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Cysteine (C)-X-C Receptor 4 undergoes
Transportin 1-Dependent Nuclear Localization
and remains functional at the Nucleus of Metastatic
Prostate Cancer Cells
Ayesha Shyamali Don-Salu-Hewage
Clark Atlanta University
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remains functional at the Nucleus of Metastatic Prostate Cancer Cells" (2013). ETD Collection for AUC Robert W. Woodruff Library.
Paper 747.
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ABSTRACT
BIOLOGICAL SCIENCES
AYESHA SHYAMALI DON-SALU-HEWAGE
MBBS, FACULTY OF MEDICINE
UNIVERSITY OF PERADENIYA
SRI LANKA 2007
CYSTEINE (CVX-C RECEPTOR 4 UNDERGOES TRANSPORTIN 1-DEPENDENT
NUCLEAR LOCALIZATION AND IS FUNCTIONAL AT THE NUCLEUS OF
METASTATIC PROSTATE CANCER CELLS
Committee Chair: Cimona V. Hinton, Ph.D.
Dissertation dated July 2013
The G-protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4)
plays an important role in prostate cancer metastasis. CXCR4 is regarded as a plasma
membrane receptor, that it transmits signals that support transformation, progression and
metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutic approaches
such as antagonists and monoclonal antibodies have focused on receptors the located at
the plasma membrane. An emerging concept for GPCRs is that they can localize to the
nucleus where they may retain function and mediate nuclear signaling. Herein, we
demonstrate that CXCR4 is highly expressed in high grade metastatic prostate cancer
tissues. Increased expression of CXCR4 is also detected in several prostate cancer cell
lines as compared to normal prostate epithelial cells. Our studies identify a nuclear pool
i
of CXCR4 and also define a mechanism for nuclear targeting of CXCR4.
A classical
nuclear localization sequence (cNLS), "RPRK", in CXCR4 can contribute to nuclear
localization. In addition, CXCR4 interacts with the nuclear transport receptor,
Transportin pi, to promote nuclear accumulation of CXCR4. Importantly, Gtti
immunoprecipitation and calcium mobilization studies indicate that nuclear CXCR4 is
functional and can participate in G-protein signaling revealing that the nuclear pool of
CXCR4 can retain function. Localization of functional CXCR4 to the nucleus may be a
mechanism by which prostate cancer cells evade treatment, thus contributing to increased
metastatic ability and poorer prognosis after tumors have been treated with therapy that
targets plasma membrane CXCR4. This study addresses the mechanism of nuclear
targeting for CXCR4 and demonstrates that CXCR4 can retain function within the
nucleus and provides important new information to illuminate what have previously been
primarily clinical observations of nuclear CXCR4.
CYSTEINE (C)-X-C RECEPTOR 4 (CXCR4) UNDERGOES TRANSPORTS 1-
DEPENDENT NUCLEAR LOCALIZATION AND REMAINS FUNCTIONAL AT
THE NUCLEUS OF METASTATIC PROSTATE CANCER CELLS
A DISSERTATION
SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
BY
AYESHA SHYAMALI DON-SALU-HEWAGE
DEPARTMENT OF BIOLOGICAL SCIENCES
ATLANTA, GEORGIA
JULY 2013
©2013
AYESHA SHYAMALI DON-SALU-HEWAGE
All Rights Reserved
ACKNOWLEDGMENTS
This research project would not have been possible without the help and support
of the kind people around me, to only some of whom it is possible to give particular
mention here. First of all, I would like to thank and express my deepest gratitude to my
advisor Dr. Cimona V. Hinton for her kindness, dedication, patience, invaluable support
and guidance during my matriculation at Clark Atlanta University, for which my mere
expression of thanks does not suffice. I would also like to thank my dissertation advisory
committee members: Drs. Anita Corbett, Jaideep Chaudhary, Joann B. Powell, and
Shafiq A. Khan for their time, effort, and valuable advice on both an academic and a
personal level. Special thanks to all the former and current members of Hinton's lab for
their support. I would also like to thank the financial, academic and technical support of
the Clark Atlanta University, the chair and the staff of the Department of Biological
Sciences and Center for Cancer Research and Therapeutic Development. Finally, and
most importantly, I would thankful to my beloved parents, husband, brother and in laws
for their endless love, support and great encouragement at all times throughout my
studies. This research was supported by the NIH/NCRR/RCMI2G12RR003062,
P201MD002285, F31CA153908, NIGMS/NIH (MBRS/RISE) 2R25GM060414 grants
and AAAS Women's International Research Collaboration (WIRC) for Minority Serving
Institutions (MSI), a National Science Foundation grant.
ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF FIGURES
vi
LIST OF TABLES
vii
LIST OF ABBREVIATIONS
viii
CHAPTER 1 INTRODUCTION
1
CHAPTER 2 LITERATURE REVIEW
6
2.1 The human prostate gland
6
2.2 Prostate cancer and metastasis
7
2.3 Chemokines and G-protein coupled receptors
10
2.4 Subcellular localization of GPCRs; against the conventional paradigm
12
2.5 Cysteine (C)-X-C receptor 4 (CXCR4)
13
2.6 The role of CXCR4 in prostate cancer
15
2.7 Subcellular localization of CXCR4
16
2.8InternalizationofCXCR4
16
2.9 Intracellular trafficking of GPCRs
17
2.10 Nuclear import of proteins and the role of nuclear localization signal
18
2.11 Functions of nuclear GPCRs
20
2.12 CXCR4 inhibitor AMD3100 and prostate cancer
21
2.13 Significance of nuclear localized CXCR4
25
CHAPTER 3 MATERIALS AND METHODS
3.1 Chemicals and reagents
27
27
iii
3.2 Cell lines and cell culture
28
3.3 Characterization of CXCR4 IgG2B (R&D systems) antibody
28
3.4 Immunohistochemistry (IHC)
29
3.5 Histomophometry measurement of staining intensity for CXCR4 in prostate
cancer tissues
30
3.6 Subcellular fractionation
31
3.7 Indirect immunocytochemistry (ICC) for CXCR4
32
3.8 Site-directed mutagenesis
32
3.9 Transient transfections
33
3.10 Expression of transportin pi (TRN1) (Karyopherin 02)
33
3.11 Immunoprecipitation
34
3.12 Short interfering RNA transfection
34
3.13 Immunoprecipitation of active Gai
34
3.14 Intranuclear calcium (Ca2+) mobilization
35
3.15 Statistical analysis
36
CHAPTER 4 RESULTS
37
4.1 CXCR4 is expressed in the nucleus of prostate tissues
37
4.2 CXCR4 is present in nuclear fractions of prostate cell lines
39
4.3 CXCR4 contains a functional classical nuclear localization sequence
39
4.4 CXCR4 demonstrated and interaction with Transportin 1
41
iv
4.5 Nuclear-associated CXCR4 is functional
42
CHAPTER 5 DISCUSSION
44
CHAPTER 6 CONCLUSION
50
APPENDIX
52
REFERENCES
69
LIST OF FIGURES
Figure
Page
1.
Schematic illustration of the anatomy of the prostate gland
2.
Schematic representation of orientation of G-protein coupled receptor in the cell
54
membrane
55
3.
Schematic illustration of CXCR4 receptor activation
56
4.
Signal transduction of CXCR4
57
5.
Immunohistochemical (IHC) staining of prostate tissues for CXCR4
58
6.
Characterization of CXCR4 IgG2B mouse monoclonal antibody
59
7.
Nuclear CXCR4 expression in prostate cancer cell lines
60
8.
Immunocytochemistry of prostate cancer cell lines for CXCR4
61
9.
Expression of GFP- CXCR4 and endogenous CXCR4 protein in PC3 cells
62
10. Localization analysis of wild type and mutated (R146A and ANLS) CXCR4
63
11. Localization analysis of wild type and mutated (R148A) CXCR4
64
12. Expression of wild type CXCR4 and mutated CXCR4
65
13. CXCR4 and TRN1 demonstrate an interaction
66
14. CXCR4 undergoes TRN1 dependent translocalization to the nucleus
67
15. Nuclear CXCR4 was functional at the nucleus
68
VI
LIST OF TABLES
Table
Page
1.
PSORT prediction of nuclear localization sequence (NLS) in CXCR4
2.
Multiple sequence alignment of the nuclear localization sequence (NLS) region in
CXCR4
52
53
VII
LIST OF ABBREVIATIONS
ANOVA
Analysis of Variance
BPH
Benign Prostatic Hyperplasia
BSA
Bovine Serum Albumin
cDNA
Complementary Deoxyribonucleic Acid
CRC
Colorectal Cancer
CXCR4
Cysteine (C)-X-C Receptor 4
DAPI
4'-6-Diamidino-2-Phenylindole
DNA
Deoxyribonucleic Acid
ERK
Extracellular signal-regulated kinase
FBS
Fetal Bovine Serum
GPCR
G-Protein Coupled Receptor
GDP
Guanosine Diphosphate
GTP
Guanosine Triphosphate
Jan
Janus Kinase
Karb2
Karyopherinb2
KSFM
Keratinocyte Serum Free Medium
MAPK
Mitogen-Activated Protein Kinase
mGluR5
Metabotropic Glutamate Receptor 5
viii
LIST OF ABBREVIATIONS
MMP
Matrix Metallo-Proteinases
mNLS
Monopartite Nuclear Localization Signal
NCBI
National Center for Bioinformatics
NIH
cNLS
National Institutions of Health
Classical Nuclear Localization Signal/Sequence
PBS
Phosphate Buffered Saline
PI3K
Phosphoinositide 3-Kinase
PIP3
Phosphatidylinositol (3, 4, 5)-Trisphosphate
PKA
Protein Kinase A
PKB
Protein Kinase B
PKC
Protein Kinase C
PVDF
Polyvinylidene Difluoride
RPMI
Roswell Park Memorial Institute
SDF1 a
SDS-PAGE
Stromal Cell-Derived Factor la
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
STAT
Signal Transducer and Activator of Transcription
TRN1
Transportin 1
VEGF
Vascular Endothelial Growth Factor
ix
CHAPTER 1
INTRODUCTION
Prostate cancer is the second leading cause of increased cancer incidence and
cancer-related deaths among men in the United States (1-2). Despite treatment, the high
mortality rates in prostate cancer are attributed to metastasis, which is the main obstacle
in prostate cancer treatment. Several molecules and mechanisms contribute to cancer cell
metastasis. For instance, chemoattractant cytokines (chemokines) enhance the metastatic
potential of prostate cancer by binding and activating a family of G-protein coupled
receptors (GPCRs) (3-6), that initiate signals to enhance cell adhesion, invasion and
movement, and subsequently, tumor survival at the new site of metastasis. GPCRs
constitute the largest family of transmembrane plasma membrane receptors (7). In
conventional GPCR signaling, receptors are localized to the plasma membrane and
influence the activity of plasma membrane-localized enzymes, ion channels, and/or
second messengers. Their activation by an appropriate ligand triggers signaling through
G-protein alpha (Ga) and/or beta-gamma (GPy) subunits (8), leading to context-dependent
outcomes, which may positively and/or negatively regulate the activity of effector
molecules in signaling cascades within the cell (9-10). Additionally, activated GPCRs
also trigger a series of molecular interactions that allow for feedback regulation of Gprotein coupling and receptor endocytosis to attenuate receptor signals (11-17). Muller et
1
2
al. (18) initially described the involvement of chemokine GPCR receptors in cancer
metastasis and Akashi et al. (19) reported that the chemokine GPCR, CXCR4, was highly
expressed in human malignant prostate cancer compared to normal prostate. Numerous
studies have documented the involvement of CXCR4 in key steps of prostate cancer
metastasis: (i) signaling (20-21); (ii) invasion and migration (22); and (iii) the
establishment of a vascular network (23). Hence, several therapies against cancer cell
metastasis have been designed to antagonize CXCR4-mediated signaling (24-25). In
conventional CXCR4 signaling, stromal cell-derived factor 1 alpha (SDFla), also known
as CXCL12, is the exclusive ligand for CXCR4 (26), which leads to activation of
multiple pathways involved in tumorigenesis: (i) G-protein coupled receptor (GPCR)
signaling; (ii) PI3K/AKT; (iii) MAPK; (iv) JAK/STAT; (v) Src kinase and (vi) HER2
(27-29).
