in middle panel of
Figure 3.3A). EphB4 expression was also most prominent on the
leading edges of the tumor with reduced signal in central areas (Figure
3.3B), which typically represent keratin pearls and metabolically inactive
cells. We confirmed the specificity of the signal by documenting loss of
signal when the antibody is first incubated with competing peptide and
by staining with commercially available antibodies directed at the C-
terminal of EphB4 (data not shown). Western blots of tissue from
primary tumor, lymph node metastases and uninvolved tissue were
probed for EphB4 expression. Representative cases are shown in
Figure 3.3C. Expression levels were quantified by comparison to signal
from normal adjacent tissue analyzed using equal amounts of proteins
and corrected for beta-actin levels. EphB4 expression was observed in
each of the tumor samples with higher expression in advanced stage
disease (Figure 3.3C and D). Stage III and IV tumors had a 2.8 and 5.5-
fold elevation in protein expression compared to normal adjacent tissue,
respectively. Furthermore, EphB4 protein levels were 7.8-fold higher
than normal tissues in nodal tumor, and all tumor-positive lymph nodes
showed EphB4 expression equal to or greater than the primary tumor.
3.4 EphB4 is overexpressed in transitional cell carcinoma of the
bladder
We prospectively collected fresh tumor and adjacent normal tissue from
patients undergoing radical cystectomy for transitional cell carcinoma of
the bladder. Tissue analysis included immunohistochemical staining of
frozen sections, and quantitative estimation of protein levels (Xia et al,
2006).
B
Figure 3.4 EphB4 is overexpressed
in transitional cell cancer of the
bladder. Frozen sections of freshly
harvested bladder tumor and
normal urothelium from patients
undergoing resection of bladder
cancer were analyzed by
immunohistochemistry to localize
EphB4 expression (A). Whereas
normal urothelium expresses little
EphB4 and high levels of EphB2,
adjacent tumor expresses high
levels of EphB4 and no EphB2.
Membrane localization of EphB4
can be clearly seen. Tissues were
analyzed by Western blotting for
expression of EphB4, EphrinB2
and EGFR (B). HUVEC lysates
were loaded as positive control for
EphB4 expression.
A
A total of 15 such cases were studied. EphB4 protein levels were
elevated in 14 of 15 tumors (93%) by Western blotting and levels varied
from 0.6- to 21.7-fold with a mean sevenfold higher expression in tumor
specimens compared to normal urothelium (Figure 3.4B, HUVEC was
25
26
loaded as positive control for EphB4). To define the tissue distribution
of EphB4, immunohistochemical staining was performed on frozen
tumor sections. Tumors that had higher EphB4 expression levels on
Western blotting also had stronger signals on staining. EphB4
expression was seen exclusively in the neoplastic urothelium (bladder
ca) while adjacent stroma and normal urothelium was uniformly
negative (Figure 3.4A). In a majority of positive sections, EphB4 signal
was restricted to the cell membrane. Again, a reciprocal relationship
was seen between expression levels of EphB4 and EphB2 in normal
and malignant urothelium.
3.5 EphB4 is overexpressed in carcinoma of the breast
We examined by immunofluorescence twelve unselected tumor
specimens (11 invasive ductal cancers and 1 DCIS) for which frozen
sections were available (Kumar et al, 2006). Serial sections were
stained using H/E and EphB4-specific monoclonal antibodies (Figure
3.5). Tumor-specific EphB4 expression was seen in 7 of 12 specimens
(58%). Both DCIS (Figure 3.5A) and invasive cancer (Figure 3.5B)
specimens showed membrane-localized expression of EphB4. EphB4
expression was restricted to tumor tissue, and was absent in adjacent
normal stroma (seen in H/E staining of a serial section). We confirmed
the specificity of the signal by pre-incubating the antibodies with
blocking peptide and demonstrating loss of signal in a serial section
(right panel in DCIS). EphB4 expression was also seen in blood
vessels, presumably of venous lineage (arrow in DCIS). Normal ductal
epithelium had no demonstrable expression of EphB4 (data not shown).
A – DCIS
B – Invasive cancer
Figure 3.5. EphB4 is
expressed in breast cancer.
Fresh frozen sections of
human breast cancer tissues
were evaluated by
immunohistochemistry for
expression of EphB4. Tumor-
specific expression was
evaluated by H/E staining of
a serial section (ductal
carcinoma in situ (DCIS- A),
and infiltrating ductal cancer
(B). Specificity of signal was
confirmed by loss of signal
following pre-incubation with
blocking peptide (BP). Arrow
in DCIS shows positive
EphB4 signal in a blood
vessel. T-tumor, S-Stroma.
3.6 EphB4 is overexpressed in pancreatic adenocarcinoma
In a pilot study, we analyzed expression of EphB4, EphB2 and
EphrinB2 in six pancreatic cancer specimens. All six specimens
showed high levels of EphB4 expression, minimal to no expression of
27
EphB2 and EphrinB2. Immunostaining showed membrane-specific
expression of EphB4 in tumor tissue, but not adjacent stroma (Figure
3.6A). Western blot analysis confirmed findings on staining. All six
tumor specimens expressed high levels of EphB4, while three of six
expressed low levels of EphB2 and EphrinB2. A representative blot of
four specimens is shown in Figure 3.6B. The intensity of staining
correlated well with level of expression on Western blotting.
Figure 3.6 A. Fresh frozen samples of
pancreatic cancer were sectioned and
stained to detect expression of EphB4,
EphrinB2 and EphB2. Two
representative cases are shown. B.
Protein lysates from fresh frozen tumor
specimens were serially probed for
expression of EphB4, EphB2, E
and β-actin.
phrinB2
3.7 EphB4 is overexpressed in ovarian and hepatocellular
carcinoma
In a pilot study to evaluate expression in ovarian and hepatocellular
cancers, we studied EphB4 expression by Western blotting. Of the ten
28
29
ovarian carcinoma samples studies, seven (70%) had high levels of
EphB4 (data not shown). Of the ten hepatocellular cancers, all
expressed EphB4. In 7 of ten samples, tumor expressed higher levels
of EphB4 compared to adjacent non-tumor bearing liver (data not
shown).
3.8 Biologically active EphB4 is expressed on several different
cancer cell lines
For use in studies in vitro, we determined the status of EphB4 protein in
several tumor cell lines. For brevity, only results from colon cancer cell
lines are shown here. Similar results were obtained in all types of tumor
cell lines studied. Seven colon cancer cell lines and one normal colonic
epithelial cell line, CCD840, were surveyed for EphB4 protein levels by
Western blotting (Figure 3.7A). Six of 7 cancer cell lines (86%)
expressed EphB4 at various levels, while normal colonic epithelium had
no detectable expression of EphB4. Four of seven cell lines (57%)
expressed EphrinB2, as did normal colonic epithelium (data not shown).
Kaposi sarcoma cell line, SLK, was loaded as negative control for
EphB4 and 293T cells stably expressing full length EphB4 as positive
control.
