<title>1. INTRODUCTION</title> <p>The regulation of the distribution of motile
cells in both normal and disease situations depends upon a variety of peptide
and nonpeptide mediators, which stimulate cell movement by both directed
(chemotaxis) and nondirected (chemokinesis) mechanisms. Amongst these mediators
are the chemokines, a class of peptide mediators that play critical roles in
normal development, regulation of the hematopoietic and immune systems in the
adult, and in repair processes such as wound healing and inflammation. Among
the different chemokines is the stromal cell-derived factor-1 (SDF-1), which is
now known as CXCL12. CXCL12 binds principally to the receptor CXCR4, although
it also acts through the more-recently-described receptor CXCR7 [##REF##16107333##1##]. This
review describes the roles of CXCL12 and CXCR4 in normal tissue functions and
in cancer, and suggests that the regulation of CXCR4 expression by PPAR<italic>γ</italic> may
emerge to be a unique avenue by which a key receptor involved in cancer cell
metastasis can be suppressed in a way that will assist with disease therapy.</p>
<p>Recommended by Dipak Panigrahy</p> <p>Chemokines are peptide mediators involved in normal development,
hematopoietic and immune regulation, wound healing, and
inflammation. Among the chemokines is CXCL12, which binds
principally to its receptor CXCR4 and regulates leukocyte
precursor homing to bone marrow and other sites. This role of
CXCL12/CXCR4 is “commandeered” by cancer cells to facilitate the
spread of CXCR4-bearing tumor cells to tissues with high CXCL12
concentrations. High CXCR4 expression by cancer cells predisposes
to aggressive spread and metastasis and ultimately to poor patient
outcomes. As well as being useful as a marker for disease
progression, CXCR4 is a potential target for anticancer therapies.
It is possible to interfere directly with the CXCL12:CXCR4 axis
using peptide or small-molecular-weight antagonists. A further
opportunity is offered by promoting strategies that downregulate
CXCR4 pathways: CXCR4 expression in the tumor microenvironment is
modulated by factors such as hypoxia, nucleosides, and
eicosanoids. Another promising approach is through targeting PPAR
to suppress CXCR4 expression. Endogenous PPAR<italic>γ</italic> such as 15-deoxy-Δ<sup>12,14</sup>-PGJ<sub>2</sub> and synthetic agonists such as the
thiazolidinediones both cause downregulation of CXCR4 mRNA and
receptor. Adjuvant therapy using PPAR<italic>γ</italic> agonists may, by
stimulating PPAR<italic>γ</italic>-dependent downregulation of CXCR4 on cancer cells, slow the rate of metastasis and impact beneficially on
disease progression.</p>
<title>2. CHEMOKINES AND THEIR RECEPTORS IN
CELL REGULATION</title> <p>Chemokines
are low-molecular-weight peptide ligands involved in the trafficking of
leukocytes and other motile cells [##REF##10699158##2##, ##UREF##0##3##]. 
There are four major groups of chemokines, the CXC, CC, C and CX3C
chemokines, categorized as such on the basis of their number and spacing of
conserved cysteine residues [##REF##10699158##2##, ##UREF##1##4##]. The nomenclature
of chemokines (e.g., “CXCL12") is made up of their subclass (CXC, CC, etc.)
followed by “L” for ligand, and a specific number [##REF##10699158##2##, ##UREF##0##3##].</p> <p>The receptors for
chemokines are cell-surface, seven-transmembrane G protein-coupled receptors [##REF##10699158##2##].
The naming of these receptors (e.g., “CXCR4") is based on the subclass of
chemokine that the receptor recognizes, followed by “R” for receptor and a
number (which need not correspond to the number assigned to its cognate ligand(s)). There are 19 well-recognized chemokine
receptors (e.g., CXCR1-6, CCR1-10, CX<sub>3</sub>CR1, and XCR1) [##REF##16107333##1##, ##REF##15914470##5##]. Many
chemokine receptors have more than one known ligand, and many chemokines can
activate more than one receptor. Thus,
there is much promiscuity in chemokine/receptor signaling.</p> <p>Chemokines bind within
the extracellular domain of the chemokine receptor, which comprises the
N-terminus and three extracellular loops [##UREF##0##3##]. 
The intracellular domain, which consists of three loops and the
C-terminus, associates with G proteins that, upon activation, lead to
inhibition of adenylyl cyclase activity [##UREF##0##3##]. Typical cellular consequences of
chemokine binding include changes in gene expression, cell polarization, and
chemotaxis (directed cell migration) [##UREF##1##4##].</p> <p>Chemokines play a
major role in regulating the migration of cells of the immune system, leading
to the modulation of immune responses. 
Their exact role depends on the expression pattern of receptors on
specific leukocyte subsets [##REF##10699158##2##] but encompasses the regulation of lymphocyte
trafficking, lymphoid tissue development, Th1/Th2 modulation, and the effecting
of inflammatory reactions. Chemokine receptors are also found on other cell
types, and play a part in stem cell recruitment and angiogenesis, in
development and wound healing [##UREF##1##4##]. When such pathways are subverted in
neoplastic cells, chemokines take over prominent roles in the metastatic
process, both in terms of the dissemination of cells from primary tumors and in
growth of the cancer at metastatic sites. As we will see, this is the case for
CXCR4.</p> <title>3. THE CHEMOKINE RECEPTOR CXCR4 AND
ITS LIGAND CXCL12 (SDF-1)</title> <p>The receptor now
known as CXCR4 was cloned in 1994, and was originally given the name leukocyte-expressed
seven-transmembrane domain receptor (LESTR) due to its abundant expression in
several leukocyte populations [##REF##8276799##6##]. It
was independently cloned by others and named “fusin” because of its ability to
act as a coreceptor for HIV fusion and entry [##REF##8629022##7##]. It further has the designation “CD184” as
part of the cluster of differentiation antigens found on activated leukocytes. LESTR/fusin/CD184
was originally considered to be an orphan receptor. However, the chemokine CXCL12, originally termed
stromal cell-derived factor 1 (SDF-1), was shown by two independent research
groups to be a ligand for LESTR/fusin/CD184, and the name CXCR4 was proposed [##REF##8752280##8##, ##REF##8752281##9##]. The <italic>CXCR4</italic> gene is constitutively expressed, and CXCR4 protein has been
detected on many leukocytes, including lymphocytes, monocytes, NK cells, and
dendritic cells; as well as on vascular smooth muscle cells, endothelial cells,
cells lining the gastrointestinal tract, microglia, neurons, and astrocytes [##REF##9599023##10##–##REF##15246052##13##].