Interestingly, GPCRs have been detected in subcellular organelles distinct from
its classical plasma membrane location (30). These organelles include the Golgi
apparatus (31), endoplasmic reticulum (32), the cytoskeleton (33) and the nucleus/nuclear
membrane (34). Hanyaloglu and von Zastrow (35) postulated that default recycling of
GPCRs by endosomes may contribute to enhanced re-delivery of GPCRs to the plasma
membrane, or to alternate organelles within the cell, without destroying their signaling
capacity. Nevertheless, these alternately-localized GPCR receptors reveal a new level of
complexity that may be important in modulating their function. An increasing number of
GPCRs have been observed within the nucleus or nuclear membrane, such as
lysophosphatidic acid receptors (36), metabotropic glutamate receptors (37), plateletactivating factor receptors (38), angiotensin 2 type I receptors (39), prostaglandin
3
receptors (40), endothelin receptors (41), gonadotropin releasing hormone type I receptor
(42) and yff-adrenergic receptors (43). Nuclear GPCRs have been suggested to regulate a
number of physiological processes, including cell proliferation, survival, inflammatory
responses, tumorgenesis, DNA synthesis and transcription (38, 44-49). Nuclear GPCRs
may be constitutively active, or activated by internal, newly synthesized ligands that are
bound for secretion (50). Subsequently, classical second messenger signaling pathways,
such as adenylyl cyclase-induced Protein Kinase A (PKA) activation (43),
phospholipase-induced release of intranuclear calcium, diacyglycerol-induced Protein
Kinase C (PKC) (41, 51), ERK1/2, p38 MAP Kinases and Protein Kinase B (PKB) (4849) have been shown to be activated by nuclear GPCRs.
Nuclear localization of proteins is dictated by nuclear import and export through
nuclear pore complexes (52). Small proteins (<30-50 kDa) can pass through the nuclear
pore by diffusion; however, most larger proteins require active transport to enter the
nucleus (53) and require assistance by transport proteins (54-56). Many proteins targeted
to the nucleus contain a classical nuclear localization signal (NLS) that is recognized by a
heterodimeric import receptor comprised of importin alpha and importin beta. Each
protein that localizes to the nucleus must possess a functional NLS or is required to bind
to cargo proteins which possess a NLS. Importin alpha recognizes the NLS in the cargo
protein while importin beta targets the import complex to the nuclear pore (57-58).
Importin beta is part of a larger family of transport receptors often termed
importins/exportins (59). Many of these receptors directly recognize cargo proteins and
target them directly to the nuclear pore (57).
In the case of this large family, the
targeting signals within the cargo proteins are often not well-defined (59).
While a putative NLS has been identified in CXCR4 (60), the function of this
nuclear targeting signal in CXCR4 has not been examined. A distinct importin-dependent
transport pathway has been implicated in the transport of C-C chemokine receptor type 2
(CCR2) (61). Favre et al. found that an engineered, HA-tagged CCR2 associated with a
member of the importin family of nuclear transport receptors, Transportinpi (TRNl) in a
CCR2-null cell line (61). An interaction of CCR2 with TRNl was required to detect
CCR2 in nuclear fractions, suggesting that CCR2 transported to the nucleus via TRNl
(61). TRNl has been implicated in GPCR internalization and desensitization (62).
Furthermore, TRNl serves as a receptor for NLS-null proteins in NLS-containing cargo
substrates (63), making it an essential protein for import through the nuclear pore
complex (64). Taken together, these studies suggest that both the classical nuclear import
machinery and TRNl are candidates that play a role in CXCR4 nuclear translation
(65). Nuclear CXCR4 protein expression has been observed in malignant hepatocellular,
colorectal, renal cell and nasopharyngeal carcinomas (66-69). These studies, however,
were reported as clinical observations, and failed to investigate the mechanisms of
CXCR4 localization or any biological function associated with the nuclear receptor.
These data are consistent with reports that have demonstrated functional GPCRs
associated with the nucleus (36, 70-71), and further contribute to ongoing cancer
therapeutic interventions against CXCR4.
Collectively, a functional nuclear CXCR4 may contribute to prostate cancer
relapse despite current antagonists and monoclonal antibodies against plasma membrane-
bound CXCR4 that may not be designed to cross the plasma membrane, which would be
required to antagonize active CXCR4 at the nucleus. Furthermore, identification of
transport pathways required for nuclear localization of CXCR4 may reveal additional
targets for therapeutic development to hinder prostate cancer metastasis and improve
patient survival. Therefore, we hypothesized that CXCR4 is translocalized to the nucleus
in a Transportinpi dependant manner and remains functional at the nucleus of metastatic
prostate cancer cells. The current studies were designed to test the following hypotheses:
1.
CXCR4 is associated with the nucleus of metastatic prostate cancer.
2.
Nuclear localization sequence "RPRK" and Transportinpi play a role in nuclear
translocalization of CXCR4.
3.
CXCR4 remains functional at the nucleus.
CHAPTER2
LITERATURE REVIEW
2.1 The human prostate gland
The prostate gland is an accessory sex gland located underneath the bladder and
wraps around the prostatic urethra (Appendix Figure 1) (72). It is composed of glands
and ducts arranged in a lobular fashion within a fibromuscular stroma. The glands and
ducts are composed of secretory cells, basal cells, neuroendocrine cells, stem cells, and
transit-amplifying cells. Moreover, the surrounding stroma is composed of fibroblasts,
myofibroblasts and extracellular matrix (73). The main function of the prostate gland is to
produce some of the substances that are found in normal semen, such as minerals and
sugar. In addition, it helps to control urination by pressing directly against the part of the
urethra that it surrounds. In a young man, the normal prostate gland is about the size of a
walnut (<30 g) (72).
During normal aging, however, the gland usually grows larger.
This hormone-related nonmalignant enlargement with aging is called benign prostatic
hyperplasia (BPH) and is very commonly associated with old age giving rise to storage
and voiding urinary symptoms such as urinary frequency, urgency, urgency incontinence,
nocturia, hesitancy, intermittency and dribbling, which often have a profound effect on
the quality of one's life (74). However, some of these urinary symptoms are similar to the
symptoms of a critical disease of the prostate, the prostate cancer (75).
6
2.2 Prostate cancer and metastasis
Prostate cancer is an uncontrollable growth of the cells of the prostate gland (76).
Importantly, it is the leading cause for increase cancer incidence and the second leading
cause of cancer-related deaths among men in the western population, including United
States (77). The incidence of and survival from prostate cancer differs between countries,
and among different ethnic groups within a country (2). In 2012, 241,740 new cases had
been diagnosed and 28,170 deaths had been estimated in the United States. These cancer
statistics are even more alarming among men of African descent who have the highest
incidence and mortality rates in the world. These findings were further strengthened by
several studies indicating that genetic predisposition plays a predominant role in the
development of prostate cancer (78-80).
While it is acknowledged that prostate cancer is a multifactorial disease, no
precise proven cause has been adduced. Some studies reported that the prostate cancer
development is associated with environmental factors and human behaviors such as
amount of dietary fibers consumed, consumption of animal fat, alcohol, history of
vasectomy, smoking, obesity, statin and non-steroid anti-inflammatory drug medication,
sexual activity and exposure to certain hormones, intake of vitamin D, E and minerals
such as Calcium, Selenium and Zinc (81-84). Interestingly, hormones such as androgens,
estrogens, insulin and IGF (insulin-like growth factor) lie on the borderline of
environmental and genetic factors and the levels of these hormones can vary according
to the individual genetic predisposition and external conditions such as hormone
substitution, obesity and comorbidities (85-86). Moreover, the conclusions of a large
number of studies on these causative risk factors as either protective or detrimental and
8
differ from one to another (87).
Prostate cancer displays a range of stages of the disease, from tumors of no
clinical impact to those that are aggressive and lethal with multiple metastases. Between
these two ends of the range are the locally advanced tumors with few metastases that
insidiously infiltrate pelvic structures causing significant morbidity in the way of urinary
urgency, incomplete voiding with risk for urinary obstruction, constipation, insomnia,
fatigue, obstructive lymphedema, sacral plexus neuropathy and pain. There are some
diagnostic tests such as Prostate-specific antigen blood test (PSA), prostate digital rectal
examination and prostate ultrasound and biopsy are available in clinical practice.
However, the incidence of prostate cancer has been rising continuously and the mortality
has remained roughly the same (88). Importantly, there are no straight forward diagnostic
methods available to identify low risk patient, therefore early detection and appropriate
treatments remain as key approaches (89). Currently, different types of treatments are
available for patients with prostate cancer. Some treatments have proven efficient in the
treatment of localized disease including radical prostatectomy, radiotherapy and hormone
therapy (androgen ablation or castration) and some are still being tested in clinical trials
for metastatic disease (90-92).
Despite numerous advances, the high mortality rate in prostate cancer is principally
due to the spread of malignant cells to many tissues other than the prostate, by a process
called metastasis. Metastasis is the main obstacle in cancer treatment and its prevention is
a key factor in cancer prognosis. Metastatic progression contributes to the majority of the
morbidity and mortality associated with prostate cancer. While only 4% of men with
prostate cancer have metastatic disease, the presence of metastases portends a poor
prognosis with a five-year survival rate of 30% (compared with 100% for loco-regional
disease) (93). Unfortunately, no treatment is currently available for the complete cure of
metastatic prostate cancer (94), which is clinically significant as cancer metastasis
correlates with the leading cause of cancer-related deaths (95). Approximately 80% of
cancer patients still die from tumor dissemination following treatment by surgery,
radiotherapy and chemical therapy (96). As a result, there is a growing interest in the
early detection and screening of men for prostate cancer, and for a greater understanding
of the mechanisms that lead to metastasis.
Metastasis is a complicated, multi-step process (97). In metastasis, malignant cells
must initially "escape" from the primary tumor, invade into the surrounding tissues and
enter into the vascular circulation. If these malignant cells able to survive in the blood
stream, they must arrest at a secondary target site, cross the vascular barrier, and migrate
into the extra vascular connective tissues. Subsequently, the tumor cells must proliferate
and thereby establish a secondary (metastatic) tumor, of which the tumor cells are the
same type of cancer cells as the primary tumor. These events involve numerous cell-cell
and cell-matrix adhesive interactions, which are mediated by cell surface adhesion
molecules (98). In the case of prostate cancer progression, cancer cells first spread to the
regional lymph nodes and then primarily to the bone. The long-observed tendency for
prostate cancer cells to metastasize into the bone is supported by clinical data and autopsy
studies (99). Moreover, skeletal metastases represent a major clinical problem for men
suffering from prostate cancer. Nearly 80% of men who die from this disease have
significant spread of the cancer to the bone (96, 100).
Numerous hypotheses have been put forth to explain the preference of
10
metastasizing prostate cancer to the bone. These include the anatomic configuration of
venous drainage system from the prostate to the spine, the presence of "leaky" sinusoids
in the bone marrow, the elaboration of "tumor attracting" chemotactic factors by bone
and other marrow cells, and the production of growth factors in the marrow that stimulate
proliferation and survival of seeded cells (101). All of these possibilities are likely to be
important determinants of metastasis of prostate cancer to bone (102). Yet, despite the
increasing understanding in each of these areas the molecular basis for how these tumors
preferentially 'home' to bone remains to be determined. The metastasis of circulating
prostate cancer cells is functionally similar to the homing behavior of hematopoietic cells
to the bone marrow where many molecules are known to regulate hematopoietic stem cell
homing, participating as both chemo-attractants and regulators of cell maturation (103).
This leads to a growing interest in elucidating the molecular mechanisms underlying
prostate cancer metastasis to the bone and in developing the therapeutic targets to prevent
prostate cancer metastasis.
2.3 Chemokines and G-protein coupled receptors
In recent studies, many chemoattractant cytokines have been reported to mediate
the events of tumor cell adhesion, migration and invasion during metastasis to specific
organs (104). Chemokines are a superfamily of chemoattracting, cytokine-like proteins
that bind to and activate a family of chemokine receptors. Over 50 chemokines have been
identified, and based on the arrangement of their first two of four-conserved cysteine
residues, the chemokine superfamily has been divided into four subfamilies: a (C-X-C), B
(C-C), y (C), and 5 (C-X-X-X-C) (3-6).