30
A
C
B
Figure 3.7 EphB4 expression was assessed in seven colon cancer cell lines and normal
colonic epithelial line, CCD840, by Western blotting using an EphB4-specific monoclonal
antibody (A). The blot was stripped and re-probed for β-actin to control for loading. HT29
cells were stained with FITC-labeled EphB4 antibody, PE-labeled EphrinB2 antibody, or
both and analyzed by FACS analysis (B). HT29 cells were treated with different
concentrations of EphrinB2/Fc fusion protein for 10 min. Cell lysates were
immunoprecipitated and probed with p-Tyrosine specific antibody 4G10. Total EphB4
pulldown was evaluated with EphB4 specific monoclonal antibody (C, upper panel). To
study time-dependent phosphorylation, HT29 cells were treated with 1µg/ml EphrinB2/Fc
for various durations of time and cell lysates analyzed similarly (C, lower panel).
HT29 cells were stained with FITC-labeled EphB4-specific monoclonal
antibody and PE-labeled EphrinB2-specific polyclonal antibody to
detect surface expression of proteins and to evaluate if different clones
of cells express the two proteins or one cell expresses both (Figure
3.7B). Nearly all cells expressed EphB4, while a third expressed
EphrinB2. Hence, the same cell expresses EphB4 and EphrinB2.
EphB4 expressed on tumor cells is functional based on receptor
31
phosphorylation in response to treatment with EphrinB2. EphrinB2/Fc-
induced EphB4 phosphorylation was dose-dependent and occurred as
early as 10 minutes after stimulation (Figure 3.7C, upper panel).
Phosphorylation was also time-dependent (Figure 3.7C, lower panel).
Phosphorylation was seen as early as 10 min after stimulation with
1 µg/ml of clustered EphrinB2/Fc, but not Fc alone, peaked at 20 min
and began to decline by 60 min.
CHAPTER IV: Expression of EphB4 is under control of
different oncogenes and tumor suppressor genes
4.1 EphB4 is upregulated by oncogenes
4.1.1 EGFR
We sought to determine if erbB receptors regulate the expression of
EphB4. EGFR is expressed at high levels by breast cancer cell line
SKBR3, while ZR75 cell line does not express EGFR. Thus, SKBR3
cells were treated with an EGFR-kinase specific inhibitor, AG1478, to
study the effect on EphB4 (Figure 4.1). A time and dose-dependent
inhibition of EphB4 expression was seen with an IC50 at 500nM. At a
dose of 1 µM AG1478, EphB4 expression was nearly completely
inhibited at 36h (Figure 4.1, left panel). AG1478 had not effect on ZR75
cells that do not express EGFR (Figure 4.1, right panel).
Figure 4.1 EGFR signaling upregulates EphB4 expression. SKBR3 and
ZR75 cells were treated for 36 hours with varying doses of an EGFR-
specific kinase-inhibitor, AG-1478, and cell lysates were analyzed by
immunoblotting for expression of EphB4.
32
33
To confirm these findings, induction of EphB4 via EGFR signaling was
examined. Serum-starved SKBR3 cells were treated with increasing
doses of EGF for 36h (Figure 4.2, left panel). A dose-dependent
increase in EphB4 expression was seen following treatment with EGF.
In order to elucidate the signaling molecules downstream to EGFR that
regulate EphB4 expression, serum-starved SKBR3 cells were treated
with 100ng/ml EGF in the presence of various inhibitors of specific
downstream molecules. The JAK-2 inhibitor, AG490 (4µM), completely
abrogated EphB4 expression. The PI3K-inhibitor Wortmannin (50nM)
also abolished EphB4 expression, while a 79% reduction in expression
levels was seen with the Akt-inhibitor, SH-5 (100nM). There was also a
75% reduction in levels of EphB4 with the src-inhibitor, PP2 (500nM).
The MEK-inhibitor, PD98059 (200nM), resulted in a 31% reduction in
expression, while the p38 MAPK-inhibitor SB203580 (5µM) had no
effect on EphB4 levels (Figure 4.2, right panel). Thus, EGFR signaling
regulates EphB4 expression via different downstream pathways, with
the JAK-STAT and PI3K-Akt pathways playing a predominant role.
Induction of EphB4 following activation of EGFR by EGF was
attenuated by specific inhibition of JAK-STAT and PI3K-AKT pathways,
while inhibition of p38 MAPK had no effect. STAT3 is a downstream
effector molecule that undergoes differential phosphorylation by JAK
Figure 4.2 EGF upregulates EphB4 signaling via JAK-STAT and PI3K/Akt.
Serum-starved SKBR3 cells were treated for various doses of EGF for 36
hours as shown and EphB4 levels in lysates analyzed on immunoblotting
(left panel). Serum-starved SKBR3 cells were treated with 100ng/ml EGF in
the presence of different kinase inhibitors for 36 hours and EphB4 levels in
lysates analyzed similarly (right panel).
and MAPK. While JAK phosphorylates a tyrosine residue at position
705, MAPK phosphorylates a serine residue at position 727. The
differential effects of JAK inhibitor and p38MAPK inhibitor suggest that
the site of STAT3 phosphorylation plays an important role in the
regulation of EphB4 expression by EGFR. PI3K signaling leads to
activation of Akt and mTOR, both of which regulate cell fate. Loss of
PTEN, which is observed in nearly half of all cancers, allows unabated
activity of PI3K and thus, downstream effects such as tumor cell
survival. The complete abrogation of EphB4 expression with a PI3K
inhibitor and Akt inhibitor provides evidence for a prominent role of this
pathway in the regulation of EphB4. It is likely that JAK-STAT and PI3K
pathways are co-dependent, as inhibition of either pathway led to a
profound reduction in EphB4 levels.
34
4.1.2 HER-2/neu
Based on these findings, we proposed that erbB2 may have similar
effect on EphB4 expression. We treated SKBR3 cells with herceptin
under conditions known to result in endocytosis and degradation of the
receptor. A 76% reduction in HER-2/neu levels with 20µg/ml herceptin
at 48h correlated with a 76% reduction in EphB4 expression (Figure
4.3, left panel). Herceptin had no effect on EphB4 expression in ZR75
cells (HER-2/neu negative, Figure 4.3, left panel).
Figure 4.3 HER2/neu signaling upregulates EphB4 expression. SKBR3 and
ZR75 cells were treated for 24 hours with varying doses of erbB2 antibody
trastuzumab (Herceptin) and cell lysates were analyzed by immunoblotting for
the proteins shown (left panel). Cell lysates from parent mouse fibroblasts (3T3
cells) stably transfected with empty vector or HER-2/neu were analyzed for
expression of EphB4 and β-actin (right panel).
Direct evidence for the role of HER-2/neu in the regulation of EphB4
was obtained by ectopic expression in the EphB4-negative mouse
fibroblast cell line, NIH-3T3. Stable transfection of HER-2/neu into these
cells resulted in expression of EphB4 protein (Figure 4.3, right panel),
while no expression was observed in the vector transfected cell line.
35
4.1.3. Akt
The protein kinase Akt is downstream to several different receptor
tyrosine kinases and is frequently mutated in many cancers. We chose
to study if Akt plays a role in upregulating EphB4 expression. An Akt-
specific kinase inhibitor significantly inhibits EGF-induced upregulation
of EphB4 (Figure 4.4, left panel). Contrarily, overexpression of wild type
Akt in an EGFR and EphB4 negative cell line induces EphB4, while a
functionally silent Akt mutant has no effect (Figure 4.4, right panel).
These results confirm that AKt can independently regulate EphB4
expression.
Figure 4.5 Akt signaling upregulates EphB4 expression. Serum-starved SCC-
15 cells were treated with 100ng/ml EGF in the presence or absence of 20µM
Akt inhibitor SH5 (Akt-i, left panel) and EphB4 expression studied by Western
blotting. Wild-type or dominant-negative mutant Akt was transfected into
SW620 cells and EphB4 levels assessed by Western blotting 24hr later (right
panel).