Until recently, CXCR4 was considered to be the only receptor for CXCL12, but
the previous orphan receptor RDC1 is now recognized as an additional CXCL12 receptor,
for which the name CXCR7 has been given [##REF##16107333##1##]. 
CXCL12 itself is widely expressed at different levels in many tissues [##REF##7490086##14##].</p> <title>4. CXCL12 AND CXCR4 IN NORMAL TISSUE FUNCTION</title> <p>The interplay between CXCL12 and CXCR4 is
critical to normal development. Indeed (and unlike mice deficient in other
chemokine/receptors) mice lacking CXCL12 or CXCR4 die in utero or shortly after birth [##REF##10699158##2##, ##REF##8757135##15##–##REF##9634238##17##]. CXCL12/CXCR4 signaling is required during the
development of the hematopoietic, cardiac, vascular, and nervous systems. Absence
of this axis in embryonic life leads to defects in bone marrow myeloid cell
formation, cardiac dysfunction due to impaired ventricular septum formation,
and developmental defects in the cerebellum and in the vasculature of the
gastrointestinal tract [##REF##8757135##15##–##REF##9634238##17##].</p> <p>In the normal
adult, CXCL12 and CXCR4 are involved in the homing and retention of
hematopoietic progenitor cells in the bone marrow. These progenitor cells express high levels of
CXCR4, and are attracted to CXCL12 produced by stromal cells in specialized bone
marrow niches [##REF##8996247##18##]. Activating mutations
of the <italic>CXCR4</italic> gene lead to aberrant
retention of myeloid cells within the bone marrow [##REF##12692554##19##]. CXCL12 also acts as a
chemoattractant for stem cells and some differentiated cells in the pathological
contexts of inflammation and tissue regeneration/repair [##REF##15888687##20##–##REF##17572689##24##]. It is this
function of controlling cell migration and homing that is subverted in cancer.</p> <title>5. CXCL12 AND CXCR4 IN CANCER
METASTASIS AND GROWTH</title> <p>In many ways, the
process of metastasis is similar to leukocyte and stem cell trafficking,
processes which involve the CXCL12/CXCR4 axis [##REF##15888687##20##]. Cancer cells that express CXCR4 exploit the
same signaling pathway, leading to homing and retention in tissues that are
rich in CXCL12.</p> <p>The foundation for
our appreciation of the role that CXCR4 and CXCL12 may play in cancer
metastasis was set in 2001, when a landmark study by Albert Zlotnik's
group demonstrated the importance of the CXCL12/CXCR4 axis in site-specific
metastasis of breast cancer [##REF##11242036##25##]. In that study, it was found that CXCR4
expression was low or undetectable in normal epithelial cells, but consistently
upregulated in breast cancer cell lines and primary breast cancer cells at both
the mRNA and protein level. Human breast
carcinoma cells that expressed high levels of CXCR4 underwent morphological
changes and migrated directionally in response to CXCL12, indicating that the
CXCR4 receptor was active. Crucially,
the ligand CXCL12 was highly expressed in tissues taken from human organ sites
to which breast cancer cells metastasize, including lymph nodes, lung, liver,
and bone marrow, but expressed at low levels in tissues that represent rare
sites of metastasis, including the kidney, skin, and muscle. The ability of MDA-MB-231 human breast cancer
cells (a cell line that is metastastic in experimental models) to migrate
towards protein extracts of lung and liver, or to produce lung and lymph node
metastasis after tail-vein injection or orthotopic implantation, was inhibited by
neutralizing anti-CXCR4 and/or anti-CXCL12 antibodies. These findings were the first to show the
biological importance of this chemokine/receptor pair in the evolution and
spread of cancer.</p> <p>Since that time,
the CXCL12/CXCR4 axis has been shown to be important in the progression and
spread of more than 25 different cancers. Our present knowledge is based on
(i) studies in cellular and animal experimental models, (ii) surveys of human tissues at
different stages of cancer progression, and (iii) population-based studies of morbidity
and survival. A summary of present data is shown in ##TAB##0##Table 1##.</p> <p>CXCR4 has been
shown to be expressed at high levels on cells of all of the major adult solid
epithelial cancers (breast, colorectal, lung, ovary, prostate, etc.). The
ability of the cells to colonize other tissues by gaining advantage from
CXCR4-dependent mechanisms depends on the presence of CXCL12 in the tissue
fluid. Various studies have shown significant CXCL12 concentrations in the
fluid-filled cavities through which many cancers disseminate, and at tissue
locations in which metastases characteristically develop. Biologically,
significant CXCL12 levels have been
found in peritoneal ascites from ovarian cancer patients [##REF##11431324##26##], pleural
effusions in lung cancer [##REF##14672915##27##], lymph nodes, bone, and lungs as well as other
tissues [##REF##11242036##25##, ##REF##12927045##28##, ##REF##11912162##29##].</p> <p>Detailed studies
of the cellular interactions involved in the metastasis of prostate cancer
cells to bone [##REF##11912162##29##] have shown that the interaction of CXCL12 with CXCR4 plays a
major role in successive steps in the metastatic process. Human osteoblasts
express CXCL12 mRNA and protein, whereas prostate cancer cells express CXCR4
mRNA and receptor. Prostate cancer cells
that have become disseminated into the circulation respond to the CXCL12-CXCR4
pathway by enhanced adherence to the bone marrow endothelium and migration
across endothelial barriers and basement membranes, ultimately adhering to
components of the bone marrow in response to a CXCL12 gradient [##REF##11912162##29##]. CXCL12
from osteoblasts has also been shown to act on CXCR4 to induce release of IL-6
from human squamous cell carcinoma cells to promote osteoclastogenesis [##UREF##2##30##].</p> <p>As well as
promoting the migration of cancer cells and their invasion through physical
barriers as well as adherence to target structures, CXCL12 can act upon CXCR4
on the cancer cells to promote cancer cell growth along with other mitogenic
factors. This has been shown in cells from colorectal [##REF##16823836##31##], prostate [##REF##12761880##32##], and
ovarian [##REF##12384559##33##] cancers. Furthermore, CXCL12 can promote cancer
dissemination indirectly by enhancing the vascular supply, since the
CXCL12/CXCR4 axis may also promote tumor angiogenesis. Vascular endothelial growth factor (VEGF) and
CXCL12 have been shown to increase angiogenesis synergistically in an in vivo Matrigel assay and to promote
proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro [##REF##15695388##34##].</p> <title>6. THE EFFECT OF CXCL12 ON CELLULAR PROCESSES</title> <p>Activation
of CXCR4 produces specific cellular changes that are consistent with a
migratory and invasive cell phenotype. Exposure of cells to CXCL12 produces upregulation
of matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 [##REF##11242036##25##, ##REF##11431324##26##, ##REF##11912162##29##, ##REF##15059909##35##–##REF##18416455##39##].