Chemokine receptors are seven transmembrane domain receptors coupled to G
11
proteins, hence designated as G-protein coupled receptors (GPCRs). These receptors
oriented in the plasma membrane with their "N terminus" and three extracellular loops to
the outside of the cell surface, while the three intracellular loops as well as the "C
terminus" in the cytoplasm (Appendix Figure 2). GPCRs constitute the largest family of
transmembrane plasma membrane receptors (7). Moreover, GPCRs account for
approximately 2% of receptor proteins in the human genome (105). Ligands directed at
GPCRs (primarily agonists and antagonists) represent the largest family of
pharmacological agents, accounting for nearly 30% of current clinical pharmaceutical
agents available (106). The binding of an agonist to a GPCR leads to a conformational
change of the receptor that promotes the exchange of guanosine diphosphate (GDP) for
guanosine triphosphate (GTP) on the G protein a-subunit and is presumed to allow the
dissociation of the G protein Ga- and Gp7 subunits (9, 107). Subsequently, the activated
Ga- and G^ subunits positively and/or negatively regulate the activity of effector
molecules in signaling cascades within the cell (9-10). Additionally, the activated GPCR
also sets a series of molecular interactions that allows for feedback regulation of G
protein coupling and receptor endocytosis (11-17). Interestingly, it has been reported that
GPCRs may potentially facilitate tumor dissemination at each of the key steps of
metastasis, including adherence of tumor cells to endothelium, extravasation from blood
vessels, metastatic colonization, angiogenesis, proliferation, and protection from the host
response via activation of key survival pathways, such as ERK/MAPK, PI3K and
JAK/STAT signaling (108-110).
2.4 Subcellular localization of GPCRs; against the conventional paradigm
The general principle concept regarding GPCRs is that these receptors are
12
localized primarily to the plasma membrane. Thus, the initial investigations into the
cellular physiology of GPCRs have focused on their role as plasma membrane-based
signaling components (30). Despite this, there is increasing evidence that GPCRs are also
present at cellular locations distinct from the plasma membrane, such as Golgi apparatus
(31), endoplasmic reticulum (32), cytoskeleton (33) and even at the nucleus (34).
Hanyaloglu et al. (35) explained that default recycling of GPCRs by endosomes may
have contributed to enhanced re-delivery of GPCRs to the plasma membrane, or to
alternate organelles within the cell, without destroying their signaling capacity.
Nevertheless, these alternately-localized GPCR receptors reveal a new level of
complexity that may be important in modulating their function.
Interestingly, GPCRs such as lysophosphatidic acid receptors, metabotropic
glutamate receptors, platelet-activating factor receptors, angiotensin 2 type I receptors,
prostaglandin receptors, endothelin receptors, gonadotropin releasing hormone type I
receptor (42) and /?-adrenergic receptors (36-41, 43) have been observed within the
nucleus or nuclear membrane. Moreover, nuclear GPCRs have been suggested to
regulate a number of physiological processes, including cell proliferation, survival,
inflammatory responses, tumorgenesis, DNA synthesis and transcription (38, 44-49).
Furthermore, nuclear GPCRs may be constitutively active, or activated by internal, newly
synthesized ligands that are formed for secretion (50). Subsequently, classical second
messenger signaling pathways, such as adenylyl cyclase-induced Protein Kinase A
(PKA) activation (43), phospholipase-induced release of intranuclear calcium,
diacyglycerol-induced Protein Kinase C (PKC) (41, 51), ERK1/2, p38 MAP Kinases and
Protein Kinase B (PKB) (48-49) have shown to be indeed activated by nuclear GPCRs.
13
2.5 Cysteine (Q-X-C receptor 4 (CXCR4)
The GPCR, Cysteine (C)-X-C receptor 4 (CXCR4) was first detected as a coreceptor for CD4+ T-cell infection in human immunodeficiency virus type 1 (111-112).
CXCR4 is broadly expressed in cells of both the immune and the central nervous systems
(113-114) and can mediate migration of resting leukocytes and hematopoietic progenitors
in response to its exclusive natural ligand, Stromal cell-derived factor la (SDFla) (115-
118). SDFla is a member of the CXC or a-chemokine subfamily and is the only known
ligand for the chemokine receptor CXCR4 (119-122). SDFla and CXCR4 are
constitutively expressed in a large number of tissues (122-126) and regulate embryonic
development (127-130). Furthermore, knockout studies revealed that these two proteins
are mandatory for various developmental processes, including chemotaxis or homing of
myeloid stem cells from the fetal liver to the bone marrow environment (128, 130).
Signal transduction by CXCR4 leads to activation of G proteins (Appendix Figure
3), phospholipase C and elevation of cytosolic free calcium (131). Stimulation of
chemokine receptors also results in activation of ERK1/2 and PI3-Kinase leading to
formation of Phosphatidylinositol (3,4, 5)-trisphosphate (PIP3) and activation of AKT
(Appendix Figure 4). In contrast to other chemokine receptors, stimulation of CXCR4
can lead to prolonged activation of these two signaling pathways (132). The involvement
of CXCR4 and of its ligand SDFla in this process makes this chemokine- receptor pair of
particular interest in investigating its role in tumor growth and metastasis. Several studies
have indicated that CXCR4 plays a significant role in neoplastic development and is
expressed in several malignant gliomas, breast tumors, certain leukemia cell lines,
endometrial cancer, Burkitt's lymphoma, neuroblastomas, multiple myeloma, pancreatic
14
cancer tissue and cell lines as well as in prostate cancer (133-139). In 2001, Muller et al.
(134) provided the first evidence related to the metastatic involvement of CXCR4/SDFla
pathway in a cancer model. Notably, it was shown that higher expression of SDFla was
found in tissues, which are preferential sites for the metastasis of breast cancer (134).
Subsequent studies have shown SDFla was found in the ascitic fluid of patients with
ovarian cancer, suggesting a possible role in the spread of cancer cells to the peritoneum
(140-141). Therefore, it is apparent that one of the most intriguing and perhaps an
important role of CXCR4 is that regulating metastasis. CXCR4 receptors may potentially
facilitate tumor dissemination at each of the key steps of metastasis, including adherence
of tumor cells to endothelium, extravasation from blood vessels, metastatic colonization,
angiogenesis, proliferation, and protection from the host response via activation of key
survival pathways such as ERK1/2, AKT or JAK/STAT, etc. (108-110). Collectively,
these studies emphasize the importance of further studies on CXCR4/SDFla pathway in
developement of therapies against cancer metastasis.
2.6 The role of CXCR4 in prostate cancer
The role of CXCR4 in prostate cancer metastasis was first observed by Taichman
et al. (142) who demonstrated that CXCR4 was expressed in prostate cancer cell lines
and biopsies, and facilitated prostate cancer metastasis to the bone (142-143). Other
studies have revealed that CXCR4 was highly expressed in human malignant prostate
tissues and SDFla enhanced prostate cancer cell adhesion to bone endothelial cells, their
trans-endothelial migration and invasion (144-145). In the bone microenvironment,
SDFla was constitutively expressed by osetoblasts, fibroblasts, and endothelial cells,
15
thus providing an attractive and favorable environment for CXCR4-expressing prostate
cancer cells. The metastatic events mediated by CXCR4 in prostate cancer are also
correlated with activation of various signaling pathways, such as the PI3K/AKT, MAPK
and JAK/STAT pathways (146). For instance, in prostate cancer, CXCR4 preferentially
activated the PI3K/AKT pathway; however, emerging studies have suggested that the
MAPK pathway was preferred to facilitate metastasis (146). Moreover, Milliken et al.
and Geminder et al. showed that activation of AKT by CXCR4 facilitated invasion by
increasing expression of matrix metalloproteinases (MMP) enzymes 1 and 13, which
degraded the extracellular matrix (147), and rearranged cytoskeletal proteins to form
lamellipodia and pseudopodia that were used for movement (148). Furthermore,
increased expression of <x5 and 03 integrins lead to increased adhesion of prostate cancer
cells to endothelial cells in a CXCR4-dependent manner (149). Collectively, considering
the role of CXCR4 in cell survival and movement, these studies have validated that
CXCR4 receptor is a logical therapeutic target for treatment of prostate cancer metastasis.
2.7 Subcellular localization of CXCR4
CXCR4 is classically a plasma membrane receptor. The nuclear location of
CXCR4 has been observed both in tumor cells (68, 150-151) and non-tumor cells (66).
Moreover, previous studies have identified a nucleus associated CXCR4 in hepatocellular
carcinoma (68), invasive ductal mammary carcinoma (150) and non-small-cell lung
cancer (151). Kato et al. described a CXCR4 expression pattern as diffuse, or focal
expression, in the nucleus of breast cancer, and also observed a significant correlation
between the rate of lymph node metastases and a nuclear presence of CXCR4 in these
tumors (150). Furthermore, colorectal cancer (CRC) cells with nuclear CXCR4
16
expression significantly metastasized to the lymph node than did those with primarily
cell membrane expression (152), which leading to the conclusion that expression of
nuclear CXCR4 predicted lymph node metastasis in CRCs. These studies, however, were
reported as clinical observations, and failed to further investigate nuclear CXCR4, its
mechanism of localization or any biological relevance associated with the receptor.
2.8 Internalization of CXCR4
The current knowledge about the orientation of CXCR4 in the cell to be
stimulated by its ligand, SDFla and become a functional receptor is that it has to be
located on plasma membrane (153). In un-stimulated cells CXCR4 is found both on the
cell surface and internally in the cytoplasm, presumably recycling to the cell surface
through endosomes (154-155). Thus, besides modulating the levels of receptors at the cell
surface, the cellular endocytic pathway plays a major role in the attenuation of ligand-
induced receptor-mediated signaling on the cell surface (156). GPCR-mediated signaling
is attenuated within minutes after ligand binding. CXCR4, like most GPCRs, is
desensitized rapidly through the action of GPCR kinases (GRKs), which phosphorylate
the receptor on several C terminal Ser/Thr residues (157). Moreover, upon SDFla
binding, CXCR4 is rapidly phosphorylated by GRKs and internalized via clathrin-coated
pits (157). It has also been shown that, phosphorylation of serines and threonines in the
COOH-terminal region are also involved in receptor internalization. Furthermore, this
process of phosphorylation of the COOH-terminal domain is essential for arrestin-
mediated uptake of seven-transmembrane domain receptors via clathrin-coated pits (158159). Interestingly, although CXCR4 is associated with heterotrimeric G-Proteins, Amara
et al. showed that internalization of CXCR4 is independent of G-protein signaling
17
because pertussis toxin, which is capable of inhibiting Gai subunits, does not inhibit the
internalization process (154).
2.9 Intracellular trafficking of GPCRs
Trafficking of GPCRs into different cellular organelles, upon stimulation with
their respective ligands, is far less studied than their endocytic trafficking, and much
work is needed to better understand their targeting to specific compartments. In most
studies, specific interacting proteins have been identified which reduced intracellular
trafficking as well as those which enhance the process such as proteins which are coexpressed with the receptors (160). Moreover, a number of GPCR mutations have also
been shown to be responsible for receptor trapping in the endoplasmic reticulum and
rerouting toward the degradation compartment (161). Interestingly, proteins which
translocalized/trafficked from the plasma membrane to the nucleus must contain, or be
bound to proteins that contain, at least one nuclear-localization signal/sequence (NLS)
(52) to allow transport towards the nucleus. Proteins lacking these recognition signals
remain cytoplasmic or may be directed to other cellular compartments (162).
2.10 Nuclear import of proteins and the role of nuclear localization signal.
Nuclear localization of a protein is determined by nuclear import and nuclear
export molecular trafficking across the nuclear membrane or nuclear envelope through
nuclear pore complexes that are composed of nucleoporins (52). In addition, it is reported
that the steady-state localization of the protein on either side of the nuclear envelope
depends on its interaction with the retention factor(s) in the cytoplasm or the nucleus to
which the protein gets attached (54). Up to date, two established mechanisms, diffusion
and active transport, facilitate the import of proteins into the nucleus. Small proteins
18
(<40-60 kDa) are capable of passing through the nuclear pore by simple diffusion, while
larger proteins use active transport mechanisms, which require assistance by transport
machinery (54-56, 163). Proteins that localize to the nucleus must possess a functional
NLS or are required to bind to cargo proteins which possess a NLS(s) (65).
NLS is a stretch of positively charged amino acids (lysines or arginines) which
label a protein for import into the nucleus. NLSs are thus highly basic. Additionally, the
NLSs are divided into two types as classical and non-classical. The classical type NLSs
consists of monopartite NLS (mNLS), containing a single cluster of 4-6
Lysine(K)/Arginine (R) amino acid residues. Bipartite NLSs, contain two clusters of
basic amino acid residues separated by 10-12 amino acids (164). Majority of non-
classical type of NLSs consist of a single 20-40 long stretch of non-basic amino acids
(165-167). Some non-classical type NLSs do not follow this rule as they consist of a
single short stretch of one or more basic amino acids, but yet distinct from the mNLS
(168-170). The NLS(s) function as a bridge which connects the protein to be translocated
and the carrier proteins of the translocation machinery. Additionally, through the NLS(s),
the cargo protein binds to the nuclear import receptor proteins called importins (importinpi in most cases and importin-a/pi complex in other cases) (52). Importin-pi then binds
to the nucleoporins of the nuclear pore complexes that subsequently translocate the cargo
protein to the nucleus. In the nucleus, importin-pi binds to RanGTP and this binding
leads to the release of the cargo protein. The high level of RanGTP in the nucleus is
maintained by its guanine nucleotide exchange factor RCC1 (52). Proteins without a
conventional NLS, but with the nucleoporin binding motif like that of importin-pi, may
directly bind the nucleoporins of the nuclear pore complexes and thus be imported to the
19
nucleus in an importin-independent manner (52). The functionality of NLSs was usually
verified by site-directed mutagenesis of the NLS and subsequent observations of the
ability of the protein to be localized to the nucleus (52, 162, 171).