4.2 EphB4 is downregulated by tumor suppressor genes
4.2.1 PTEN
Given that Akt regulates EphB4 expression and PTEN is an important
regulator of Akt, we investigated whether the relatively high expression
36
of EphB4 is related to PTEN by re-introducing wild-type PTEN into
cancer cells. Cells were co-transfected with a truncated-CD4
expression plasmid to allow selection of transfected cells. Transfected
cells were then sorted for the co-transfected truncated CD4 marker. We
found that the expression of EphB4 in PC3 cells was reduced by 75%
or more by the re-introduction of wild-type PTEN (Figure 4.6 A).
B
A
C
Figure 4.6 PTEN downregulates EphB4 expression. Cells were co-transfected
with truncated-CD4 and p53 or PTEN or vector only. 24 h later CD4-sorted cells
were collected, lysed and analyzed sequentially by Western blot for the
expression of EphB4 and β-actin (A). EphB4 expression was studied in anterior
(AP), ventral (VP) and dorsolateral (DLP) lobes lobes of prostate glands of age-
and background-matched wild type (Wt) and PTEN -/- mice by western blotting
(B). Representative immunofluorescence study of dorso-lateral gland is shown (C)
Conversely, prostate glands of PTEN knock-out mice showed
significantly increased EphB4 expression compared to age and
background matched wild type mice both by Western blotting (Figure
4.6B) and immunohistochemistry (Figure 4.6C).
37
4.2.2 p53
Mutations in p53 are frequently seen in several epithelial cancers. We
were hence interested in determining if p53 contributes to the
expression of EphB4. The HCT-116 colon cancer cells express low
levels of EphB4. Stable p53 knockout cells express over 2-fold higher
EphB4 levels (Figure 4.7). We introduced wild-type p53 into p53
knockout cells along with the truncated cell surface receptor CD4 for
sorting transfected cells. EphB4 expression declined by over 65% in
positively selected p53 transfected cells, while no change was observed
in cells transfected with empty vector (Figure 4.7). These results
suggest that p53 regulates EphB4 expression either directly or via
downstream effects.
Figure 4.7 p53 downregulates
EphB4 expression. HCT116 cells
and its p53 knockout mutant clone
were evaluated for expression
EphB4. Knockout cells were co-
transfected with truncated-CD4
and p53 or empty vector only. 24h
later CD4-sorted cells were lysed
and analyzed sequentially by
Western blot for the expression of
EphB4 and p53.
Wild-type p53 interacts with several signaling pathways that provide
survival signals to cells, including the PI3K and MAPK pathways. It is
thus likely that loss-of-function mutation in p53 leads to activation of Akt
and thus, induces EphB4 expression.
38
4.2.3 APC
APC is an important mediator of the canonical Wnt-catenin signaling
pathway. APC is frequently deleted in several familial colon cancers.
We chose to evaluate if EphB4 plays a role in APC signaling. HT29
colon cancer cells have mutated APC. Re-introduction of wild-type APC
significantly downregulated expression levels of EphB4 on Western blot
(Figure 4.8 left panel). Conversely, non-tumor bearing colonic
epithelium of APC
min
mice expresses higher levels of EphB4 than
normal colonic epithelium of age-and gender-matched wild-type litter
mates (Figure 4.8 right panel). Adenomas from the APC
min
mice
express even higher levels of EphB4.
Figure 4.8 APC downregulates EphB4 expression. HT29 colon cancer cells
have mutated APC. Cells were co-transfected with truncated-CD4 and wild type
APC or vector only. 24 h later CD4-sorted cells were collected, lysed and
analyzed sequentially by Western blot for the expression of EphB4 and APC
(left panel). Colon epithelium from age and background-matched wild type and
APC
min
mice were analyzed for EphB4 levels by Western blotting (right panel).
39
Thus, expression of EphB4 is tightly controlled by several oncogenes
and tumor suppressor genes (Figure 4.9). It is likely that several other
genes also regulate EphB4 levels and the exact downstream pathways
that orchestrate this expression are currently being investigated. In any
event, such a tight control suggests a prominent role for EphB4 in tumor
biology.
Figure 4.9 Regulation of EphB4 expression. EphB4 expression in cancer cells is
upregulated by several oncogenes and downregulated by tumor suppressor
genes. Data suggesting positive feedback loop from EphB4 to Akt (green arrow) is
from Kumar et al, 2006
40
41
CHAPTER V: EphB4 provides survival signals to tumor
cells in vitro and in vivo
Focused overexpression of EphB4 in tumor cells, driven upward by
oncogenes and downward by tumor suppressors, points to a likely
significant role for EphB4 in tumor cells. We elected to elucidate this
functional role of EphB4 in tumor cells. To this end, targeted EphB4
knockdown was achieved using EphB4-specific siRNA and antisense
ODN.
5.1 EphB4 knockdown inhibits cell viability in vitro
Various EphB4 specific anti-sense phosphorothioate-modified
oligodeoxynucleotide (ODN) and siRNA were synthesized. The most
active antisense ODN and siRNA that knock down EphB4 expression in
transiently transfected 293T cell line were chosen (data not shown).
EphB4-siRNA corresponding to the sequence 5’-GGU GAA UGU CAA
GAC GCU GUU-3’ and 3’-UUC CAC UUA CAG UUC UGC GAC-5’ was
used for RNA interference. Control siRNA was generated by mutating
three bases in this sequence to effectively abrogate EphB4 knockdown.
This mutated siRNA (siRNA ∆) had a sequence 5’-AGU UAA UAU CAA
GAC GCU GUU-3’ and 3’-UUU CAA UUA UAG UUC UGC GAC-5’.
Additionally, siRNA directed against GFP with sequence 5’-CGC UGA
CCC UGA AGU UCA TUU-3’ and 3’-UUG CGA CUG GGA CUU CAA
GUA-5’ was also used as a negative control. EphB4-specific siRNA, but
42
not mutated siRNA ∆ or siRNA directed against GFP, caused a dose-
dependent decrease in EphB4 protein levels (Figure 5.1A and data not
shown). 48hours after transfection of 100nM EphB4-siRNA, an 89%
reduction in protein expression was observed. Concomitant with fall in
EphB4 levels, cell viability (measured at 48 hours of EphB4-siRNA
treatment) declined in a dose-dependent manner. Treatment with
100nM EphB4-siRNA, but not siRNA ∆, reduced cell viability by 77%
(Figure 5.1B, left panel) in the EphB4-positive SCC15 cell line, while no
effect was seen in the EphB4-negative SCC4 cell line (Figure 5.1B,
right panel). EphB4-positive cell lines from various cancers studied
showed a significant reduction in cell viability following treatment with
EphB4-specific siRNA (Figure 5.1C). In all cases, mutated siRNA had
not effect and similarly, EphB4-negative cells lines had no loss in
viability with either siRNA.