In addition, CXCL12 enhances adhesion to components of the extracellular matrix
such as fibronectin, laminin, and collagen types I/III [##REF##12239174##37##, ##REF##15592929##40##], or to other
cell types (e.g., endothelial or bone marrow stromal cells) [##REF##11912162##29##, ##REF##11591806##41##, ##REF##15548713##42##]. These
changes are mediated in large part by integrin signaling [##REF##11912162##29##, ##REF##14583470##43##, ##REF##15806155##44##]. Many
signaling pathways are activated by CXCL12 downstream of CXCR4 in cancer
cells. For example, CXCL12 has been
shown to increase ERK1/2 phosphorylation [30, 31, 49, 0.70, 76.78, 79], Akt
phosphorylation [50, 77.88], and PI3K activation [##REF##15235108##45##].</p> <title>7. CXCR4 IN BREAST CANCER</title> <p>CXCR4 is expressed
at a low level in normal breast epithelium but becomes more strongly expressed
in the early stages of carcinogenesis, showing both a more intense immunohistochemical
staining pattern and an altered cellular localization in studies of human
ductal carcinoma in situ (DCIS)
[##REF##16344916##46##, ##REF##15026622##47##]. An extensive tissue microarray study of 1808 invasive breast
carcinomas and 214 pre-invasive breast samples linked to clinical data has
shown that the level of CXCR4 expression can be linked to tumor progressivity
(tumor grade and lymph node status) and to other prognostic factors such as
HER2 expression and hormone receptor (ER and PR) negativity, as well as to
patient survival [##REF##16344916##46##]. These observations in human tissues have led to the view
that CXCR4 provides a selective advantage to newly formed neoplastic cells in
the early primary breast tumor as well as being important to later invasion and
metastasis [##REF##15246052##13##, ##REF##16344916##46##–##REF##16671092##48##]. This is consistent with observations in mouse models of
breast cancer in which interventions affecting CXCR4 reduced both growth of the
primary tumor and metastasis [##REF##15574767##49##].</p> <p>Prominent CXCR4
expression is a feature of all major histological forms of invasive breast
cancer, including ductal, lobular, mucinous [##REF##16344916##46##], and the distinctive and
highly aggressive inflammatory form of the disease [##REF##17351259##50##]. Several independent
studies have shown that the extent and pattern of CXCR4 expression is related
to axillary lymph node involvement in different forms and stages of breast
cancer [##REF##12927045##28##, ##REF##16239110##51##–##REF##17763975##53##]. CXCR4 positivity has also been noted as a key feature of
breast carcinoma metastasis to bone [##REF##17938257##54##] and brain [##REF##17717635##55##]. The power of CXCR4 as
a marker for lymph node metastasis can be greatly increased by concurrently
examining the expression of additional markers such as VEGF, MMP-9, and CCR7 [##REF##17306924##38##, ##REF##16115904##56##].
Furthermore, CXCR4 is also one of a subset of markers (the others are uPAR,
S100A4, and vimentin) that define highly aggressive and invasive breast
carcinoma cells that are associated with malignant pleural or peritoneal
effusions in breast cancer patients [##REF##17450528##57##]. CXCR4 expression is therefore a general
marker for the spread of breast cancer to its secondary sites, and for aggressive
stages of the disease.</p> <p>There is evidence
not only for the use of CXCR4 as a general marker for the progression and
metastasis of breast cancer, but also for the identification of individual tumor
cells as they are homing from the primary tumor to secondary sites as patients
develop metastatic disease. Individual CXCR4-expressing tumor cells have been
found in the peripheral blood of breast cancer patients [##REF##15914200##102##], and CXCR4
expression in breast cancer has been associated with the presence of individual
tumor cells in the bone marrow of patients [##REF##16132577##103##].</p> <title>8. CXCR4 IN COLORECTAL CANCER</title> <p>CXCR4 is
abundantly expressed by colorectal carcinoma cells [##REF##10525044##104##, ##REF##10419917##105##]. The involvement of CXCR4 expression in
colorectal cancer progression was first shown by Roos and colleagues [##REF##12839981##71##]. CT-26 mouse colon carcinoma cells were
transfected with CXCL12 extended with a Lys-Asp-Glu-Leu (KDEL) sequence. The KDEL receptor functions to retain
resident endoplasmic reticulum (ER) proteins, which contain a C-terminal KDEL
sequence, in the ER. With this
“intrakine approach," CXCL12-KDEL binds to the KDEL receptor and is retained in
the ER, and CXCR4 protein which binds to CXCL12 is also retained in the ER,
preventing its expression at the cell-surface [##REF##12839981##71##, ##REF##11457880##106##]. This approach was first developed as a
strategy to reduce HIV infection [##UREF##3##107##]. After intrasplenic injection, CXCL12-KDEL-transfected CT-26 cells, which had reduced cell-surface CXCR4
protein expression, did not form liver metastases, whereas control cells did [##REF##12839981##71##]. The incidence of lung metastasis was also
reduced with CXCL12-KDEL-transfected cells, and survival was increased. Interestingly, unlike Zlotnik's group, who
had suggested that CXCR4 expression was necessary for the movement of tumor cells
to secondary sites [##REF##11242036##25##], Zeelenberg and colleagues found that CXCR4 expression
was not required for migration of CT-26 colorectal tumor cells to the lungs,
but rather for tumor expansion at secondary sites [##REF##12839981##71##]. Therefore, these authors concluded that CXCR4
is necessary for the outgrowth of colon cancer micrometastases.</p> <p>Ottaiano
and colleagues found that CXCR4 was overexpressed in human colorectal carcinoma
tissues compared to normal tissues [##REF##15592929##40##]. 
Cell-surface CXCR4 protein was also expressed at high levels on SW620,
SW48, and SW480 colorectal carcinoma cells, and at moderate levels on Caco-2
and LoVo cells. CXCL12 enhanced the
chemotaxis of SW480 cells as well as their adhesion to fibronectin and collagen
type I/III, and both effects were blocked with an anti-CXCR4 neutralizing
antibody. CXCL12 also induced
cytoskeletal changes, proliferation, and ERK1/2 phosphorylation in SW480 cells. Similarly, Schimanski and colleagues found that
SW480, SW620, and HT-29 colorectal carcinoma cells expressed CXCR4 protein, as
did colorectal carcinoma tissue samples [##REF##15755995##72##]. 