Among the proteins which are responsible for nuclear cytoplasmic shuttling of
proteins, Transportin 1 (TRN1) is one of the protein which have been shown to be
involved with GPCRs (172). TRN1 is the protein product of the TNP01 gene and also
known as M9-interacting protein or karyopherin P2 (Karb2), which is an 890-amino acid
long protein (173). Karyopherin 0s (Karbs) are also known as importins and exportins. In
humans, ten Karyopherin Ps have been shown to carry diverse macromolecular substrates
into the nucleus and one of these Karyopherin import pathways has been known to be
responsible for the import of a significant group of RNA processing proteins (174-176).
Moreover, binding of a substrate to import and export Karyopherin Ps is dependent on the
NLS and nuclear export signal. During nuclear import of proteins, the Karyopherin p-
protein cargo complex translocates into the nucleus through its association with the
nuclear pore complex. Once inside the nucleus the Karyopherin P-protein complex
dissociates, which is dependent on RanGTP binding to the import Karyopherins. The free
Karyopherin Ps are then recycled into the cytoplasm to be available for a new cycle of
cargo import (177). Interestingly, TRN1 also serves as a receptor for NLS-null proteins in
NLS-containing cargo substrates (63), making it an essential protein for import through
the nuclear pore complex (64). While GPCRs at the nucleus is an emerging concept, the
mechanism(s) of their transport in human disease has not been reported. Interestingly,
HA-tagged chemokine receptor, CCR2, an engineered chemokine receptor, associated
with TRN1 in a CCR2-null cell line, HEK 293 (human embryonic kidney cells); the
20
knocking down of TRN1 excluded HA-tagged CCR2 from nuclear fractions (172).
Additionally, TRN1 is involved in GPCR desensitization (62), making it an ideal
candidate to study its involvement in CXCR4 translocation to alternate, internal
organelles (65). However, whether CXCR4 interacts with the TRN1 and uses the same
mechanism to translocate to the nucleus is not known.
2.11 Functions of nuclear GPCRs
The most important, but less focused aspect of nuclear GPCRs is their functional
status at the nucleus. It has generally been assumed that the signal transduction cascades
are initiated at the plasma membrane and not at the nuclear membranes. Recent studies
have shown that the nuclear envelope plays a major role in signal transduction cascades
(178). Several other plasma membrane receptors (both GPCRs and non-GPCRs), also
been observed alternatively in the nucleus and nuclear membrane, have shown to
maintain the functional integrity of the receptor (70, 179-180). The plasma membrane
GPCR, mGluR5 (Metabotropic Glutamate Receptor 5) has also been observed at the
nuclear membrane and has been shown to induce signaling cascades that is known to alter
gene transcription and regulate many paradigms of synaptic plasticity in primary cultured
striatal neurons (70). Moreover, vascular endothelial growth factor receptorl (VEGFR1),
a tyrosine kinase receptor, was found at the nuclear membrane of breast cancer cells and
is protected them from apoptosis (179). Additionally, some GPCRs have shown to initiate
signaling cascades that are quite distinct from those activated by the same receptors
resident at the cell surface (181). One of the important functions found with nuclear
GPCR is the increased intra-nuclear calcium (Ca2+) levels (36, 44, 182). Intra-nuclear
Ca2+ is derived from either cytoplasmic Ca2+ shuttling to the nucleus (183), nuclear
21
lumen (184) and nucleoplasmic reticulum (185). It has been shown that the amplitude and
the duration of this increased Ca2+affects gene transcription (186). At the plasma
membrane, CXCR4 participates in GPCR signaling, which plays a significant role in
tumor cell survival. Upon ligand activation, CXCR4 uncouples with its G-proteins, and
subsequently results in Ca2+ release from intracellular reservoirs (187). However, nuclear
localized CXCR4 is capable of mobilizing Ca2+is not known.
2.12 CXCR4 inhibitor AMD3100 and prostate cancer
A considerable amount of effort has been put forth into the development of
CXCR4 antagonists over the years for HIV infection, and now for cancer metastasis
(188). Chemokine receptor antagonists, the largest group of inhibitors, impede the
binding of natural chemokine ligand(s) to their cognate chemokine receptors (189). This
group of antagonists include neutralizing antibodies, such as MAB171 and 12G5 antiCXCR4 (190), and small-molecule inhibitors (191), such as cyclams (bi- and mono-) and
noncyclams inhibitors (AMD11070, CTCE-9908, RCP168 and FC131).
Cyclams are a group of organic, aromatic or nonaromatic macrocyclic ring-
structured compounds capable of binding to metal cations, which are made up of more
than one kind of atoms, such as carbon and nitrogen atoms. They are divided into two
groups, monocyclams and bicyclams, based on the number of macrocyclic ring structures
in the compound (192). Among the cyclams, some bicyclam compounds have been
identified as specific small-molecule antagonists to target CXCR4. The compound
AMD3100, also known as Plerixafor, is the first nonpeptide, small-molecule bicyclam
that is a highly specific CXCR4 antagonist that acts by competing against SDFlct (193).
AMD3100 consists of two monocyclam (1,4, 8,11-tetraazacyclotetradecane) units
22
connected by an aliphatic or aromatic linker. AMD3100 has reduced oral bioavailability
and is categorized as a therapeutic agent. It has to be administered by intraveneous
infusion, thus prevailing a severe limitation to its clinical applicability as a long-term
therapeutic agent (189). It was originally identified as an anti-HIV agent and is the most
potent and specific X4-HIV strain entry inhibitor of the bicyclams that have been
described to date (194-197). The target of action was initially thought to be the viral
envelope glycoprotein gpl20; however, the direct target of action was shown to be
towards the co-receptor CXCR4, which was used by T lymphotropic HIV strains to enter
the cells (198). The supportive evidence from clinical studies have demonstrated that
CXCR4 blockade by AMD3100 in HIV-infected patients selectively inhibited X4-HIV
strain, but not R5-HIV strains, by inhibiting HIV cell entry by binding to and inhibiting
the function of SDF1 (199). The initial (phase I) clinical trial undertaken with AMD3100
has been shown to increase white blood cell counts, which was an unexpected side effect
in HIV patients (200). It was determined that AMD3100 is a competitive antagonist of
SDF1 binding, with partial agonist properties (200-201). The poor oral bioavailability
and significant increment in white blood cell counts with repeated dosages limited its
clinical use in HIV therapy (202). AMD3100 has been approved for clinical use in
patients with leukemia, since it plays a significant role in stem cell mobilization in
leukemia patients (203-204).
The significance of antitumor activity of AMD3100, both in vitro and in vivo, was
also evidenced by the ability of AMD3100 to inhibit infiltration of acute lymphoblastic
leukemia and ovarian cancer cells (25, 205). Several studies have evaluated the role of
AMD3100 in combination with other therapies in certain cancer types. For example, the
23
combined therapy of AMD3100 and local irradiation has been shown to induce a
significant tumor growth delay and increased curability in brain, lung, and breast tumors
(24, 206). In addition, orthotopic U87 gliomas have shown a growth inhibition after
chemotherapy (BCNU) with AMD3100, however, there had been no effect when they
have been given as a single therapy (207). Thus, these studies emphasize the importance
of understanding the role of CXCR4/ SDFla pathway in different tumors in pre-clinincal
studies to design and optimize inhibitors which could act in combination with other
therapies in cancer patients. There are few studies available to indicate the usage of
AMD3100 in prostate cancer. In 2008, Zhang et al. first reported the effects of AMD3100
on CXCR4 inhibition of perineural invasion in prostate cancer cells using in vitro
experiments, animal models and human prostate cancer tissues (208). The study reported
that SDFla induced matrix metalloproteinases in prostate cancer cells, which eventually
led to perineural invasion, resulting in infiltration and metastasis by degrading the matrix
around the tumor and the nerve tissue. The study confirmed that AMD3100 was capable
of inhibiting this invasion by prostate cancer cells by blocking the effects of SDFla on
CXCR4, and eventually the downstream signal transduction pathways (208). AMD3100,
inhibits CXCR4 and does not interact with any of the other chemokine receptors, CXCRl
through CXCR3, or CCR1 through CCR9 (197). There is evidence to suggest that
AMD3100 may be a weak agonist. However, AMD3100 has been shown not to cause
CXCR4-mediated calcium flux, G-protein activation, and chemotaxis (209).
Apart from invasion, studies have shown that AMD3100 antagonized migration
of prostate cancer cells (20). Frigo et al. described that AMD3100 completely inhibits
migration of LNCaP cells(a prostate cancer cell line) toward SDFla (210). AMD3100
24
inhibitory effect on prostate cancer cell migration via CXCR4/SDF1 axis has been
supported by another study in 2010, which has been conducted using a population of cells
commonly referred to as adipose tissue-derived stromal or stem cells (ADSC) which are
recruited towards the prostate cancer cells due to CXCR4/SDFlcc signaling (211). These
cells participate in the expansion of pre-existing vasculature and/or formation of new
blood vessels in the tumor site. These newly formed blood vessels then feed the growth
of cancer cells (212-213). Migration of CXCR4 expressing ADSC cells (211, 214)
towards PC3-conditioned medium that contains SDF1 was dose-dependently inhibited by
AMD3100 and thus further emphasized the importance of AMD3100 in inhibition of
prostate cancer growth and metastasis. There are supportive evidences to indicate the role
of AMD3100 as an inhibitor of CXCR4/SDFla pathway, not only in prostate cancer
cells, but also in prostate cancer progenitor cells that are CD133+ and CD44+. Previous
studies have shown that CD133+ and CD44+ cells are putative stem cell populations in
the prostate gland (215-219), which have self-renewing and multipotent abilities with
strong tumorigenic potential in vivo (215, 217, 220). Moreover, the mRNA expression
analysis revealed that the chemokine receptor CXCR4 was highly upregulated and the
activated CXCR4/SDFla axis in these progenitor cells played a role in self-renewal,
differentiation, cell adhesion, and tumorigenicity. The mouse xenograft studies revealed
that the inhibition of CXCR4 by AMD3100 is beneficial in targeting of prostate cancer
progenitors in vivo, suggesting that inhibition of CXCR4/SDFla pathway in prostate
cancer progenitors could lead to more effective cancer treatment and may provide
synergistic antitumor activity with conventional therapy. The study observed that
inhibition of CXCR4 with AMD3100 could specifically inhibit proliferation of prostate
25
progenitor population in PC3 and DU145 prostate cancer cell lines in vitro and in vivo
and may provide a new approach to deplete prostate cancer stem cells (221).
2.13 Significance of nuclear localized CXCR4
Nucleus associated CXCR4 protein expression has been observed as an incidental
finding in previous studies which failed to investigate CXCR4 at the nucleus, its
mechanism of localization or any biological relevance associated with the receptor (66-
69). Consistent with other reports that have demonstrated functional GPCRs associated
with the nucleus, a functional CXCR4 at the nucleus will further contribute to ongoing
cancer therapeutic interventions against CXCR4. Importantly, a functional nuclear
CXCR4 may contribute to prostate cancer relapse despite current antagonists against
plasma membrane-bound CXCR4 (179), as current therapeutics are intended for the
plasma membrane receptor and may not be designed to cross the plasma membrane to
antagonize CXCR4 that may be localized and active at the nucleus. Additionally,
identification of a transport molecule associated with CXCR4, such as TRN1, is equally
important as it provides another target for therapeutic development to hinder metastasis
and improve cancer patient survival.
CHAPTER 3
MATERIALS AND METHODS
3.1 Chemicals and reagents
Cell culture supplies and kanamycin sulfate (61-176-RG) were from MediaTech.