For application in vivo, we generated several antisense ODN. The most
effective anti-sense ODN used, AS-10M, spanned nucleotides 1980-
1999 with a sequence ATG GAG GCC TCG CTC AGA AA. Scrambled
ODN containing random nucleotide sequence and a similar CpG site,
TAC CTG AAG GTC AGG CGA AC, was used as control. AS-10M
treatment, but not scrambled ODN treatment, also resulted in a
A
Cancer type % cell death with EphB4 KO
Squamous cell carcinoma
(Head and Neck)
85%
Breast adenocarcinoma 80%
Prostate adenocarcinoma 80%
Colon adenocarcinoma 76%
Bladder adenocarcinoma 80%
Ovarian adenocarcinoma 90%
Pancreatic adenocarcinoma 65%
Hepatocellular carcinoma 75%
C
B EphB4-positive EphB4-negative
43
Figure 5.1 EphB4 provides survival signals to cancer cells in vitro. SCC
cells were transiently transfected with Lipofectamine 2000 alone (control),
EphB4-specific siRNA (EphB4-siRNA) or mutated EphB4 siRNA (EphB4-
siRNA ∆). 48 hours later, 20 µg whole cell lysates were analyzed by
immunoblotting for EphB4 and β-actin levels (A). 1×10
4
SCC15 cells
(EphB4 positive, B, left panel) or SCC4 cells (EphB4 negative, B, right
panel) were transfected with Lipofectamine alone, mutated EphB4 siRNA ∆
or EphB4-siRNA and plated in a 48-well plate. Cell viability was assessed
by MTT assay at 48 hours and survival expressed as percentage of
absorbance relative to untreated cells. Several different EphB4-expressing
cancer types exhibit significant loss in tumor cell viability with 100nM of
EphB4-siRNA (C). In each case, mutated siRNA had no effect.
significant dose-dependent loss in EphB4 expression with an 84% loss
in receptor expression at 72 hours of treatment (Figure 5.2 A). Again,
knockdown of EphB4 expression resulted in a significant reduction in
cell viability with a 72% reduction in cell viability at 72 hours at a dose of
10µM AS-10M (Figure 5.2 B, left panel). The IC50 for AS-10M was at
6µM. AS-10M had no effect on cell lines that do not express EphB4
(Figure 5.2B, right panel).
A
B
EphB4-positive EphB4-negative
Figure 5.2 AS-mediated knockdown of EphB4 inhibits cancer cell survival.
SCC15 cells were treated with varying doses of scrambled ODN or EphB4-
specific ODN (AS-10M). 72 hours later, 20 µg whole cell lysates were
analyzed by immunoblotting for EphB4 and β-actin levels (A). 1×10
4
SCC15
cells (B, left panel) or SCC4 cells (B, right panel) were treated with
scrambled or AS-10M ODN. Cell viability was assessed by MTT assay at 7
hours and survival expressed as percentage of absorbance relative to
untreated cells.
2
44
45
5.2 EphB4 knockdown inhibits cell viability in vivo
We evaluated the effect of EphB4 knockdown in vivo on tumor growth
in murine tumor models of several tumor subtypes. Mice were injected
with tumor cells subcutaneously in the flank region (xenografts) or in the
region of tumor origin (orthotopic model). On day four after injection,
mice were randomly divided into three groups after ensuring uniform
tumor sizes and treatment begun with vehicle (PBS), scrambled ODN
(S), or AS-10 ODN (AS-10M) administered intraperitoneally daily at a
dose of 10mg/kg. Treatment with AS-10M led to significant reductions
in tumor volumes in all tumor types. As an example, head and neck
squamous cell cancer xenograft is shown in Figure 5.3. The animals
had no clinical evidence of toxicity, fed well and maintained normal
weight and activities. Gross and histologic examination of various
organs at sacrifice showed no abnormalities (data not shown). IL-12
levels in blood samples of mice in the various treatment groups at
sacrifice were comparable as were TNF- α levels (data not shown). At
the time of sacrifice, spleens were harvested from mice in all treatment
groups and evaluated. The spleen weights were comparable between
the three groups and histologic examination revealed no change (data
not shown). No expression of EphB4 was discernable in the antisense
ODN-treated tumors by Western blot in comparison to high levels seen
in vehicle or scrambled ODN-treated tumors (Figure 5.3 B). These
A
Cancer type % tumor reduction with EphB4 KO
Squamous cell carcinoma
(Head and Neck)
80%
Breast adenocarcinoma 72%
Prostate adenocarcinoma 65%
Colon adenocarcinoma 62%
Bladder adenocarcinoma 75%
Ovarian adenocarcinoma 82%
D
C
46
Figure 5.3 EphB4 provides survival signals to cancer cells in vivo. 5 X 10
6
SCC-15 cells
were implanted in the flank of 10-12-week old, male Balb/C athymic mice (n=8 per group,
experiment repeated twice) and tumor volume measured as length X (width)
2
X 0.52. Mice
were administered vehicle alone (control), or 10 mg/kg scrambled (S) or EphB4-specific
antisense ODN (AS-10M) intraperitoneally daily starting day 4 after tumor implantation.
Tumor volumes were statistically significantly smaller in the AS-10M group compared to the
control and scrambled ODN groups at all time points of measurement beginning day 7 (A).
Animals were sacrificed at four weeks and 20 µg tumor lysates were analyzed by Western
blotting for EphB4 and β-actin levels (B). Gross and histologic pictures of tumors from the
three groups (C). Several different EphB4-expressing cancer types exhibit significant loss in
tumor growth with 10mg/kg AS-10 (D). In each case, scrambled ODN had no effect.
47
results confirm that the observed reduction in tumor growth with EphB4-
specific antisense ODN is a direct effect of targeted EphB4 knockdown
and not mediated by pleiotropic cytokine responses.
Thus, EphB4 functions as a survival factor for EphB4-positive tumor
cells in vitro and in vivo. Targeting EphB4 in EphB4-positive tumors
may have a clinical benefit by directly interrupting survival cues and
inhibiting tumor invasion. In addition, based on knockout mice studies,
EphB4 knockdown in tumors may also block angiogenesis that supports
tumor growth. Our studies in animal models confirm these hypotheses
and demonstrate that antisense-oligonucleotides that target EphB4
expression effectively block the growth of tumors and can induce
regression of established tumors in mice (data not shown). They further
suggest an additional anti-angiogenic effect of EphB4 knockdown in
vivo by documenting reduction in tumor microvasculature (data not
shown).
48
CHAPTER VI: EphB4 inhibits apoptotic pathways in
tumor cells
Tyrosine kinase receptors can promote tumor cell survival in many
ways, and evasion of natural death mechanisms is uniformly noted in
several cancer types. Given that EphB4 knockdown profoundly impairs
tumor cell viability, we speculated that EphB4 may inhibit apoptotic
pathways in tumor cells. We therefore undertook to study apoptosis in
tumor cells following knockdown of EphB4.