CXCL12 induced the chemotaxis of SW480 and SW620 cells. Kim and colleagues found that in patients with colorectal cancer with liver
metastases, higher CXCR4 expression was found on metastatic tissues compared to
the primary tumor [##REF##15837989##73##]. Furthermore,
elevated CXCR4 expression in colorectal cancer is associated with disease
progression and reduced survival [##REF##15592929##40##, ##REF##15755995##72##, ##REF##15837989##73##, ##REF##16675573##75##].</p> <title>9. THE UTILITY OF CXCR4 AS A MARKER OF
TUMOR PROGRESSION</title> <p>CXCR4
expression has been associated with disease progression, increased recurrence,
and reduced survival in many cancer types, as listed in ##TAB##0##Table 1##. As pointed out
earlier, CXCR4 protein expression is detectable in the majority of cases of DCIS
of the breast, whereas CXCR4 levels are very low in adjacent normal breast
epithelium [##REF##16344916##46##]. This suggests that the acquisition
of CXCR4 expression may occur very early in malignant transformation, suggesting
its potential as a biomarker. As indicated earlier, it has been suggested that
CXCR4 expression may be useful as an indicator of prognosis [##REF##16115904##56##, ##REF##15837989##73##].</p> <p>Although
mutations in the <italic>CXCR4</italic> gene have not
been reported in the context of cancer, patients with a single nucleotide
polymorphism in the 3′ untranslated region of the <italic>CXCL12</italic> gene had reduced incidence of long distance metastasis of
epidermoid non-small cell lung cancer (NSCLC) [##REF##16155757##108##].</p> <title>10. PRECLINICAL EFFICACY OF
ANTI-CXCR4 TREATMENTS</title> <p>Several studies have
demonstrated the efficacy of strategies designed to reduce CXCR4 expression or
inhibit its activity in preclinical models of cancer development and metastasis. A neutralizing anti-CXCR4 antibody prevented
metastasis of MDA-MB-231 breast cancer cells in mice [##REF##11242036##25##] and in another study reduced
tumor growth after intraperitoneal (IP) injection of Namalwa non-Hodgkin's lymphoma cells [##REF##12036921##86##]. Interestingly, a neutralizing antibody
against CXCR4 also inhibited the growth of subcutaneous tumors derived from
pancreatic cancer cells that did not themselves express CXCR4, probably because
of the ability of the antibody to block CXCR4 on tumor vasculature [##REF##15994964##109##].</p> <p>CXCR4 peptide
antagonists have also proven effective in preclinical cancer models. The CXCR4 peptide antagonist 4F-benzoyl-TN14003
inhibited lung metastasis of MDA-MB-231 breast cancer cells [##REF##12935890##110##], and
4F-benzoyl-TE14011 reduced pulmonary metastasis of B16-BL6 melanoma cells [##REF##15207725##111##]. Murakami and colleagues assessed the
contribution of CXCR4 to the metastatic process by transducing B16 murine
melanoma cells with CXCR4, followed by IV injection in syngeneic B57BL/6 mice [##REF##12499276##112##]. CXCR4 expression in this context led to
increased pulmonary metastasis, which was reduced with the CXCR4 peptide
antagonist T22. Liang and colleagues showed that TN14003 itself, which is a 14-mer peptide CXCR4 antagonist,
inhibited in vitro invasion of
MDA-MB-231 breast cancer cells and lung metastasis after tail vein injection of
these cells, without causing any toxicity [##REF##15205345##113##].</p> <p>Small molecule (nonpeptide)
inhibitors of CXCR4 have also been tested in preclinical cancer models. Rubin and colleagues showed that the
noncompetitive CXCR4 antagonist AMD3100 inhibited tumor growth after
intracranial implantation of Daoy medulloblastoma cells and U87 glioblastoma
cells [##REF##14595012##63##] and also inhibited peritoneal carcinomatosis and ascites formation
after IP inoculation of NUGC4 human gastric carcinoma cells [##REF##16489019##78##]. In a different approach, blocking the
mammalian target of rapamycin (mTOR) pathway downstream of CXCR4 was shown to
suppress processes involved in the peritoneal dissemination of gastric cancer [##REF##18375114##114##].</p> <p>Liang and
colleagues also showed the preclinical efficacy of anti-CXCR4 treatments using
an RNA-silencing molecular approach [##REF##15705897##115##]. 
MDA-MB-231 breast cancer cells transfected with siRNA oligonucleotides
to knock down CXCR4 were injected into the tail veins of SCID mice. Mice received twice-weekly IV injections of
siRNA oligonucleotides to maintain CXCR4 knockdown. The control mice all developed lung metastases,
whereas only one of six mice receiving CXCR4 siRNA-transfected cells and
followup injections with CXCR4 siRNA developed metastases. Stable knockdown of
CXCR4 expression in 4T1 murine breast carcinoma cells using short hairpin RNA
reduced orthotopic tumor growth and lung metastasis [##REF##15574767##49##]. Similarly, MDA-MB-231
cells that had undergone stable knockdown of CXCR4 did not form tumors or lung
metastases after orthotopic injection into mammary fat pads of SCID mice,
whereas CXCR4-positive cells were tumorigenic [##REF##15472715##116##]. NSCLC 95D lung cancer cells in which CXCR4
was knocked down using antisense technology also formed lung metastases in
fewer mice after SC injection compared to CXCR4 positive cells [##REF##16322285##88##]. Finally, manipulations of CXCR4 expression
have become possible using microRNAs (miRNAs), which are endogenous short RNAs
with the ability to repress the translation of target mRNAs [##REF##12600936##117##–##REF##16713585##119##]. The
approach of expressing a synthetic miRNA against CXCR4 mRNA to knock down CXCR4
expression has been used successfully in MDA-MB-231 breast cancer cells, HeLa
cervical carcinoma cells, and U2-OS osteosarcoma cells [##REF##16287976##118##, ##REF##15937477##120##, ##REF##17889832##121##]. Reduced
CXCR4 expression in the breast cancer model was accompanied by reduced
migration and invasion of the cells in
vitro and fewer lung metastases in
vivo [##REF##17889832##121##]. These studies show the importance of CXCR4 expression in
both primary and secondary tumor growth.</p> <title>11. CLINICAL ASSESSMENT OF
CXCR4-TARGETED REAGENTS</title> <p>The bicyclam
compound AMD3100 was developed as a small molecule CXCR4 antagonist [##REF##12815382##122##]. Although this compound has not yet been fully
assessed in clinical trials to determine its therapeutic potential in cancer, it
has been examined in small trials in the context of HIV treatment and
hematopoietic progenitor cell mobilization [##REF##10817726##123##–##REF##15752146##128##]. One trial with AMD3100 reported one patient
with thrombocytopenia, two patients with premature ventricular contractions,
and several patients with paresthesias [##REF##15385732##126##]. 