SDFla (3 00-28A) was from PeproTech. The following reagents and human antibodies
were from Cell Signaling: 10X cell lysis buffer (9803); mouse anti-rabbit IgG (5127);
anti-CD44 (156-3C11); anti-GFP (2956S); and anti-Gai (5290). Anti-CXCR4 (MAB172)
was from R&D Systems. Anti-Topoisomerasel (SC-271285), anti-Lamin A/C (SC20681), Fusin (H-l 18)-CXCR4 (SC-9046), Fusin (4G10)-CXCR4 (SC- 53534), antiFibronectin IgG2B (SC-271098), anti-GFP (SC-9996), Protein A/G Plus-Agarose beads
(SC-2003), anti-Karyopherin p2/ Transportin pi (SC-166127), Karyopherin p2 siRNA
(h) (SC-35737) and DAPI (SC-3598) were from Santa Cruz Biotech. NE-PER Nuclear
and Cytoplasmic Extraction Kit (78833), Protease Inhibitor Cocktail Kit (78410) and Halt
TM Phosphate Inhibitor Cocktail (78420) were from Thermo Scientific. Anti-aTubulin
(T5168), Anti-pActin (A5441) Triton X-100 (T8532) and propidium iodide (P4170) were
from Sigma-Aldrich. Prostate disease spectrum tissue array (PR8011) was purchased
from Biomax. JetPRIME® Polypus transfection reagent (114-07) was from VWR
International. Nonidet P-40 Substitute (Ml 58) was from BioExpress and FluoForte
Calcium Assay Kit (ENZ-51016) was from Enzo Life Sciences.
26
27
3.2 Cell lines and cell culture
PC3, DU145,22RV1 human prostate cancer cell lines, RWPE1 human prostate
cell line and 293T human embryonic kidney cell line were obtained from American Type
Culture Collection (ATCC). PC3, DU145, 22RV1 and 293T cells were maintained in
complete RPMI media: RPMI1640 containing 10% fetal bovine serum (FBS), 1% nonessential amino acids and 1% antibiotic-antimycotic at 37°C in 5% CO2. The RWPE1
cells were maintained in keratinocyte serum free medium (KSFM) containing 50 mg/ml
gentamycin, 0.05 mg/ml bovine pituitary extract (BPE), and 5 ng/ml Epidermal growth
factor (Invitrogen) at 37°C in 5% CO2. All cells were maintained at 60% to 80%
confluency.
PC3 cells were originally isolated from a prostate vertebral metastasis (222-223),
while DU145 cells were obtained from prostate brain metastasis (223-224). 22RV1 cells
were from a human prostate carcinoma epithelial cell line derived from a xenograft that
was serially propagated in mice (223, 225), and the RWPE1 normal human prostate
epithelium cells were isolated from a white male donor (223, 226).
3.3 Characterization of CXCR4 IgG2B (R&D systems) antibody
The specificity of the anti-human CXCR4 mouse monoclonal antibody (R&D
Systems) for the CXCR4 protein was determined by immunoprecipitation and western
blot analysis using CXCR4-positive PC3 and CXCR4-null HEK 293T whole cell lysates.
Briefly, PC3 and 293T cells (5xlO6) were grown on 100 mm dishes in complete media
overnight, followed by incubation in RPMI only (serum-starvation) for 24 hrs. Cell were
washed with IX phosphate-buffered saline (PBS) and harvested in IX lysis buffer. Equal
protein concentrations were estimated by Bradford assay (BioRad) and equal amounts of
28
PC3 and HEK 293T cell lysates were assessed for western blot analysis with CXCR4IgG2B mouse monoclonal antibody. Beta-actin was used as a loading control.
Subsequently, 1 mg of supernatant was immunoprecipitated (IP) with CXCR4-IgG2B
mouse monoclonal antibody or Fibronectin-IgG2B mouse monoclonal antibody overnight
at 4°C (Santa Cruz; 1 ug per 250 ug of protein), followed by incubation with Protein A/G
Plus-Agarose beads for 2 hrs at 4°C. Protein-bound agarose beads were separated from
lysates by a series of 3 washes with IX PBS and centrifugation (max speed/2 min/room
temperature (RT). Proteins in Laemmli buffer were separated by 10% SDS-PAGE,
transferred to polyvinylidenefluoride (PVDF) membranes and probed for CXCR4-IgG2B
(1:1000). To confirm that PC3 cells expressed Fibronectin, 25 \ig of whole cell lysate
was harvested for western blot analysis.
3.4 Immunohistochemistry (IHC)
IHC analysis was performed on a prostate disease spectrum tissue array (Biomax)
ranging from normal to high grade metastatic tissues. The array consisted of 80 total
tissue cores including adenocarcinoma, metastatic, hyperplasia, chronic inflammation,
adjacent normal tissue and normal tissue. Each individual core had a diameter of 1.5 mm
and a thickness of 0.5 um. Briefly, formalin-fixed, paraffin-embedded specimens were
retrieved in washes of xylene, ethanol, and antigen retrieval solution, pH 6.0, (Biocare
Medical) at 125°C for 30 sec. Specimens were neutralized in 0.3% hydrogen peroxide for
15 min at room temperature (RT), washed with IX PBS in a humidified chamber and
blocked with blocking solution (5% normal goat serum/Tris-buffered saline/Tween-20;
TBST) for 30 min. CXCR4 was detected with a mouse anti-human CXCR4 monoclonal
antibody (R&D Systems; 1:1000) in blocking solution overnight at 4°C, followed by a
29
biotinylated affinity purified goat anti-mouse IgG (H+L) secondary antibody (Vector
.Laboratories;!: 1000), in blocking solution for 30 min at RT. Specimens were washed
thoroughly between incubations, developed in diaminobenzidine (Vector Laboratories)
for 3 min at RT, and counterstained the nuclei with Meyer's hematoxylin for 1-3 min.
Specimens were rinsed in deionized water for 5 min and subjected to dehydration in
graded up series of ethanol followed by incubation in xylene, prior to mounting on slides.
A negative control tissue sample was prepared by incubating in biotinylated affinity
purified goat anti-mouse IgG (H+L) antibody, only, as described above. The specimens
were analyzed and photographed by Dr. Dezhi Wang (227) at the Center for Metabolic
Bone Disease Core Laboratory, UAB School of Medicine, Birmingham, Alabama. The
distribution of positive cells for CXCR4 was recorded to portray the diffuse or focal
nature of the positive cells as sporadic (positive cells <5%); focal (positive cells >11%
but less than 50%); or diffuse (positive cells >50%) according to the average density of
positive cells for CXCR4 (DAB stained), to see the obvious difference in strength of
CXCR4 expression.
3.5 Histomophometry measurement of staining intensity for CXCR4 in prostate
cancer tissues
The average density of positive cells (DAB stained) was measured by using
Bioquant® Image Analysis Software (RtmBometrics) and an Olympus BX51 Microscope
with a Q-Imaging camera. The software analyzed an average group of pixels, and
returned a data value based on the color value of the pixels in stained samples. Three
random fields of prostate tissues were selected at a magnification of (400X) for each
section based on the size of the tissue. In each random area, those cells (a group of
30
pixels) that were stained positively (brown) with the CXCR4 antibody were selected by
the thresholding tool of the software. The specimen light source is known to affect
density measurement; therefore, all sections were measured utilizing the same
background correction supplied by Bioquant® Image Analysis Software.
3.6 Subcellular fractionation
Prostate cancer and normal prostate epithelial cells (lxlO6) were serum-starved
for 3 hrs (22RV1 and RWPE1) or 24 hrs (PC3 and DU145), prior to treating with SDFla
(100 ng/ul) for 30 min. Subcellular fractionations were performed per the manufacturer's
instructions (Thermo Scientific). Briefly, cells were lysed in a series of buffers and
centrifugation steps to obtain a non-nuclear fraction and an intact nuclear pellet, followed
by further lysing to isolate nuclear proteins. Forty to one hundred micrograms of nuclear
and non-nuclear fractions were separated by SDS-PAGE electrophoresis and transferred
to PVDF membranes. Expression of CXCR4 or GFP-CXCR4 fusion protein was detected
with anti-human CXCR4 antibody (R&D Systems; 1:1000) or a mouse monoclonal GFP
antibody (Santa Cruz; 1:500). Anti-topoisomerase I (Santa Cruz; 1:1000) and anti-CD44
(Cell Signaling; 1:1000) antibodies were used to ensure the integrity of fractions and as
loading controls. X-ray films were scanned and Quantity One software program was used
for densitometry analysis.
3.7 Indirect immunocytochemistry (ICC) for CXCR4
22RV1, DU145 and PC3 cells (3xl05) were plated on glass coverslips (Fisher),
serum-starved as described, prior to treatments with SDFla (100 ng/\il). Cells were fixed
with ice-cold 100% methanol for 5 min at -20°C and washed with IX PBS. Non-specific
proteins were blocked in blocking solution (3% normal donkey serum/1% BSA/0.1%
31
Triton X-100 in IX PBS) for 30 min at RT, prior to incubating with CXCR4 (R&D
Systems, 1:100), Lamin A/C (Santa Cruz, 1:100), or GFP mouse monoclonal antibody
(Santa Cruz, 1:100) in blocking solution at 4°C overnight. Secondary detection was with
Cy3-conjugated donkey anti-mouse IgG or FITC conjugated anti-rabbit IgG (Jackson
Immuno Research, 1:1000) in blocking solution at RT for lhr, followed by three washes
in IX PBS. In some cases, nuclei were stained with propidium iodide (1 ug/ul) or DAPI
(1:250) in IX PBS prior to mounting in Aqua-Polymount (Polyscience, Inc). Images were
taken at Georgia Institute of Technology, Atlanta, GA with a 63x Plan-Apochromat
63x/1.40 Oil DIC objective on a Zeiss LSM-510 UV Confocal Microscope at excitation
488nm for FITC and 543nm for Cy3 or at Clark Atlanta University, Atlanta, GA with
Axiovision software 4.8.2 on a Zeiss Axio Imager.zl fluorescence microscope at 40X
magnification at excitation 470nm for FITC, 358nm for DAPI and 551nm for Cy3.
3.8 Site-directed mutagenesis
The R146A and R148A amino acid substitutions within the putative cNLS and
deletion of the cNLS were created in a plasmid encoding GFP-CXCR4 using the Quik
Change XL Site-Directed Mutagenesis Kit (Stratagene). The plasmid; pEGFPNl-CXCR4
served as the template (60). The forward and reverse primers of R146A, R148A and the
deleted NLS were (Integrated DNA Technologies): (i) R146A: FWD5'-
CACGCCACCAACAGTCAGGCACCAAGGAAGCTGTTGGCTG-3', REV 5'CAGCCAACAGCTTCCTTGGTGCCTGACTGTTGGTGGCGTG-3'; (ii) R148A:
FWD5^-CCAACAGTCAGAGGCCAGCGAAGCTGTTGGCTGAAA-3\ REV 5XTTTCAGCCAACAGCTTCGCTGGCCTCTGACTGTTGG-3*; and (iii) NLS deletion:
FWD5'-CGCCACCAACAGTCAGCTGTTGGCTGAAAAGG-3' and REV5'-
32
CCTTTTCAGCCAACAGCTGACTGTTGGTGGCG-3'.
The resultant plasmids were pEGFPNl-CXCR4RJ46A, pEGFPNl-
CXCR4R148A and pEGFPNl-CXCR4ANLS. Positive CXCR4 mutant clones were
selected with kanamycin and further purified by maxi-prep (Omega Bio-tek). The
presence of desired mutations and absence of any additional mutations were confirmed
by DNA sequencing on an ABI3130x1 Gene Analyzer Sequencer at Morehouse School
of Medicine, Atlanta, GA.
3.9 Transient transfections
Transient transfections were performed with 2 ug of concentrated DNA and
jetPRIME® Polypus transfection, per the manufacturers' instructions. Briefly, PC3 cells
were incubated with jetPRIME®-DNA complexes in 15% FBS/RPMI for 4 hrs and the
media was replaced with 15% FBS in RPMI for an additional 18 hrs, prior to serum-
starvation (24 hrs). Cells were then harvested for respective experiments.
3.10 Expression of transports pi (TRN1) (Karyopherin p2)
Serum-starved PC3 cells (5xlO6) were treated with SDFla for 30 min prior to
harvesting 60 ug of whole cell lysates for western blot analysis. Expression of
Transportin pi was detected with a mouse monoclonal antibody (Santa Cruz; 1:1000); aTubulin or (3-Actin was used as a loading control.
3.11 Immunoprecipitation
One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4
(Santa Cruz; 1 ug per 250 ug of protein) overnight at 4°C, followed by incubation with
Protein A/G Plus-Agarose beads (Santa Cruz) for 2hrs at 4°C. CXCR4-bound agarose
beads were separated from lysate by a series of 3 washes with PBS and centrifugation at
33
maximum speed for 1 min at 4°C. Proteins were processed for western blot analysis for
TRN1 (Santa Cruz; 1:1000) and subsequently reprobed for CXCR4 with rabbit antiCXCR.4 (Santa Cruz, 1:500) antibody followed by incubation with mouse anti-rabbit IgG
(Cell Signaling) secondary antibody. Thirty micrograms of the supernatant obtained after
incubation with agarose beads were also separated by 10% SDS-PAGE, and processed
for western blot analysis for CXCR4 as described in characterization of CXCR4
antibody.