6.1 EphB4 protects tumor cells from apoptosis in vitro and in vivo
The accumulation of nucleosomes in the cytoplasm is a hallmark of
apoptosis. We quantitated cytoplasmic nucleosomes by ELISA in
EphB4-positive tumor cells following treatment with EphB4 siRNA. A
dose-dependent increase in DNA fragmentation was observed with
EphB4-siRNA but not mutant siRNA ∆ (Figure 6.1A). Treatment with
EphB4-siRNA at a dose of 100nM for 36hr resulted in a 16-fold
induction of apoptosis, while EphB4 siRNA ∆ had no effect. We then
wished to determine the effect of EphB4 on cell cycle regulation in
tumor cells. Knockdown of expression of EphB4 by specific siRNA
resulted in accumulation of cells in sub-G0 phase (as assessed by
FACS analysis), suggestive of apoptosis (Figure 6.1B). Treatment of
cells for 16 hours with EphB4-siRNA results in accumulation of 30.7%
C
Figure 6.1 EphB4 protects cancer cells from apoptosis in vitro. SCC15 cells were
transiently transfected with Lipofectamine alone (control) or EphB4-specific siRNA
(EphB4-siRNA) or mutated siRNA (EphB4-siRNA ∆). Apoptosis was analyzed by
ELISA for cytoplasmic nucleosomes using whole cell lysates 36h later (A). Cell cycle
analysis of SCC15 cells treated with 100nM EphB4-specific siRNA (EphB4 siRNA) or
mutant siRNA (EphB4 siRNA ∆) for the duration of treatments indicated (B). The % of
cells in G0, G1, S, G2, and M phase are indicated. SCC15 cells treated with 100nM
EphB4-specific siRNA (EphB4 siRNA) or mutant siRNA (EphB4 siRNA ∆) for 36h.
Cells were stained with Annexin-FITC and propidium iodide and analyzed on dual
fluorescence scales (C)
A
B
49
50
cells in sub-G0 population, compared to 7.8% of mock transfected cells
(Figure 6.1B, left panel). In contrast, EphB4-siRNA ∆ had no discernable
effect on cell cycle distribution. Prolonged (36 hours) treatment with
100nM EphB4-siRNA results in accumulation of 80% of the cells in sub-
G0 while mock transfected cells showed only 1.9% of cells in the sub-
G0 population. In order to confirm the induction of apoptosis following
EphB4 knockdown in tumor cells, we stained these cells with Annexin-
FITC and propidium iodide to study cell membrane flipping without loss
in cell membrane integrity. Whereas 6% and 4% of mock- and EpB4-
siRNA ∆ transfected cells, respectively, showed membrane flipping,
45% EphB4-siRNA transfected cells demonstrated membrane flipping
consistent with the induction of apoptosis (Figure 6.1C). Hence, we
were able to confirm in vitro that EphB4 knockdown does indeed induce
apoptosis.
We then wanted to show if this phenomenon carried over in vivo as
well. To that end, we harvested EphB4-AS treated tumors and
performed immunohistochemical evaluation (Figure 6.2). AS-10 treated
tumors, in comparison to control tumors, showed an 86% reduction in
Ki-67 positive cells consistent with reduction in tumor proliferative index.
This was associated with a 15-fold increase in apoptosis by TUNEL
assay. Thus, EphB4 knockdown causes tumor cell death by induction of
apoptosis in vitro and in vivo
Figure 6.2 EphB4 protects cancer cells from apoptosis in vivo. 5 X 10
6
SCC-
15 cells were implanted in the flank of the ten- to twelve-week old, male
Balb/C athymic mice. Mice were administered vehicle alone (control), or 10
mg/kg scrambled ODN or EphB4-specific antisense AS-ODN
intraperitoneally daily starting day 4 after tumor implantation for six weeks.
5 µ sections of formalin-fixed paraffin embedded tumors were analyzed by
immunohistochemistry for Ki-67 (top row) and for apoptosis by TUNEL
(bottom row) with in situ apoptosis staining kit.
6.2 EphB4 interrupts predominantly the extrinsic apoptotic
pathway
Apoptosis can be orchestrated by two major pathways in cells. The cell-
membrane originating extrinsic pathways requires activation of
caspase-8, while the mitochondrial intrinsic pathway proceeds under
control of casapse-9 activation without the involvement of caspase-8.
We wanted to identify the exact apoptotic pathway that EphB4
interrupted in cancer cells. This was achieved by measuring activation
of caspase-8 and caspase-9 by a colorimetric caspase activation assay.
EphB4-siRNA treatment led to a 7-fold increase in casapse-8 activity
and a modest 2.5-fold increase in caspase-9 activity (Figure 6.3A),
51
A
B
Figure 6.3 EphB4 interferes with apoptotic pathways in vitro. SCC15 cells were
transiently transfected with Lipofectamine alone (control) or EphB4-specific siRNA
(EphB4-siRNA) or mutated siRNA (EphB4-siRNA ∆). Caspase-8 and caspase-9
activation was assayed colorimetrically 36h later and expressed as percent
activity compared to lipofectamine-treated cells (A). Expression levels of various
anti-apoptotic proteins in 20 µg whole cell lysates were analyzed by
immunoblotting (B). Band intensity relative to β-actin is shown below the blots.
while EphB4 siRNA ∆ had no such effect. Similar results were obtained
with EphB4-antisense ODN (data not shown). Overexpression of anti-
apoptotic proteins such as Mcl-1, Bcl-2, Bcl-x
L,
and FLICE-inhibitory
protein (FLIP) occurs commonly in cancers in order to protect tumor
cells from death. We therefore evaluated the effect of EphB4
52
53
knockdown on these anti-apoptotic proteins (Figure 6.3B). Concomitant
to fall in EphB4 levels with EphB4-siRNA in tumor cells, a reduction in
levels of Mcl-1 and Bcl-x
L
by 45% and 88%, respectively, was
observed. EphB4 knockdown had no demonstrable effect on
expression of FLIP and Bcl-2. EphB4 knockdown thus alters cell
viability predominantly through induction of apoptosis pathways (mainly
caspase-8 activation) and to a smaller extent via downregulation of
select anti-apoptotic proteins (Mcl-1 and Bcl-x
L
).
In order to confirm the induction of the membrane originating caspase-8
mediated extrinsic apoptotic pathways following EphB4 knockdown in
tumors in vivo, we studied caspase-8 and caspase-9 activity in tumors
in the three treatment groups (Figure 6.4A). Similar to our data in vitro,
EphB4 knockdown with AS-10 resulted in a 4-fold induction in caspase-
8 activity, while scrambled ODN had no effect. In addition, EphB4
knockdown also resulted in a 2.3-fold induction in caspase-9 activity in
vivo. We believe that the anti-angiogenic effect of EphB4 knockdown
results in significant tumor cell ischemia that activates intrinsic apoptotic
cascades as well. Further, we performed a western blot analysis on
tumor sample proteins using antibodies that recognize cleaved and
activated casapse-8 products as well as native uncleaved protein
(Figure 6.4B). Following EphB4 knockdown in tumors, there was
increase in levels of the intermediate and active cleavage products of
caspase-8 demonstrating that EphB4 inhibits activation of the extrinsic
apoptotic pathways in tumors.
A
B
Figure 6.4 EphB4 interferes with apoptotic pathways in vivo. 5 X 10
6
SCC-15 cells
were implanted in the flank of the ten- to twelve-week old, male Balb/C athymic
mice. Mice were administered vehicle alone (control), or 10 mg/kg scrambled
ODN or EphB4-specific antisense AS-ODN intraperitoneally daily starting day 4
after tumor implantation for six weeks. Tumor lysates were analyzed for caspase-
8 and 9 activation using colorimetric assays (A). Western blot was run to evaluate
cleavage products consistent with casapse-8 activation (B).
Thus, EphB4 provides survival advantage to tumor cells by interfering
with apoptotic pathways in vitro and in vivo. In particular, EphB4
knockdown activates capase-8 and induces apoptosis, suggesting that
54
55
the predominant site of action of EphB4 is at the cell membrane.