AMD3100 did not reduce viral load in HIV patients [##REF##12815382##122##], but did
effectively increase hematopoietic progenitor cell mobilization [##REF##15020611##124##, ##REF##15890685##125##, ##REF##15900288##127##, ##REF##15752146##128##]. However, the mechanisms of action are under
debate and may be unrelated to inhibition of CXCR4 as was first presumed.</p> <title>12. REGULATION OF CXCR4 EXPRESSION BY
FACTORS WITHIN THE TUMOR</title> <p>Zeelenberg and
colleagues found that CT-26 murine colon carcinoma cells grown in vitro expressed CXCR4 mRNA, but
cell-surface protein levels were not detectable [##REF##12839981##71##]. When the same cells were freshly isolated
from lung or liver metastases or from intrasplenic tumors, cell-surface
expression was strongly upregulated. 
This elevated expression was lost after 2–4 days in culture, indicating
that it was not due to selection of a subpopulation of cells with a high CXCR4
expression. The authors concluded that
CXCR4 expression was induced by the in
vivo tumor microenvironment. Although
others have shown that metastatic cells maintain high CXCR4 expression when
cultured in vitro [##REF##17671223##129##], and
indeed CXCR4 has been suggested as a cancer stem cell biomarker [##REF##18371365##130##], as
discussed below there is substantial evidence indicating that CXCR4
expression is nevertheless influenced by the tumor microenvironment. Additionally,
aberrant activation of signaling pathways within cancer cells, such as those
initiated through HER2, can also contribute to elevated CXCR4 expression [##REF##15542430##131##].</p> <p>Multiple features
and factors present in the tumor microenvironment have been shown to regulate
CXCR4 expression on tumor cells and other cell types. One such feature is hypoxia [##REF##13679920##97##, ##REF##14597738##132##]. Solid tumors tend to be hypoxic due to
structural abnormalities in their vasculature [##REF##15254862##133##]. Staller and colleagues were the first to
demonstrate the involvement of hypoxia in the regulation of CXCR4 expression [##REF##13679920##97##]. Their goal was to identify genes regulated by
the von Hippel-Lindau tumor suppressor protein
(pVHL) in renal cell carcinoma cells. pVHL is often inactivated in renal cell
cancer (RCC) leading to constitutive activation of hypoxia-inducible factor-1
(HIF-1) target genes. In a microarray
analysis, they found that CXCR4 mRNA expression was suppressed by the reintroduction of functional pVHL into
pVHL-deficient A498 RCC cells, an effect that was due to inactivation of HIF-1. CXCR4 protein was also downregulated,
resulting in reduced migration of RCC cells towards CXCL12. Hypoxia increased CXCR4 mRNA expression in
HEK-293 human embryonic kidney cells and primary human proximal renal tubular
epithelial cells, and a hypoxia response element (HRE) was identified within the
CXCR4 promoter [##REF##13679920##97##]. The authors
speculated that intratumoral hypoxia may lead to increased CXCR4 expression in
diverse types of solid tumors, increasing metastasis to distant organs. Shioppa and colleagues found that hypoxia
increased CXCR4 mRNA and cell-surface protein expression in several cell types,
including monocytes, human monocyte-derived macrophages, tumor-associated
macrophages, HUVECs, CAOV3 ovarian carcinoma cells, and MCF-7 breast carcinoma
cells, leading to increased migration towards CXCL12 due to the activation of
HIF-1 [##REF##14597738##132##].</p> <p>The hypoxic environment within tumors also
leads to high extracellular levels of adenosine (adenine-9-<italic>β</italic>-D-ribofuranoside),
a nucleoside that is involved in energy metabolism and comprises the core
structure for adenine nucleotides. The concentration of adenosine in the
extracellular fluid of solid tumors is about 100-fold that of adjacent normal
tissue [##REF##9205063##134##]. Adenosine concentrations in tumors reach levels that can act on
any of four subtypes of adenosine-selective, G-protein-coupled receptors: A1,
A2a, A2b, and A3 [##UREF##4##135##]. Adenosine receptors of all four known subtypes are
expressed differentially on different cell types within the tumor, including
stromal cells, endothelial cells, and infiltrating leukocytes. We have shown
that through such receptors, adenosine can have protumor effects directly on
cancer cells and also indirectly via other
supporting/infiltrating cells [##REF##11992407##136##–##REF##16611738##139##]. Adenosine also acts through A2a and
A2b adenosine receptors on human colorectal carcinoma cells to upregulate CXCR4
mRNA expression up to 10-fold, and selectively increase cell-surface CXCR4
protein up to 3-fold [##REF##16823836##31##]. This increase in cell-surface CXCR4 enables the
carcinoma cells to migrate toward CXCL12 and enhances their proliferation in
response to CXCL12.</p> <p>One of the further
major factors that allows tumor expansion is vascular endothelial growth factor
(VEGF), which is also produced in response to hypoxia and which promotes
neovascularisation of the tumor. The angiogenic effect of VEGF increases the
supply of nutrients and blood-borne growth factors to allow growth of the
tumor. There is significant interplay between the roles of VEGF and CXCR4 in
tumor expansion. Concomitan high expression of CXCR4 and VEGF has been
observed in colorectal [##REF##17641542##74##, ##REF##16675573##75##], breast [##REF##17306924##38##], and ovarian [##REF##15695388##34##] cancers, as well
as in glioma [##REF##17611402##140##] and osteosarcoma [##REF##16528367##91##], in each of which it has been linked
to increased angiogenesis, invasion, and/or metastasis. Clinical studies have
shown that although VEGF and CXCR4 both predispose to lymphatic involvement and
nodal metastasis in colorectal cancer, they work through different regulatory
strategies [##REF##17641542##74##]. Their collaborative role in angiogenesis parallels a similar
joint action in noncancer processes involving neovascularisation (e.g., [##REF##17522382##141##]), and it has been suggested in
the context of tumor angiogenesis that their actions may be synergistic [##REF##15695388##34##].