3.12 Short interfering RNA transfection
Transient transfection of Transportin pi specific siRNA (Santa Cruz) was
performed on PC3 cells plated on glass coverslips using JetPRIME®. Briefly, cells
(2*105) were plated in 35 mm, 6 well dishes and transfected with 50 nM Transportin plsiRNA (Santa Cruz) in 15% FBS/RPMI media at 37°C in 5% CO2 for 24 hrs. Transfected
cells were serum-starved for 24 hrs, prior to immunocytochemistry analysis.
3.13 Immunoprecipitation of active Gai
Serum-starved PC3 cells (5xlO6) were treated with SDFla for 30 min prior to
harvesting for immunoprecipitation. Briefly, cells were washed in IX PBS and gently
scraped inNP-40 lysis buffer (IX PBS pH 7.4, 0.1% Triton X 100, 0.1% NP40 and IX
cocktail inhibitor). After 30 min incubation on ice, the lysate was centrifuged at 600 rcf/5
min /4°C). The supernatant was gently decanted, and the nuclear pellet was resuspended
in lysis buffer, 10 times the volume of the nuclei pellet, and sonicated on ice for 3 sec.
The lysate was centrifuged at 600 rcf/5 min/4°C, and 1 mg of supernatant was
immunoprecipitated for CXCR4 (mouse monoclonal, Santa Cruz) overnight at 4°C,
followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at
34
4°C. CXCR4-bound agarose beads were separated from lysate by a series of 3 washes
with NP40 lysis buffer and centrifugation (5000 rpm/2 min/RT). The final wash was with
IX PBS. Beads were processed for western blot analysis and membranes were probed for
Gai (Cell Signaling; 1:1000). Subsequently, the blots were reprobed for CXCR4 with
rabbit anti-CXCR4 (Santa Cruz, 1:500) antibody followed by incubation with mouse antirabbit IgG (Cell Signaling) secondary antibody. Topoisomerasel (Santa Cruz) and antiCD44 (Cell Signaling) were used to assess the purity of nuclear lysates.
3.14 Intranuclear calcium (Ca2+) mobilization
Serum-starved PC3 cells (2.5x105) were harvested to obtain intact nuclei in NP-40
lysis buffer as described above, prior to performing assay per the manufacturer's
instructions (Enzo Life Sciences). Briefly, untreated isolated nuclei were resuspended in
100 ul of FluoForte dye-loading solution (Enzo Life Sciences) for 45 min at 37°C and 15
min at RT, then centrifuged at 600 rcf/5 min/RT. Solutions of AMD3100 (100 ng/ul) and
pertussis toxin (PTX) (200 ng/ml) were prepared in calcium free, phenol free RPMI.
Nuclear samples were resuspended in 100 ul of AMD3100 and PTX, aliquoted into
black-walled, clear bottom 96well plates, and incubated for 1 hr. Next, the SDFla was
added to samples in plates, (final dilution 100 ng/ul) and incubated for 30 min at RT.
Intranuclear calcium mobilization was determined by the intensity (increase) of
fluorescent (FluoForte)-bound Ca2+ in the media. Results were measured on a microplate
reader at excitation at 490 nm and emission at 525 nm. Each sample was prepared in
triplicate per experiment, and performed at least three times.
3.15 Statistical analysis
Where applicable, data were analyzed by a paired student's t-test or ANOVA
35
using GraphPad Prism (GraphPad) software. P values less than 0.05 were considered
significant.
CHAPTER 4
RESULTS
4.1 CXCR4 is expressed in the nucleus of prostate tissues
Previous domain analysis of CXCR4 suggested that CXCR4 contains a nuclear
targeting signal between amino acids 90-170 (228). A bioinformatics analysis using
PSORTIINLS prediction software (http://psort.ims.u-tokyo.ac.jp/) revealed a putative
nuclear localization sequence, "RPRK" (60, 229-231) between amino acids 146-149
within CXCR4 (Appendix Table 1). In addition, a HomoloGene/NCBI database search
for the cNLS within CXCR4 revealed that "RPRK" motif is conserved among species,
including chicken, mouse, chimpanzee and others (Appendix Table 2). Moreover,
CXCR4 has been detected in the nucleus of several cancer tissues (67, 69). Based on
these data, we tested whether CXCR4 protein could be detected within the nucleus of
prostate tissues (Appendix Figure 5). Using a prostate tissue microarray ranging from
normal to high-grade metastatic lesions, we detected positive immunoreactivity for
CXCR4 in prostate samples. Positive immunoreactivity was detected as sporadic
(CXCR4 positive cells <5%), focal (CXCR4 positive cells >11%, but less than 50%), or
diffuse (CXCR4 positive cells >50%), compared to the average total density of positive
cells for CXCR4 (DAB stained). Samples with immunohistochemical scores of negative,
weak or moderate staining, with sporadic to focal distributions, were considered to have
36
37
'low' expression, whereas diffuse distributions of staining were considered to have 'high'
expression for CXCR4. Staining intensity was sporadic to focal in the nucleus of low
grade prostate tissues (See Appendix Figure 5), while high grade malignant tissues
(Appendix Figure 5), displayed an increased staining intensity and diffuse expression of
CXCR4 throughout the tissue compared to low grade. In both low and high grade tumors,
a fraction of CXCR4 clearly co-localized with the nucleus. Staining intensity for CXCR4
was weak, or even null, in normal tissues (See Appendix Figure 5).
We have reported that PC3 cells were positive and HEK 293T cells were null,
respectively, for CXCR4 protein (20). To ensure the specificity of CXCR4 monoclonal
antibody (MAB172IgG2b) used to detect its corresponding protein in the nucleus of
prostate tissues, we re-analyzed PC3 and HEK 293T cells for CXCR4 with CXCR4
antibody (MAB172IgG2b) by western blot analysis (Appendix Figure 6A). Similar to
our previous studies, CXCR4 was detected in PC3 but not in 293T whole cell lysates
(Appendix Figure 6A).
Subsequent analysis of cell lysates by immunoprecipitation with MAB172 IgG2b
followed by western blot analysis with MAB172 IgG2b detected CXCR4 only in PC3
cell lysates (Appendix Figure 6B). To further confirm the specificity of MAB172IgG2b
to CXCR4, PC3 cell lysates were subjected to immunoprecipitation with Fibronectin
IgG2b, an isotype control, followed by western blot analysis with MAB172 IgG2b
(Appendix Figure 6C). Fibronectin was not detected by IP with CXCR4, but was detected
by western blot with a Fibronectin antibody (Appendix Figure 6C).
38
4.2 CXCR4 is present in nuclear fractions of prostate cell lines
We used biochemical fractionation to confirm the nuclear localization of CXCR4
detected in our tissue staining. Prostate cancer cell lines were fractionated into nuclear
and non-nuclear samples for detection of CXCR4 by western blot analysis (See Appendix
Figure 7). We found that normal prostate epithelial cells (RWPE1) were null for CXCR4
(232); however, three CXCR4-expressing prostate cancer cell lines (22RV1, DU145 and
PC3) dually expressed CXCR4 in both nuclear and non-nuclear fractions independent of
SDFla stimulation (Appendix Figure 7). The purity of subcellular fractions was
confirmed by expression of CD44, a non-nuclear marker (233), and topoisomerase 1, a
nuclear marker (234), which ruled out the possibility that expression of CXCR4 observed
in nuclear fractions was due to contamination.
We further confirmed CXCR4 in nuclei of prostate cancer cell lines by indirect
immunohistochemistry (ICC) (Appendix Figure 8). In all three cell lines, we found
CXCR4 to be ubiquitously expressed throughout the cells, or as distinct foci around the
nucleus, at the plasma membrane and in the cytoplasm independent of SDFla treatment.
Collectively, these observations suggest that CXCR4 is expressed at the plasma
membrane, but also associated with the nucleus of prostate cancer cells.
4.3 CXCR4 contains a functional classical nuclear localization sequence
Nuclear-localized plasma membrane receptors must contain a classical nuclear
localization sequence (cNLS) to permit transport to and/or into the nucleus (235). To
determine whether the PSORT II-predicted NLS, «146rprk149" was functional and
contributed to nuclear expression of CXCR4, we evaluated the intracellular distribution
of wildtype GFP-tagged CXCR4 (pEGFPNl-CXCR4, GFP-CXCR4 fusion protein), two
39
mutated fusion proteins in which Arginine 146 and 148 were separately mutated to an
Alanine (CXCR4R146A and CXCR4R148A. respectively), as well as a fusion protein
where the cNLS was deleted (CXCR4ANLS).
Plasmids encoding GFP-CXCR4 were
transfected into PC3 cells and examined by immunocytochemistry (Appendix Figure 9).
The localization pattern of GFP-CXCR4 at the plasma membrane and in the cytoplasm
was consistent with endogeneous CXCR4 (Appendix Figure 9). Previous studies have
reported an expression pattern for GFP-CXCR4 similar to our observation in other cancer
cell lines (236-237). Wild-type GFP-tagged CXCR4 was localized predominantly at the
plasma membrane, with some localization at the nucleus in untreated cells (Appendix
Figure 10). However, an increase in punctate staining was observed at the
nucleus/nuclear membrane upon treatment with SDFla. Interestingly, both
CXCR4R146A and CXCR4R148A (Appendix Figure 10 and 11 respectively) were
detectable at the nucleus, suggesting that neither R146A nor R148A mutations in the
NLS were sufficient to inhibit CXCR4 localization to the nucleus. To further examine the
requirement of this cNLS to localize CXCR4 to the nucleus, we deleted the NLS,
"146RPRK149", within pEGFPN 1-CXCR4 (CXCR4ANLS). We detected CXCR4ANLS
at the plasma mebrane and diffusely throughout the cytosol, similar to wild-type GFPCXCR4, but we did not detect CXCR4ANLS at the nucleus (Appendix Figure 10). To
further confirm that CXCR4ANLS was excluded from the nucleus, PC3 cells were
transiently transfected with wildtype GFP-CXCR4 or CXCR4ANLS then fractionated
into nuclear and non-nuclear samples for analysis by western blot analysis (Appendix
Figure 12). Consistent with immunocytochemistry observations, we found that wild type
GFP-CXCR4 and CXCR4ANLS were both detectable in non-nuclear fractions, while
40
only GFP-CXCR4 was detected in nuclear fractions.
Collectively, these data suggest
that the "RPRK" motif may be involved in localization of CXCR4 to the nucleus in
prostate cancer.
4.4 CXCR4 demonstrated and interaction with Transportin 1
We identified a putative cNLS motif that could be critical for CXCR4 nuclear
localization; however, the motif "RPRK" is not a typical classical NLS (58). In fact, such
sequences can also mediate direct binding to other transport receptors. Among the
different molecules that are involved in the transport of various cargos to the nucleus,
members of the karyopherin beta (P) family contribute directly or indirectly to the nuclear
shuttling of molecules (238). Transportin pi (TRNl), also known as Karyopherin p2, is a
transport molecule of the importin-P family that has been linked to desensitization (62)
and nuclear-cytoplasmic shuttling of receptors (172, 239-243). To test whether TRNl
was involved in CXCR4 transport to the nucleus, we first established that PC3 cells
expressed TRNl by western blot analysis (Appendix Figure 13A); HEK 293T cells
served as a positive control for TRNl expression. Next, we tested for an interaction
between CXCR4 and TRNl. We immunoprecipitated CXCR4 from whole cell lysates
and tested for co-purification of TRNl by western blot analysis (Appendix Figure 13B).
To test whether the association between CXCR4 and TRNl led to CXCR4 translocation
to the nucleus, we decreased TRNl protein expression by Transportin pi-siRNA, then
determined CXCR4 localization by ICC. We first confirmed an effective Transportin pisiRNA mediated depletion of TRNl by western blot analysis (Appendix Figure 14A). As
previously described in control cells, CXCR4 localized to the plasma membrane, in the
cytoplasm and in distinct foci around the nucleus. When TRNl expression was
41
diminished, CXCR4 was undetectable around the nucleus, even in the presence of SDFla
(Appendix Figure 14B).