Several apoptotic signals originate at the cell membrane, some of the
better elucidated ones being the FAS-ligand/FAS pathway, the TNF-
α/TNFR pathway and TRAIL/death receptor pathway. EphB4 may
interrupt one or more of these pathways due to downstream signaling-
mediated effects. Signaling can result from stimulation by EphrinB2 in
an autocrine or paracrine manner depending on cell type, or due to self-
aggregation and activation due to high receptor density. Alternatively,
EphB4 may function in a signaling-independent mechanism, wherein
interaction of EphB4 with certain components of apoptotic pathways
can interrupt apoptotic signals. Such a model has been demonstrated in
liver cells in which Fas-L binding to Fas is blocked by the extra-cellular
domain of the c-met receptor and thereby, apoptosis is inhibited (Wang
et al, 2002).
56
CHAPTER VII: Extra-cellular domain of EphB4 inhibits
TRAIL-mediated cell death in tumor cells in a signaling-
independent fashion
EphB4 provides direct survival cues to tumor cells by predominantly
inhibiting the extrinsic capase-8 mediated apoptotic pathway. Loss of
EphB4 results in activation of caspase-8, and to lesser extent caspase-
9, indicating that inhibition of EphB4 expression permits intrinsic signals
to induce cell death. However, the exact molecular mechanism by
which EphB4 interrupts caspase-8 activation has not been established.
Tumor cells often express death receptors which induce apoptosis upon
activation of the death receptor by its cognate ligand, TRAIL. Reduction
of EphB4 and induction of the death receptor pathway may serve to
inhibit tumor cell survival. We began by studying if such a synergy
exists between EphB4 knockdown and induction of extrinsic apoptotic
pathways.
7.1 EphB4 knockdown sensitizes tumor cells to TRAIL-induced
cell death
In order to study if EphB4 had an effect on the sensitivity of cells to
extrinsic death receptor pathways, active EphB4-siRNA was combined
with TRAIL or FAS-ligand. Treatment of SCC15 cells with 10nM EphB4-
57
siRNA, but not EphB4-siRNA ∆, resulted in a marked increase in TRAIL-
induced loss in cell numbers (Figure 7.1A, left panel). For example,
treatment with 50ng/ml TRAIL reduced cell number in EphB4-siRNA
transfected cells by 75% in contrast to 30% reduction seen in mock
transfected cells. FAS-ligand, in contrast, had no effect on SCC15 cells
alone or in combination with EphB4 siRNA (data not shown). This is
consistent with our data showing the lack of FAS receptor, but presence
of death receptors, DR4 and DR5 in SCC15 cells (data not shown).
Complete EphB4 knockdown even at maximum doses of 100nM did not
appreciably alter levels of TRAIL, or its receptors DR4, DR5 or DcR1
(Figure 7.1A, right panel). Thus, the effect of EphB4 knockdown on
sensitivity of SCC15 cells to TRAIL does not result from alterations in
expression levels of this protein. Whereas SCC15 cells have some
sensitivity to TRAIL, HT29 colon cancer cells are relatively resistant to
TRAIL-induced apoptosis with 250ng/ml TRAIL having no effect on cell
survival (Figure 7.1B). Introduction of EphB4-siRNA increased TRAIL-
induced loss of cell viability in a dose-dependent fashion (Figure 7.1B,
left panel). Whereas 20nM EphB4-siRNA alone resulted in 10% cell
death and 125ng/ml TRAIL alone had no effect, a combination of
125ng/ml TRAIL with 20nM EphB4-siRNA resulted in 50% cell death.
Combination of TRAIL with EphB4 siRNA ∆, however did not alter cell
viability (Figure 7.1B, right panel). The increased sensitivity to TRAIL-
58
A. SCC15 cells (partially sensitive to TRAIL)
B. HT29cells (not sensitive to TRAIL)
C
Figure 7.1 EphB4 inhibits death receptor pathway. SCC15 cells were transfected
with vehicle (Lipofectamine), 10nM EphB4-specific (EphB4 siRNA) or mutant
siRNA (mEphB4 siRNA ∆). 24hr later, 1X10
4
cells were treated overnight with
varying doses of TRAIL. Cell number was assessed by MTT assay (A) and
membrane flipping by dual-color FACS following Annexin-FITC and PI staining
(C). Levels of TRAIL and its receptors was assessed by Western blot (A, right
panel). Transfected HT29 cells were exposed to various doses of TRAIL for 16
hours and cell survival assessed by MTT assay (B).
59
induced apoptosis after a partial knock-down of EphB4 is consistent
with a role of EphB4 in protection from TRAIL-induced apoptosis. About
12% of parent SCC15 cells demonstrated AnnexinV positivity in the
absence of propidium uptake. TRAIL (100ng/ml) increased this number
to 26% and EphB4-siRNA (20nM) to 21%. A combination of the two
however, resulted in 64% cells undergoing membrane flipping
consistent with a synergistic induction of apoptosis (Figure 7.1C).
Because EphB4 interrupts apoptosis originating at the cell membrane
as does TRAIL, induction of caspase-8 is the primary event following
EphB4 knockdown and stimulation with TRAIL. If this was indeed the
case, addition of a caspase-8 specific inhibitor should be able to
reverse cell death resulting from TRAIL and EphB4 knockdown. We
confirmed this by adding caspase-8 specific and caspase-9 specific
inhibitors in the above experiment. Whereas TRAIL (100ng/ml) resulted
in 25% cell death in parent SCC15 cells, addition of a caspase-8
specific, but not casapse-9 specific, inhibitor fully reversed this cell
death (Figure 7.2, left panel). In the presence of EphB4 siRNA 10nM,
100ng/ml TRAIL resulted in 50% cell death. Addition of a caspase-8
inhibitor recovered all but 10% cell death, whereas caspase-9 inhibitor
was able to recover only 10% of cell death (Figure 7.2, right panel),
confirming that the observed phenomena resulted from a preferential
induction of extrinsic caspase-8 mediated apoptosis.
Figure 7.2 EphB4 inhibits extrinsic apoptotic pathway. SCC15 cells were
transfected with vehicle (parent cells), or 10nM EphB4-specific (EphB4 siRNA).
24hr later, 1X10
4
cells were treated overnight with varying doses of TRAIL in the
presence of a casapse-8 specific or caspase-9 specific inhibitor. Cell viability was
assessed by MTT assay
7.2 Forced expression of EphB4 renders tumor cells resistant to
TRAIL
Given that EphB4 knockdown sensitizes TRAIL-resistant cells to TRAIL,
we evaluated if forced expression of EphB4 will render an EphB4-
negative, TRAIL-sensitive cell line resistant to TRAIL. A549 cells do not
express EphB4 (Figure 7.3A) and are partially sensitive to TRAIL-
induced apoptosis. These cells were co-transfected with the truncated
CD4 receptor (to allow sorting of transfected cells) and full length
EphB4 expression vector or null vector. Transfected cells were sorted
using magnetic beads coated with anti-CD4 antibodies and purity of
transfected cells was cross-verified with fluorescent EphB4 monoclonal
antibody to be 91% (data not shown). Cells transfected with EphB4, but
not null vector, expressed EphB4 protein (Figure 7.3A, right panel). We
60
A
Figure 7.3 Forced expression of EphB4 renders TRAIL resistance to tumor cells.