It is not surprising that these two entities are closely linked; VEGF receptors
and CXCR4 have common regulatory pathways. For example, interference with Notch
signalling leads to downregulation of both VEGF receptor 2 and CXCR4 [##REF##18339870##142##].</p> <p>The
relationship between VEGF and CXCR4 is complex. Firstly, VEGF can promote CXCR4
pathways. VEGF is present in high levels in tumors and may upregulate CXCR4
expression on tumor cells, as has been demonstrated in glioma [##REF##15967572##143##] and breast
cancer [##REF##12499259##144##]. In the case of tumor cells, this upregulation of CXCR4 by VEGF
can happen through an autocrine mechanism [##REF##12499259##144##]. VEGF can also upregulate CXCR4
on the endothelial cells that may be involved in angiogenesis during tumor
expansion [##REF##10233851##145##, ##REF##17075581##146##].</p> <p>Conversely,
the ability of CXCR4 to signal through PI3K/Akt and ERK1/2 provides a route
through which VEGF expression may be regulated by CXCR4 [##REF##17559806##147##–##REF##16005185##149##]. Binding of CXCL12 to CXCR4 has been shown to
increase cellular secretion of VEGF in ovarian cancer [##REF##17697577##150##], breast cancer [##REF##17559806##147##],
prostate cancer [##REF##16005185##149##, ##REF##15180966##151##], and malignant glioma [##REF##16084492##152##]. This phenomenon
parallels the ability of the CXCL12/CXCR4 axis to stimulate VEGF secretion in
normal lymphohematopoietic cells [##REF##11553854##153##]. One might therefore expect a large part of the antitumor activity of
CXCR4 antagonists to be mediated through reduced secretion of VEGF. Indeed,
interference with the CXCL12-CXCR4 pathway has been shown to cause
downregulation of expression of VEGF [##REF##18416455##39##]. However, blocking the CXCL12/CXCR4
axis in vivo can inhibit tumor
growth and angiogenesis without producing alterations in VEGF pathways [##REF##15994964##109##].</p> <p>Other
growth factors whose levels are elevated in tumors may also enhance
CXCR4-dependent mechanisms. Tumors have high levels of tumor necrosis factor-<italic>α</italic>
(TNF-<italic>α</italic>), derived primarily from tumor-associated macrophages (TAMs) [##REF##8119765##154##–##REF##16951987##156##]. TNF-<italic>α</italic> itself, or macrophages that serve as a source of TNF-<italic>α</italic>, are
able to increase CXCR4 mRNA and cell-surface protein expression on ovarian
cancer cells [##REF##16288025##157##] and astroglioma cells [##REF##11160334##158##]. A significant correlation
between TNF-<italic>α</italic> and CXCR4 expression was found in ovarian cancer biopsies [##REF##16288025##157##]. The increase in CXCR4 at a cellular level
appears to be due to TNF-<italic>α</italic>-induced activation of NF-<italic>κ</italic>B signaling and is
associated with enhanced migration towards CXCL12 [##REF##16288025##157##]. Therefore, TAMs may contribute to increased
CXCR4 expression on cancer cells via
production of TNF-<italic>α</italic>.</p> <p>Finally,
polypeptide growth factors that are associated with the extracellular matrix,
and indeed components of the extracellular matrix itself, can upregulate CXCR4
on cancer cells. Transforming growth
factor-<italic>β</italic> (TGF-<italic>β</italic>) increases cell-surface CXCR4 protein expression on human
melanoma cells [##REF##15059909##35##] and we have recently found that FGF-2 upregulates CXCR4 on
human colorectal cancer cells (Bseso B and Blay J, manuscript in preparation). Furthermore, type-I collagen and the
preparation Matrigel, which is a secreted ECM rich in laminin [##REF##15975825##159##], also
increase levels of CXCR4 on melanoma cells [##REF##15059909##35##]. Therefore, interactions with matrix proteins
within tumors may also increase CXCR4 expression.</p> <title>13. THE ROLE OF CYCLOOXYGENASE-2
AND PGE<sub><bold>2</bold></sub> IN CANCER </title> <p>The shift to
malignancy in epithelia and indeed the progression to invasion and metastasis
are associated with increased expression of the enzyme cyclooxygenase-2 (COX-2)
[##REF##7926468##160##–##REF##15623626##163##]. High COX-2 expression is in
cancer is often associated with reduced patient survival [##REF##15623626##163##]. The immediate
effect of high COX-2 expression is increased prostaglandin synthesis, particularly
prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) [##REF##11278747##164##], which in experimental
models is associated with the production of vascular loops and arches and
evidence of abnormal vessel function [##REF##14688410##165##], a phenotype consistent with tumor
angiogenesis. Observations of increased
expression of angiogenic regulatory genes, including VEGF, ang-1, and ang-2 are
consistent with this view [##REF##15930264##166##]. Furthermore, nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit
cyclooxygenases, reduce both tumor incidence and microvessel density in
COX-2-expressing mice [##REF##15930264##166##] and reduce cancer progression in preclinical models
and clinical trials [##REF##11900248##167##]. Indeed, NSAIDs
and COX-2 inhibitors reduce the relative risk of developing colorectal cancer
by 40–50% [##REF##11900248##167##–##REF##15887126##169##].</p> <p>Tumor-promoting
effects of COX-2 overexpression appear to be due in large part to increased PGE<sub>2</sub> production [##REF##8228569##170##–##REF##12455057##173##]. Associated with the increase in COX-2, there is a
decreased expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an
enzyme involved in the inactivation of PGE<sub>2</sub>, in cancer compared to normal tissues [##REF##15542609##174##], as well as
upregulation of cytosolic PLA<sub>2</sub> (cPLA<sub>2</sub>), which increases the supply of arachidonic acid substrate
for COX-2 [##UREF##6##175##–##REF##12048163##177##]. In addition to promoting angiogenesis, PGE<sub>2</sub> also stimulates cancer cell proliferation [##REF##7548186##178##, ##REF##15753380##179##], promotes cell migration [##REF##14499627##180##],
and causes transactivation of polypeptide growth factor receptors [##UREF##7##181##].</p> <title>14. OTHER PROSTAGLANDINS IN CANCER</title> <p>Prostaglandins
together with the thromboxanes are classed as prostanoids, and belong to a larger group of compounds
referred to as eicosanoids [##REF##11729303##182##]. The main prostanoids apart from PGE<sub>2</sub> are prostaglandin F<sub>2<italic>α</italic></sub> (PGF<sub>2<italic>α</italic></sub>), prostaglandin D<sub>2</sub> (PGD<sub>2</sub>), prostaglandin I<sub>2</sub> (PGI<sub>2</sub> or