4.5 Nuclear-associated CXCR4 is functional
Finally, we sought to determine whether CXCR4 receptors at the nucleus are
functional. Upon ligand stimulation and activation, GPCRs, including CXCR4, dissociate
from a trimer of G-proteins (Ga and GpY), and initiate secondary signaling pathways, such
as increased cyclic AMP, increased intracellular calcium levels, and others (244). Thus, a
reduction in the G-alpha protein, Gai, associated with CXCR4 represents an active state of
a receptor. Therefore, we co-immunoprecipitated CXCR4 and Gai and determined Ga;
expression levels by Western blot analysis, to assess whether nuclear-associated CXCR4
was active. Whole cells were stimulated with SDFla then harvested to isolate intact
nuclei (Appendix Figure 15A). Nuclei were lysed, then immunoprecipitated with antiCXCR4, prior to immunobloting to assess associated Gaj. In untreated cells, we observed
a basal level of Gai expression, which decreased upon treatment with SDFla, suggesting
that nuclear-associated CXCR4 is functional and can respond to SDFla (Appendix
Figure 15B).
We further tested the functionality of nuclear-associated CXCR4 by assessing
whether the receptor stimulated release of intra-nuclear Ca2+. Isolated intact PC3 cells
nuclei were incubated with Ca2+-binding fluorescent probe (FluoForte® dye), then
incubated with CXCR4 antagonist, AMD3100, or Gai/Gao inhibitor pertussis toxin (PTX),
separately for one hour, followed by treating with SDFla. Calcium mobilization was
determined by an increase in fluorescent probe in the media, and measured at ex=490
nm/em=525 nm. We observed a significant increase in intra-nuclear Ca2+ release from
42
nuclei stimulated with SDFla, compared to untreated samples (Appendix Figure 15C).
Furthermore, AMD3100 antagonized CXCR4 function, which resulted in decreased Ca2+
levels, to a level even lower than SDFla-treated samples. PTX prevents G-proteins from
interacting with GPCRs, thus interfering with intracellular communication. As such, we
did not observe an increase in Ca2+-mobilization in PTX-treated nuclei, even in the
presence of SDFla, supporting that the intra-nuclear Ca2+ surge was evoked by CXCR4,
yet sensitive to PTX inhibition. Our results are in agreement with earlier studies that
observed functional GPCRs associated with the nucleus, and that these nuclear GPCRs
mobilized Ca2+ (36, 182, 245).
CHAPTER 5
DISCUSSION
This study established that CXCR4 receptor protein is expressed in prostate
cancer cells and is associated with the nuclei in these cells. Additionally, we provide
insight into a specific nuclear protein import mechanism that contributes to nuclear
localization of CXCR4. Importantly, CXCR4 responded to its ligand, SDFla, at the
nucleus. Nuclear localization of CXCR4 may be a mechanism which prostate cancer
cells employ to survive, even after the insult of chemotherapy. Therefore, antagonizing
nuclear transport pathways and/or the action of nuclear CXCR4, could provide a rational
approach to prevention and management of prostate cancer.
Prostate cancer mortality is often a result of metastasis to secondary organs.
CXCR4 is involved in the metastatic spread of primary tumor cells through activation of
requisite pathways, which signal to downstream targets for invasion and movement
through the vasculature, the establishment of a blood supply at the new tumor site and
inhibition of immunosurveillance mechanisms that will destroy the new tumor (21).
Taichman et al. initially observed that CXCR4 facilitated prostate cancer metastasis to
the bone, the primary site of distal prostate cancer colonization (22). SDFla was
constitutively expressed in the bone marrow by osetoblasts, fibroblasts, and endothelial
cells, which directed cell migration, by attracting prostate cancer cells that expressed
43
44
CXCR4 on the plasma membrane. To date, CXCR4, like all GPCRs, is regarded as a
plasma membrane receptor. However, an emerging concept is that GPCRs are
translocated to the nucleus and other intracellular organelles, possibly after
internalization (246). Wright et al. reported that endogenous al-adrenergic receptors (alARs) localized to and signaled at the nuclei in adult cardiac myocytes (247), and in 2012,
the group described that al-AR nuclear localization drove the formation of receptor
oligomers and regulated signaling in adult cardiac myocytes (248), suggesting that
GPCRs at the nucleus exhibited the same behavior as their plasma membrane
counterparts.
The mode of CXCR4 intracellular localization to the nucleus, and subsequent
functions, are not well understood. The generally accepted view of CXCR4 localization
and signaling centers upon a plasma membrane-localized receptor that is activated by an
extracellular ligand, SDFla. This ligand then initiates intracellular signals through G-
proteins that support favorable responses for tumor development. Following activation
and subsequent signaling, the known concept of events is that CXCR4 is rapidly
internalized from the plasma membrane to attenuate signaling communication, and then
recycled back to the plasma membrane as an inactivated receptor, or sorted to the
lysosome for degradation; both processes are important for receptor signal termination
(249-251).
Consistent with our findings reported here, GPCRs that have been detected at
the nucleus were reported to have originated from the plasma membrane (36, 43, 70,
252).
The results of this study support an alternately-localized and functional CXCR4
receptor. As assessed by decreased Ga; expression and Ca2+ mobilization in the presence
45
of SDFla, CXCR4 can participate in signaling at the nucleus. Nuclear CXCR4 was
initially observed in diverse tumor tissues (68-69, 150-151, 253) and correlated with
significant predictors for poor overall malignant survival (253), lymphovascular invasion
(254) and lymph node metastasis (152). Our findings extended these observations
further, as providing evidence that: (i) CXCR4 is present at the nucleus of prostate tumor
tissues and cell lines; (ii) nuclear CXCR4 contains a functional cNLS which excluded
CXCR4 from the nucleus when deleted; and (iii) CXCR4 associates with TRN1 and
depletion of TRN1 decreased localization of CXCR4 to the nucleus.
To date, considerable evidence supports the presence of GPCRs at the plasma
membrane, or within the perinuclear/nuclear compartments of cells, following ligand
activation (36, 70, 255-256). It is well known that CXCR4 is highly expressed in
malignant prostate cancer cells (257) and is involved in metastasis (20-21, 23, 142);
however, few reports have observed a subcellular localization for CXCR4 other than the
plasma membrane and endosomes in tumor tissues. We observed positive staining for
CXCR4 in the nucleus of prostate cancer tissues; furthermore, the amount of nuclei
positively stained for CXCR4 increased with the grade of the tumor. In prostate cancer
cell lines, unexpectedly, we detected CXCR4 in nuclear fractions of untreated cells;
nuclear CXCR4 expression increased with SDFla stimulation. Sun et al. demonstrated
that prostate cancer cells expressed SDFla mRNA and secreted a biologically active
protein (143). Perhaps the nuclear expression of CXCR4 in untreated cells may result
from autocrine signaling (258-259). Endogenous SDFla ligand secreted by prostate
cancer cells may act upon plasma membrane-localized CXCR4 in an autocrine manner,
resulting in receptor internalization and subsequent nuclear targeting of CXCR4. Our
46
results do not support a correlation between increased nuclear CXCR4 expression and
prostate cancer metastasis, or the clinical relevance of nuclear CXCR4 expression in
predicting prostate cancer prognosis/ survival. However, Woo et al. predicted that high
expression of nuclear CXCR4 in hormone receptor negative breast cancer was associated
with the high possibility of lymph node metastasis (260). The origin and functional
relevance of nuclear CXCR4, and the precise mechanisms involved in its nuclear
translocation, have yet to be established. Wang et al. presumed the presence of a non-
traditional, nuclear localization sequence, "RPRK", within CXCR4, using C-terminal
deletant plasmid constructs to reveal expression of CXCR4 at the nucleus (228). Nuclear
localization sequences are a stretch of positively charged, highly basic amino acids
(lysines or arginines) (261) and are divided into two types: (i) the classical monopartite
type containing a single cluster of 4-6 lysine(K)/arginine (R) amino acid residues and the
(ii) the bipartite type contains two clusters of basic amino acid residues separated by 1012 amino acids (164). The majority of non-classical types of NLSs consist of a single 2040 long stretch of non-basic amino acids (165-167). Some non-classical NLSs do not
follow these rules, as they consist of a single short stretch of one or more basic amino
acids that are distinct from the monopartite NLS (65, 162, 168-170), such as the motif
"RPRK". Ren et al. reported that mammalian target of rapamycin (mTOR) contained
"RPRK", and upon its deletion, onion cells lost nuclear expression of mTOR (230).
Transportinpi studies on plasma membrane GPCRs have reported that they are
functional and able to initiate signaling at the nucleus (36, 43, 70, 182). Of particular
interest, the importins P, to which TRNl belongs, were involved in nuclear localization of
GPCRs, such as angiotensin 1, fibroblast growth factor receptors and CCR2 chemokine
47
receptor (56, 172, 242, 262-264). The known concept of GPCR signaling is that signal
transduction cascades are initiated at the plasma membrane, but not at the nuclear
membrane. However, studies have demonstrated that the nuclear envelope plays a major
role in signaling cascades. For instance, GPCR-associated heterotrimeric G proteins
(265), and downstream signaling molecules, such as adenylate cyclase (266),
phospholipase C (178) and phospholipase D (265), have been localized at the nucleus.
Moreover, nuclear membranes have been shown to possess signaling molecules such as
1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate (178), further confirming the
role that nuclear membranes play in signal transduction. We observed that nuclear
CXCR4 was associated with Gaj in untreated samples; Gai dissociated from CXCR4 upon
SDFla stimulation, compared to untreated samples, further confirming that nuclear
CXCR4 is functional. Most studies that have reported functional nuclear GPCRs
observed an increase in intra-nuclear Ca2+ levels upon stimulation with an appropriate
agonist (36, 44, 182), emphasizing an importance of nuclear GPCRs to cells, since
nuclear Ca2+ plays pivotal roles in nuclear functions (cell division, proliferation, protein
import, apoptosis, and gene transcription) (267). Among the various reports where
nuclear calcium was mobilized from organelles, such as the cytoplasm (183), nuclear
lumen (184) and nucleoplasmic reticulum (185), the latter two have been suggested to
increase/enhance signals initiated in the cytoplasm, and/or generate their own transient
Ca + signals (184, 268). Additionally, the amplitude and duration of calcium signals have
also been shown to differentially control activation of transcription factors (186). For
instance, transcription factors, such as NF-kB and c-Jun are activated by transient
increases in Ca2+(186). Taken together, data herein, and by our colleagues, emphasize a
48
secondary GPCR signaling network at the nucleus, which may ensure, or enhance, the
communication and coordination of numerous biochemical signals that are critical in
regulating tumorigenic paradigms within the cell.
CHAPTER 6
CONCLUSION
CXCR4 is regarded as a plasma membrane receptor, where it transmits signals
that promote metastasis, and consequently, therapeutics have been designed to inhibit
CXCR4 signaling at the plasma membrane. Herein, the data presented provide the first
clear evidence that CXCR4 located at the nucleus of advanced metastatic prostate cancer
cells can function as a ligand-responsive receptor. Furthermore, this study also provide
evidence for the involvement of the nuclear localization sequence "RPRK" within
CXCR4 and the transport receptor Transportin pi in translocating CXCR4 to the nucleus.
Lee et al. (269) described a unique survival system in breast cancer cells by which VEGF
acted as an intracrine survival factor through its binding to nuclear VEGFR1. A similar
survival system is present in tumor cells that express CXCR4 at the nucleus, and secrete
SDFla from the same cell in an autocrine fashion. This study could have significance
therapeutic development; if the CXCR4 receptor can initiate signaling from inside the
cell, the receptor may evade chemotherapeutic agents that are designed to antagonize the
plasma membrane receptor and/or cannot pass through the hydrophobic regions of the
plasma membrane to reach nuclear CXCR4 receptors. Therefore, antagonizing the action
of nuclear CXCR4 could provide a rational complementary approach to the prevention
and management of prostate cancer metastasis. A nuclear form of CXCR4 may be a
49
50
possible mechanism that prostate cancer cells employ to survive. Most chemotherapeutic
molecules are designed to target the extracellular region of plasma membrane-bound
CXCR4 and are not designed to pass through non-polar regions of the plasma membrane
to reach internal, functional receptors. Therefore, these studies on CXCR4 translocation
to the nucleus may contribute to the current knowledge of prostate cancer cell migration
and survival which may improve therapeutics that prevent and manage metastasis.
APPENDIX
Table 1. PSORT prediction of nuclear localization sequence (NLS) in CXCR4.
Gene
Nuclear localization
Amino acid sequence
Position
sequence
CXCR4
RPRK
Cellular
orientation
Arg-Pro-Arg-Lys
146-149
Cytoplasmic
PSORT is a NLS prediction server to determine the NLS scores of amino acid residues of
a protein. It receives amino acid sequence information of a source, e.g., human CXCR4,
as inputs and subsequently analyzes the input sequence by applying stored rules for
various sequence features of known protein sorting signals. Finally, it reports possible
sequences for the input protein to be localized at each candidate site with additional
information. The human CXCR4 sequence (NCBI assession number NPOO 1008540)
was searched by the server, which provided a NLS score for each of the 356 residues
comprising CXCR4. http://psort.hgc.jp/
51
52
APPENDIX
Table 2. Multiple sequence alignment of the nuclear localization sequence (NLS)
region in CXCR4.