A549 lung cancer cells were transfected with full-length EphB4 expression vector
or null vector along with a truncated CD4 receptor that was used to sort
transfected cells. Transfected and sorted cells were cultured in the presence of
varying doses of TRAIL and cell survival assessed on day 3 (A, left panel). EphB4
expression in transfected cells was confirmed by immunoblotting (A, right panel).
A549 lung cancer cells were transfected with full-length EphB4 expression vector
or null vector along with GFP expression vector. Cells were cultured in the
presence of 100ng/ml TRAIL. Number of GFP-positive cells was counted daily for
three days and averaged over ten random high-power fields by a blinded observer
B
then studied the sensitivity of EphB4 or null vector transfected cells to
TRAIL-induced apoptosis (Figure 7.3A, left panel). Ectopic expression
of EphB4 in A549 cells protected them from TRAIL-induced apoptosis.
61
62
TRAIL at 100ng/ml, for example, resulted in 40% loss of viability in
vector-transfected cells, while EphB4 transfected cells were completely
resistant to TRAIL-induced apoptosis. Alternatively, cells were co-
transfected with EphB4 expression vector or null vector and GFP
expression vector. The pool of cells was cultured in the presence of
TRAIL (100ng/ml). The number of GFP positive cells was counted in
ten independent high power fields daily for five days. There was a
steady increase in TRAIL-resistant GFP-positive cells when they were
co-transfected with EphB4 expression vector, but not null vector (Figure
7.3B). This confirms that overexpression of EphB4 protects TRAIL-
sensitive cells from TRAIL. The question that was then raised was
whether EphB4-signaling was responsible for this resistance to TRAIL.
In order to address this question, we generated a signaling-deficient
mutant of EphB4, the EphB4-eGFP (Figure 7.4A, cartoon on left).
7.3 EphB4-eGFP mutant protein
The EphB4-eGFP mutant protein is generated by deleting the
cytoplasmic tail of EphB4 from just past the transmembrane region and
replacing it with GFP. Such a protein functions in a unique fashion
(Figure 7.4A, cartoon on right). Wild-type EphB4 itself signals into the
EphB4-expressing cell (forward signaling), and also stimulates
EphrinB2 to signal into EphrinB2 expressing cells (reverse signaling).
A
63
C
B
E D
Figure 7.4 EphB4-eGFP mutant protein characterization. In order to delineate the
role of signaling down EphB4, a mutant protein with the cytoplasmic tail replaced by
GFP was generated (A, cartoon on left), which was anticipated to allow reverse
signaling without forward signaling (A, cartoon on right). The mutant protein was
detected by Western blot as an 80KDa band (B). Parent 293 cells (293-empty) have
no autofluorescence (C, upper panel) and do not have detectable staining with PE-
labeled anti-EphB4 antibody on FACS (C, lower panel). Transfection with wild-type
EphB4 (293-EphB4) fluoresces with PE only, while 293 cells transfected with GFP
(293-GFP) demonstrate fluorescence in th entire cell and on the GFP axis alone on
FACS. Cells transfected with EphB4-eGFP (293-B4-GFP) fluoresce at the cell
membrane only and on both PE and GFP axes on FACS. Upon treatment with
2µg/ml EphrinB2/Fc, parent cells show low levels of phosphorylation, which is
unchanged upon GFP transfection (D). There is a significant increase in total
amount and amount of phosphorylated EphB4 upon transfection with wild-type
vector, whereas EphB4-eGFP protein itself does not undergo phosphorylation and
inhibits phosphorylatio of wild-type protein. Equal numbers of cells from the four
transfections were mixed with solution containing equal amount of alakaline
phosphatase tagged EphrinB2. Cells were pelleted and AP activity tested in the
pellet (E). Both wild-type and EphB4-eGFP transfections significantly increases B2-
AP binding.
EphB4 knockdown abolishes both forward and reverse signaling.
Whereas EphB4-eGFP protein is incapable to forward signaling
because it lacks kinase and tyrosine residues, it is still capable of
reverse signaling. We confirmed expression of the protein on Western
blot using antibodies directed against the extra-cellular domain of
EphB4 (Figure 7.4B). Wild type EphB4 migrates at 127kDa, whereas
EphB4-eGFP migrates at about 108KDa. Whereas parent cells and
EphB4-wt transfected cells have no autofluorescence, and the entire
cell fluoresces when transfected with GFP expression vector, EphB4-
eGFP transfected cells have membrane associated fluorescence
confirming membrane targeting of the mutant protein (Figure 7.4C lower
panel). We further confirmed membrane expression by FACS analysis
(Figure 7.4C, upper panel). Parent cells (293-Empty) do not fluoresce
64
65
on green or red axes. Cells transfected with wild type EphB4 (293-
EphB4) fluoresce on red axis only when stained with PE-tagged EphB4-
specific antibodies. Cells transfected with GFP expression vector (293-
GFP) fluoresce on the green axis only. To the contrary, cells
transfected with EphB4-eGFP (293-B4-GFP) fluoresce on both axes.
We then sought to confirm if this mutant protein was indeed signaling
deficient. The same four set of cells were stimulated with 0.5µg/ml
EphrinB2/Fc and tyrosine phosphorylation assessed after
immuoprecipitation of EphB4 from whole cell lysates (Figure 7.4D).
Parent cells and GFP transfected cells expressed low levels of
phsophorylable EphB4. Following wild-type EphB4 transfection, the
amount of total and phosphorylated protein significantly increased.
However, following transfection with EphB4-eGFP expression vector,
not only was the mutant protein not phosphorylated, it also inhibited
phosphorylation of endogenous wild-type EphB4. This may result from
the formation of functionless heterodimers between wild-type and
mutant protein. EphB4-eGFP mutant protein thus functions in a
dominant negative fashion for forward signaling. Lastly, we had to
confirm if this protein was still capable of binding EphrinB2. To this end,
the four sets of cells were mixed with EphrinB2-AP and following
centrifugation and wash, alkaline phosphatase activity was detected in
the cell pellet (Figure 7.4E). As expected, wild type and GFP
66
transfected cells had minimal AP activity, but both EphB4 wild type and
mutant protein had very high activity. Thus, EphB4-eGFP protein
functions in a dominant negative fashion for forward signaling without
impacting reverse signaling.
7.4 The extracellular domain of EphB4 is sufficient to provide
resistance to TRAIL
We then sought to determine if EphB4 signaling was involved in
protection from TRAIL. We used the SCC4 cell line that lacks EphB4
expression and is partially sensitive to TRAIL. EphB4 wild-type or
EphB4-eGFP mutant protein expression vector (or an empty vector)
was co-transfected into this cell line with a truncated CD4 expression
plasmid, which allows for sorting of transfected cells using CD4
antibody-coated magnetic beads. TRAIL was added to sorted cells and
cell number studies showed that EphB4 expression was associated
with complete resistance to TRAIL, while cells transfected with empty
vector maintained TRAIL sensitivity similar to parent cells (Figure 7.5A).
Interestingly, mutant EphB4-eGFP was also capable of inhibiting
TRAIL-induced apoptosis indicating that EphB4 functions in a signaling-
independent manner to provide survival cues to cancer cells.
We then used the EphB4-eGFP mutant protein in colo-205 colon
cancer cells that by themselves express very low levels of EphB4 and
are very sensitive to TRAIL. Colo-205 cells were transfected with the
mutant protein and cultured overnight with varying amounts of TRAIL.
Figure 7.5 EphB4-extracellular domain
is sufficient to provide resistance
against TRAIL-induced apoptosis.