prostacyclin), and thromboxane A<sub>2</sub> (TXA<sub>2</sub>). As well as
reflecting changes in COX-2, cPLA<sub>2</sub>, and inactivating enzymes, the
levels of different prostanoids in tumors can be modulated by altered expression
of specific prostaglandin synthases [##REF##12738712##183##]. Prostaglandins can also be metabolized
nonenzymatically to form a range of products both in the body and in cell
culture. PGD<sub>2</sub> can be
converted to cyclopentenone J-series prostaglandins, including prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), 9-deoxy-Δ<sup>9</sup>,Δ<sup>12‐13,14</sup>-dihydro-PGD<sub>2</sub> (Δ<sup>12</sup>-PGJ<sub>2</sub>), and 15-deoxy-Δ<sup>12,14</sup>-PGJ<sub>2</sub> (15dPGJ<sub>2</sub>); PGE<sub>2</sub> can be converted to prostaglandin A<sub>2</sub> (PGA<sub>2</sub>) [##REF##6578214##184##–##REF##1789996##186##]. The tumor microenvironment therefore has a rich
and varied content of eicosanoid mediators.</p> <title>15. PROSTAGLANDIN EFFECTS ON CANCER CELLS</title> <p>Although the major
focus of attention has been on PGE<sub>2</sub>, a range of eicosanoids acts to
restrain tumor growth. Indeed the PGE<sub>2</sub> metabolite PGA<sub>2</sub> reduces
cell number and induces apoptosis and cell cycle changes in both human breast
cancer cells and human epithelial cervical carcinoma cells [##REF##12618334##187##].</p> <p>More notably, PGD<sub>2</sub> and its series of derivatives have anticancer effects. PGD<sub>2</sub> itself can reduce the growth of 
carcinoma cells [##REF##9697269##188##]. However, other studies have shown that the nonenzymatic
breakdown of PGD<sub>2</sub> to sequential metabolites (##FIG##0##Figure 1##) may be required
for growth inhibition and that the latter metabolites are the active
eicosanoids [##REF##3857041##189##–##REF##15498850##194##]. PGD<sub>2</sub> therefore can act independently of its DP
receptors by its metabolism through a dehydration reaction to prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), Δ<sup>12</sup>-PGJ<sub>2</sub>, and then to
15-deoxy-Δ<sup>12,14</sup>-prostaglandin J<sub>2</sub>(15dPGJ<sub>2</sub>) [##REF##6578214##184##].
This reaction occurs in cell culture media, both in the presence and absence of
serum [##REF##6578214##184##, ##REF##3857041##189##, ##REF##11786541##195##]. Therefore, it is
possible that many effects noted in
vitro with PGD<sub>2</sub> are actually due to the formation of J-series
prostaglandins. Frequent replacement
with fresh medium containing PGD<sub>2</sub> in such circumstances can
eliminate the response, while the addition of the metabolite(s) themselves
leads to growth inhibition in a shorter timeframe than PGD<sub>2</sub> itself [##REF##3857041##189##].
Some workers have proposed that Δ<sup>12</sup>-PGJ<sub>2</sub> is the key
metabolite [##REF##3857041##189##]; but in fact all of the successive J-series prostaglandins,
that is, PGJ<sub>2</sub>, Δ<sup>12</sup>-PGJ<sub>2</sub>, and 15dPGJ<sub>2</sub>,
are able to reduce proliferation and induce apoptosis of cancer cells [##REF##10506103##190##]. Furthermore, the end metabolite 15dPGJ<sub>2</sub> is active against many cell types, including colorectal carcinoma cells [##REF##10076568##191##, ##REF##11950812##192##],
prostate carcinoma cells [##REF##10984506##193##], and Burkitt lymphoma cells [##REF##15498850##194##], suggesting
that 15dPGJ<sub>2</sub> may be the crucial mediator.</p> <title>16. THE ROLE OF 15dPGJ<sub><bold>2</bold></sub> AND ITS ACTION ON PPAR<italic>γ</italic>
</title> <p>15dPGJ<sub>2</sub> is an agonist for the nuclear receptor peroxisome proliferator-activated
receptor <italic>γ</italic> (PPAR<italic>γ</italic>) [##REF##8521497##196##, ##REF##8521498##197##], and activation of PPAR<italic>γ</italic> may account for the
growth inhibitory effects of 15dPGJ<sub>2</sub>. PPAR<italic>γ</italic> activation results in
its heterodimerization with the retinoid X receptor (RXR), binding to
peroxisome proliferator response elements (PPREs) on DNA, and subsequent
activation of target gene expression [##REF##15629253##198##]. 
PPAR<italic>γ</italic> is aberrantly expressed in some cancer types [##REF##9472692##199##], and in many
cases its activation leads to cell death or differentiation [##REF##10076568##191##, ##REF##9797355##200##, ##REF##9671760##201##]. This
action of 15dPGJ<sub>2</sub>, and by extension its precursors PGD<sub>2</sub>,
PGJ<sub>2</sub>, and Δ<sup>12</sup>-PGJ<sub>2</sub>, may underlie the
major action of these eicosanoids on cell growth. For example, 15dPGJ<sub>2</sub> reduces the growth of PC-3 human prostate cancer cells through the activation
of PPAR<italic>γ</italic> [##REF##16024620##202##]. However, in addition to
direct growth-inhibitory effects, 15dPGJ<sub>2</sub> may also exert anticancer
effects by reducing expression of protumor proteins. For example, 15dPGJ<sub>2</sub> inhibits
phorbol ester-induced VEGF and COX-2 expression in SW620 human colorectal
carcinoma cells [##REF##15289320##203##].</p> <title>17. 15dPGJ<sub><bold>2</bold></sub> CAUSES DOWNREGULATION OF
CXCR4 ON CANCER CELLS</title> <p>In
our studies of the possible effects of these different prostaglandins on CXCR4,
we focused upon the expression of the mature protein and furthermore restricted
our quantitation exclusively to the receptor that is displayed to the external
environment at the cell surface [##REF##16823836##31##]. Cell-surface CXCR4 reflects functional
receptor that is coupled to cellular responses [##REF##16823836##31##] rather than the very large
intracellular pool of inaccessible receptor protein [##REF##15755995##72##].</p> <p>Although
PGF<sub>2<italic>α</italic></sub> (to some extent) and PGE<sub>2</sub> (as well as its product
PGA<sub>2</sub>) have some ability to modulate CXCR4 levels, by far the most
potent prostaglandins in this regard are PGD<sub>2</sub> and its derivatives [##REF##17707368##204##]. Prostaglandin D<sub>2</sub> and the J-series
prostaglandins used at low micromolar concentrations cause substantial loss of
CXCR4 from the surface of HT-29 human colorectal carcinoma cells [##REF##17707368##204##]. In particular, 15dPGJ<sub>2</sub> completely
eliminates cell-surface CXCR4 at a concentration of 10<sup>−5</sup> M in vitro, and has significant effects
after a single dose of 300 nM, about 100-fold less than for PGF<sub>2<italic>α</italic></sub> [##REF##17707368##204##].