Species
Multiple sequence alignment
Human
130-
(NV 003458.1)
ISLDRYLAIVHATNSORPRKLLAEKVVYVGVWIPALL
LTIPDFIFANV- 177
Mouse
132-
(NP 034041.2)
ISLDRYLAIVHATNSORPRKLLAEKAVYVGVWIPALL
LTIPDFIFADVSQ-181
Norway rat
127-
(NP 071541.2)
ISLDRYLAIVHATNSQRPRKLLAEKAVYVGVWIPALL
LTIPDIIFADV-174
Dog
131-
(NP 001041491.1)
ISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALL
LTIPDFIFANV--178
Chicken
140-
(NP 989948.2)
ISLDRYLAIVHATNSQRPRKLLAEKIVYVGVWLPAVL
LTVPDIIFAST--187
Chimpanzee
130-
fNP 001009047.1)
ISLDRYLAIVHATNSORPRKLLAEKVVYVGVWIPALL
LTIPDFIFANV--177
Sequences of input organisms are compared using HomoloGene database (NCBI) and
subsequently matched into groups using a taxonomic tree built from sequence similarity;
highly related organisms are matched up first.
53
APPENDIX
Figure 1
.- central /t»ir
transition
p:u»titx
sphincl?r
Adult human
(sagittal section)
Figure 1. Schematic illustration of the anatomy of the prostate gland. Schematic
illustration of the anatomy of the prostate gland. The base of the prostate is located at the
bladder neck. The urethra bisects the prostate and consists of three zones, namely central
zone, transition zone and peripheral zone (270).
54
APPENDIX
Figure 2
Extracellular
Cell membrane
loops
COOH
Intracellular
Terminus
G-Protein»
loops
Figure 2. Schematic representation of orientation of G-protein coupled receptor in
the cell membrane. Chemokine receptors are seven transmembrane domain receptors
coupled to G proteins (Ga, Gp and Gy), hence designated as G-protein coupled receptors
(GPCRs). These receptors oriented in the plasma membrane with their "N terminus" and
three extracellular loops to the outside of the cell surface, while the three intracellular
loops as well as the "C terminus" in the cytoplasm (7).
55
APPENDIX
Figure 3
Figure 3. Schematic illustration of CXCR4 receptor activation, (i) unstimulated
receptor CXCR4, (ii) Stromal cell derived factor la (SDFla) binds with CXCR4, (iii)
conformational change of CXCR4 leading to exchange of GDP bound to Ga with GTP,
(iv) dissociation of the G-protein Ga- and G(3y subunits (9, 107).
56
APPENDIX
Figure 4
MI2
riuinoliivis
Transcription
Cell survival
Transcription
Gene expression
Cell proliferation
Gene expression
Chemotaxis
tea
Figure 4. Signal transduction of CXCR4. Signal transduction by CXCR4 leads to
activation of G proteins, which resulted in activation of ERKl/2 and PI3 Kinase
pathways and thus facilitate tumor dissemination through stimulating gene transcription,
cell proliferation and survival and chemotaxis within the cell (108, 131-132).
57
APPENDIX
Figure 5
Normal prostate tissue
Low grade prostate cancer
tissue
High grade prostate cancer
tissue
High grade prostate cancer
tissue
Figure 5. Immunohistochemical (IHC) staining of prostate tissues for CXCR4. A
human prostate tissue array, ranging from normal to high-grade prostate cancer, was
evaluated by IHC for CXCR4 expression using standard methods. Samples were
evaluated at magnification of 40X, using a Q-Imaging camera of Olympus BX51
Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate
tissues demonstrated weak or undetectable brown staining for CXCR4 (positive
cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate
tissue (grade 2, stage II, T2N0M0, adenocarcinoma) demonstrated random/focal positive
staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating
low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4,
stage IV, T4N1M1, adenocarcinoma) demonstrated diffuse/intense staining (positive cells
>50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents
50um.
58
APPENDIX
Figure 6
B
WB
IP: CXCR4
WB: CXCR4
PC3
'45KDa
CXCR4
■42KDa
293T
45K0a
PC3
IP: Fibronectin
WB: CXCR4
CXCR4
WB
45KDa
Fibronectin
250KDa
Figure 6. Characterization of CXCR4 IgG2B mouse monoclonal antibody. A,
CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4
protein by western blot analysis in PC3 (CXCR4 positive) or HEK 293T (CXCR4 null)
cell lines. B, CXCR4 antibody was evaluated for specificity to CXCR4 protein by
immunoprecipitation for CXCR4 and western blot analysis for CXCR4. C, CXCR4
IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation
with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and
western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by
western blot analysis. Beta-actin was used as a loading control.
59
APPENDIX
Figure 7
Norwiuelear
Cil
SDF1o
CXCR4
Nuclear
30Min
1
Cll
30Min
J|[
I 45K0a
1
|80KOa
CD44
Topol
lOOKDa
RWPE1
22RV1
Non-nuclear
SOF1a
Cll
30MPn
Ctl
I'500"
30Min
£
CXCR4
CO44
.
1
| 1G1M
80K0a
g sow
!
IG000
I
- —.,
4SKDa 1
lOOKDaJ2™1
Topol
Muctear fraction
Non-nuclear tuition
Nuclear
■
1
i
1
1
1
MOO
1
g
C.
v.i
!. ■_•
..a
DU145
Non-nuclear
Nuctear fraction
Non-nueiear fraction
Nuclear
I
4SKOa ~|
80KOa DU145
lOOKOa
I
|
| 1O0BB
mm
1WK3
i """i
.CW5 |
\
■
s
s
a
0
[
!
C;
:.».-■
PC3
Non-nuclear
SDF1G
Cll
30Min
Nuclear
Cll
Nan-nucloar fraction
Nuclear fraction
30Min
♦
CXCR4
45KDa
116000
CO44
80KDa
liGSOO
Topol
«■»
100KDa
Figure 7. Nuclear CXCR4 expression in prostate cancer cell lines. Normal prostate
epithelial (RWPE1) and prostate cancer (PC3, DU145,22RV1) cells were stimulated with
SDFla (100 ng/ul) prior to subcellular fractionation into non-nuclear and nuclear
fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and
anti-Topoisomerasel (Topo 1, nuclear) were used as markers for fractionation purity and
as loading controls. The bar graphs are quantitative results of the band density
representing expression of CXCR4 in each fraction. Data represents mean + S.E. from
three independent experiments. *, P<0.05.
60
APPENDIX
Figure 8
22RV1
CII
DU145
CM
PC3
3OMin
SDFla
CXCR4
Lamin
Figure 8. Immunocytochemistiy of prostate cancer cell lines for CXCR4. Prostate
cancer cells were stimulated with SDFla (100 ng/ul), fixed with methanol, blocked then
incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal
antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary
mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated antirabbit antibody. Images were obtained using a Zeiss LSM-510 UV Confocal Microscope
using the 63x Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for
FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and
cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclearassociated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate colocalization of CXCR4 with the nucleus (yellow/orange). Scale bar represent 50 (am.
61
APPENDIX
Figure 9
CXCR4
GFP-CXCR4
GFP-CXCR4
GFP-CXCR4*
♦ DAPI
*CXCR4
DAPI*CXCR4
GFP-CXCR4
Figure 9. Expression of GFP- CXCR4 and endogenous CXCR4 protein in PC3 cells.
GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPNl
transfected PC3 cells were stimulated with SDFla, fixed with methanol, blocked then
incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-
conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue).
Images were taken at 40X maginification using Axiovision software 4.8.2 with a Zeiss
Axio Imager.zl fluorescence microscope at ex=470 nm for FITC, ex=358 nm for DAPI
and ex=551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous
CXCR4 (red) with GFP-tagged CXCR4 (green).
62
APPENDIX
Figure 10
PESFPN1-CXCR4
PEGFPN1-CXCR4R146A
PEGFPN1-CXCR4ANUS
SDFIa
Select*'
Figure 10. Localization analysis of wild type and mutated (R146A and ANLS)
CXCR4. The expression of Wild type CXCR4 (CXCR4-pEGFPNl), NLS-mutant
CXCR4(pEGFPNl-CXCR4R146A,)and NLS deleted CXCR4 (CXCR4ANLS) in PC3
cells were analysed by Immunocytochemistry assay. Nuclei were stained with propidium
iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Images
were obtained with a Zeiss LSM-510 UV Confocal Microscope using the 63x Plan-
Apochromat 63x/1.40 Oil DIC objective at ex=488 nm for FITC and ex=543 nm for Cy3.
Scale bars represent 50 urn.
63
APPENDIX
Figure 11
WlhlfyprfXCIM
t'\CK4R148A-pEGFKM
\Mlilly|ir('X('K4
CWR4RI4SA j>EUH>NI
C.\CR4GH>
Figure 11. Localization analysis of wild type and mutated (R148A) CXCR4. The
expression of wild type CXCR4 (CXCR4-pEGFPNl) and NLS-mutant of CXCR4
(pEGFPNl-CXCR4R148A,) in PC3 cells were analysed by Immunocytochemistry assay.
Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion
protein GFP-CXCR4 (green). Images were taken at 40X maginification using Axiovision
software 4.8.2 with a Zeiss Axio Imager.zl fluorescence microscope at ex=470 run for
FITC and ex=551 run for Cy3.
64
APPENDIX
Figure 12
Nuclear fraction
Non-nuclear fraction
\Mld4ypeCXCR4
Wild-type CXCR4
CXCR4ANLS
Ctl
SDF1o
CD44
Topol
72KDa
GFP
BOKDa
CD44
lOOKDa
Topol
30Min
♦
m
CXCR4ANLS
Ctl
30Min
♦
72KOa
SOKDa
mm
100KOa
Figure 12. Expression of wild type CXCR4 and mutated CXCR4. Wild type CXCR4
(CXCR4-pEGFPNl) and NLS deleted CXCR4 (CXCR4ANLS) transfected cells were
stimulated with SDFla prior to subcellular fractionation into non-nuclear and nuclear
fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-
CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerasel (Topo 1, nuclear) were used
as markers for fractionation purity and as loading controls.
65
APPENDIX
Figure 13
PC3
293T
Ctl
30Min
SDF1a(30Min)
TRN1
<-80KDa
aTubulin
SOKDa
Ctl
SDF1a
30Mln
PC3 supernatant
♦
Ctl
TRN1
-80KDa
CXCR4
4SKDa
30Min
♦
CXCR4
<—45KDa
Figure 13. CXCR4 and TRNl demonstrate an interaction. ,4, Sixty micrograms of
total protein were analyzed for TRNl expression by western blot analysis using a TRNl
specific antibody. Alpha-tubulin served as a loading control. B, One milligram of PC3
whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDSPAGE. Immunocomplexes were probed with anti-TRNl or anti-CXCR4 to ensure that
CXCR4 interacted with TRNl and was immunoprecipitated, respectively. Thirty
micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by
SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4
immunoprecipitation.
66
APPENDIX
Figure 14
JA(nM)
TRNl
JWiciin
n
in
as
mm-m*mm■Mar
m *
Figure 14. CXCR4 undergoes TRNl dependent translocalization to the nucleus.
A andB, Cells were transiently transfected with TRNl-specific siRNA to determine an
effective concentration (A), prior to harvesting for immunohistochemistry with antiLamin A/C and anti-CXCR4 (B). Images were taken using Zeiss Axio Imager.zl
fluorescence microscope at 40X magnification at excitation 470 nm for FITC and 551 nm
for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus
(yellow/orange). Scale bar represents 50
67
APPENDIX
Figure 15
SWto
_
c>aai.i
-[
A.
I
A.
1
SDFlu
KMD3100
PTK
Figure 15. Nuclear CXCR4 was functional at the nucleus. A, Representative light
images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at
20X magnification. B, Whole cells were treated with SDFla prior to isolation and lysis
of intact nuclei. Nuclear lysates (1 mg) were immunoprecipitated with anti-CXCR4 and
separated by SDS-PAGE.
Immunocomplexes were probed for Gai (first row) or CXCR4
antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerasel
(Topol, nuclear) were used as markers for fractionation purity and as loading controls. C,
PC3 nuclei were isolated, incubated with FluoForte dye Ca2+ probe, followed by
incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDFla
for 30 min. An increase in fluorescent-bound Ca2+ was measured on a microplate reader
at ex=490 nm/em=525 nm.
68
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