SCC4 cells were transfected with wild
type EphB4 or EphB4-eGFP
expression vector or null vector along
with a truncated CD4 receptor that was
used to sort transfected cells.
Transfected and sorted cells were
cultured in the presence of varying
doses of TRAIL and cell survival
assessed on day 3 (A). Colo-205 colon
cancer cells were transfected with
EphB4-eGFP expression vector.
Transfected cells were cultured in the
presence of different amounts of TRAIL
as shown (B). Number of GFP positive
cells relative to total number of cells
was evaluated 16h later.
A
B
As expected, a significant reduction in total cell number was seen with
increasing doses of TRAIL (Figure 7.5B). The transfection efficiency
quantified by number of GFP positive cells was about 10%. Cells
67
68
cultured in the absence of TRAIL continued to have a similar
percentage of GFP positive cells. However, with increasing doses of
TRAIL, the vast majority (over 90%) of surviving cells were GFP
positive. This again confirms that the extracellular domain of EphB4 is
capable of inducing resistance to TRAIL.
7.5 The extracellular domain of EphB4 provides resistance to
TRAIL in vivo
We then sought to determine of forced expression of EphB4 provides
resistance to TRAIL in vivo. Parent colo-205 cells and colo-205 cells
stably expressing the EphB4-eGFP mutant protein were implanted in
flanks of mice (Figure 7.6). Mice were treated with 5mg/kg TRAIL for
five days after implantation of tumor cells. At three weeks, mice bearing
parent cells and treated with TRAIL had 46% smaller tumors compared
to untreated mice bearing parent cells. Stable expression of EPhB4-
eGFP protein resulted in 93% increase in tumor sizes. This effect may
can result from the pro-angiogenic effect of overexpression of the extra-
cellular domain of EphB4 that is capable of engaging and stimulating
tumor vascular EphrinB2. To ascertain the effect of TRAIL on these
tumors, mice implanted with cells stably expressing EphB4-eGFP were
treated with TRAIL. TRAIL treatment resulted in only 13% smaller
tumors in this case confirming that overexpression of the extracellular
domain of EphB4 provides resistance to TRAIL in vivo as well.
Figure 7.6 Extracellular domain of EphB4 provides resistance to TRAIL in
vivo. 1X10
7
parent colo-205 colon cancer cells or cells stably transfected
with EphB4-eGFP expression vector were implanted in the flank of 10-12-
week old, male Balb/C athymic mice (n=6 per group) and tumor volume
measured as length X (width)
2
X 0.52. Mice were administered 5mg/kg
TRAIL intraperitoneally daily from day 1 through 5 after implantation.
Animals were sacrificed at three weeks
In sum, EphB4 provides survival advantage to tumor cells by interfering
with apoptotic pathways, predominantly the membrane-originating,
extrinsic pathway. In particular, the extracellular domain of EphB4 is
sufficient to inhibit TRAIL-induced apoptosis in a signaling-independent
fashion. The importance of death receptor participation in EphB4
regulated cell death was determined by the use of a caspase-8
inhibitor, which, as expected, blocked TRAIL-induced cell death, but
blocked cell death induced by knockdown of EphB4 as well.
69
70
CHAPTER VIII: Conclusions and future directions
The identification of novel molecular targets and understanding their
interaction with multiple pathways that influence outcome is required to
design rational therapies for cancer. Receptor tyrosine kinases are
attractive molecular targets well suited for diagnosis, prognostic
stratification and therapy in cancer. The early success with drugs like
trastuzumab that targets HER2/neu receptor (Slamon et al, 1989)
stands testimony to the proof of principle. Patient-targeted biological
therapy holds promise as the next wave in cancer therapy.
As part of this work, we demonstrate that EphB4 is a novel therapeutic
target in several epithelial cancers. EphB4 is frequently overexpressed
in epithelial cancers, although normal cells of the same lineage express
little to no protein. Such an event appears to be exclusive to the EphB4
protein and not to other members of the Eph family of receptors or their
cognate Ephrin ligands. Focused overexpression of EphB4 is achieved
under tight regulation by oncogenes and tumor suppressors. EphB4
provides survival advantage to tumor cells via attenuation of inherent
cell death pathways and by up-regulation of anti-apoptotic proteins.
EphB4 interrupts, in a signaling-independent fashion, TRAIL-mediated
cell death. This unique function makes EphB4 an exclusive, ideal and
novel target for cancer therapy.
71
That said, answers to several key questions remain and will help us
understand better the functions of this fascinating protein.
Understanding the repertoire of cancers overexpressing EphB4 and the
extent of expression will help zero in on candidates more likely to
respond to EphB4-targeted therapeutics. Promoter analyses to expand
on regulators of protein expression and the pathways involved will
identify molecules upstream of EphB4 that may also be potential
targets. Understanding the stage of cancer progression at which EphB4
is induced and how that alters cancer growth and advancement is
critical to define more clearly the biological significance of EphB4 in
cancer. Lastly, the unique capability of EphB4 to inhibit TRAIL needs
further dissection. Studies to identify domain-specific interaction, both
biochemical and biological, are likely to be informative and exciting, but
more importantly, allow for more rational design of therapeutics.
The identification of a focused overexpression of EphB4 and its unique
ability to interfere with TRAIL-mediated cancer cell death is a novel
area of scientific research. While adding depth and breadth to our
understanding of cancer biology, it brings a new and promising
dimension to our fight to conquer cancer.
72
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Abstract (if available)
Abstract
An increasing body of evidence suggests overexpression of Eph receptor tyrosine kinases in tumors. However, their function in tumor biology is unknown. This thesis proposal evaluates the expression and biological significance of EphB4 in eight different epithelial cancers. Consistently, membranous expression of EphB4 was observed at much higher levels in tumor tissue compared to adjacent normal tissue, with a trend towards increased expression in higher grade, stage and proliferative front of tumors. EphB4 expression was upregulated by oncogenes such as EGFR, HER-2/neu and Akt, and downregulated by tumor suppressors such as PTEN, p53 and APC. EphB4 provides direct survival signals to tumor cells both in vitro and in vivo by interrupting extrinsic apoptotic pathways. Expression of the extracellular domain of EphB4 is sufficient for tumor cells to overcome apoptotic signals from the TRAIL-death receptor pathway. These results confirm that targeted overexpression of EphB4 on epithelial tumor cells provides a survival advantage to tumors by interrupting death-receptor-mediated apoptosis.
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Targeting vessel maturation: an anti-angiogenesis based cancer therapy
Asset Metadata
Creator
Subramanyan, Ram Kumar
(author)
Core Title
Over-expression of EphB4 by cancer cells provides survival advantage by interrupting death receptor-mediated apoptotic pathways
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Pathobiology
Publication Date
10/09/2006
Defense Date
06/09/2006
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
angiogenesis,apoptosis,death receptor,EphB4,OAI-PMH Harvest,TRAIL
Language
English
Advisor
Gill, Parkash S. (
committee chair
), Epstein, Alan L. (
committee member
)
Creator Email
rsubrama@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m89
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UC1156397
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etd-Subramanyan-20061009 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-17746 (legacy record id),usctheses-m89 (legacy record id)
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etd-Subramanyan-20061009.pdf
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17746
Document Type
Dissertation
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Subramanyan, Ram Kumar
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Repository Name
Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
angiogenesis
apoptosis
death receptor
EphB4
TRAIL