The time course of the decline in cell-surface CXCR4 protein is slow, reaching
a maximum only after 48–72 hours (##FIG##1##Figure 2##). The concentrations of prostaglandins
that are needed to cause downregulation after a single dose likely grossly
overestimate the steady-state levels that would cause such a response, as we
have found in other studies with labile metabolites [##REF##16823836##31##, ##REF##15215186##138##]. We estimate that the
effect of 15dPGJ<sub>2</sub> on CXCR4 is achievable with concentrations of
15dPGJ<sub>2</sub> present in vivo.</p> <p>As
can be seen in ##FIG##1##Figure 2##, the response to 15dPGJ<sub>2</sub> occurs more rapidly
than that to PGJ<sub>2</sub>, which in turn has a more rapid onset than PGD<sub>2</sub>.
We further found that each of these prostaglandins does suppress CXCR4 mRNA
expression and that the effect of 15dPGJ<sub>2</sub> again occurs earlier than
that of PGD<sub>2</sub> [##REF##17707368##204##]. The different relative kinetics of the
downregulation of CXCR4 for the J-series prostaglandins are consistent with
data on the conversion of PGD<sub>2</sub> through to 15dPGJ<sub>2</sub> [##REF##3857041##189##] pointing
to 15dPGJ<sub>2</sub> as the key factor in controlling the levels of functional
CXCR4. PGD<sub>2</sub> produces similar downregulation of CXCR4 in other cell
types such as the T47D human breast carcinoma cell line (Richard CL, Blay J,
unpublished observations), suggesting that this may be a common phenomenon. The
downregulation of CXCR4 expression by 15dPGJ<sub>2</sub> differs from 15 dPGJ<sub>2</sub>-mediated
downregulation of other proteins, including cyclin D1 and estrogen receptor <italic>α</italic>,
which has been shown to occur through protein degradation rather than through
changes in transcription [##REF##12615709##205##].</p> <title>18. 15dPGJ<sub><bold>2</bold></sub> DOWNREGULATES CXCR4
PRIMARILY VIA PPAR<italic>γ</italic>
</title> <p>The main target
for 15dPGJ<sub>2</sub> is the nuclear receptor PPAR<italic>γ</italic> [##REF##8521497##196##, ##REF##8521498##197##]. We found that
the ability of 15dPGJ<sub>2</sub> to downregulate CXCR4 occurred primarily
through this pathway. The effect of 15dPGJ<sub>2</sub> was mimicked by PPAR<italic>γ</italic>
agonists such as rosiglitazone (##TAB##1##Table 2##, [##REF##7768881##206##]), and antagonized or blocked by
the PPAR<italic>γ</italic> antagonists GW9662 and T0070907 [##REF##17707368##204##], which are irreversible
inhibitors of PPAR<italic>γ</italic> [##REF##11877444##207##, ##REF##12022867##208##]. A minor part of the downregulatory activity of
15dPGJ<sub>2</sub> was due to the inhibition of NF<italic>κ</italic>B since the 15dPGJ<sub>2</sub> analogue CAY10410 (9,10-dihydro-15-deoxy-Δ<sup>12,14</sup>-prostaglandin J<sub>2</sub>)
[##REF##15755849##209##, ##REF##15695504##210##], which retains the ability to act on PPAR<italic>γ</italic> but lacks the ability of
15dPGJ<sub>2</sub> to inhibit NF<italic>κ</italic>B, was less potent than 15dPGJ<sub>2</sub> [##REF##12022867##208##]. It is the cyclopentenone structure of 15dPGJ<sub>2</sub> (not present in CAY10410) that confers an ability to inhibit NF<italic>κ</italic>B [##REF##10781090##211##].
Consistent with a role for this structure, cyclopentenone itself (but not
cyclopentane or cyclopentene) caused downregulation of CXCR4 [##REF##17707368##204##]. Furthermore,
since PGA<sub>2</sub> possesses the cyclopentenone configuration [##REF##10638762##212##], this
explains the ability of PGA<sub>2</sub> (and that of PGE<sub>2</sub>) to
downregulate CXCR4, although it does not contain the <italic>α</italic>,<italic>β</italic>-unsaturated ketone
moiety necessary to activate PPAR<italic>γ</italic> signaling [##REF##15695504##210##].</p> <p>The
existence of a mechanism of 15dPGJ<sub>2</sub>-induced CXCR4 downregulation
may, in evolutionary terms, be an extension of the anti-inflammatory effects of
15dPGJ<sub>2</sub>. Late in the inflammation process the prostaglandin profile
shifts from a PGE<sub>2</sub>-rich state to a PGD<sub>2</sub>-rich (and
therefore 15dPGJ<sub>2</sub>-rich) state, leading to the resolution of
inflammation [##REF##16084489##213##]. Reduced CXCR4
expression may be an additional mechanism by which 15dPGJ<sub>2</sub> attempts
the resolution of inflammation.</p> <p>It
is clear that this mechanism is not operative in the context of metastatic
tumors, because CXCR4 levels are characteristically high (##TAB##0##Table 1##). Unlike PGE<sub>2</sub> which is present in elevated concentration in tumors [##REF##8228569##170##–##REF##12455057##173##], 15dPGJ<sub>2</sub> levels are likely low in tumors compared to normal tissue. Levels of its precursor
PGD<sub>2</sub> are low in tissues of familial adenomatous polyposis, a
condition that predisposes to colorectal cancer [##REF##9512123##172##], and have been negatively
correlated with hepatic metastasis in tumor tissues taken from patients with
colorectal cancer [##REF##9697269##188##]. The enzyme involved in PGD<sub>2</sub> synthesis, PGD
synthase (PGDS), is decreased in cerebrospinal fluid of brain cancer patients
compared to patients without disease [##REF##9844724##214##]. There is a contested report of
levels of 15dPGJ<sub>2</sub> being decreased during breast cancer progression,
with the lowest levels being detected in metastatic disease [##REF##12455057##173##]. Finally,
mechanisms to sequester or eliminate 15dPGJ<sub>2</sub> may be upregulated in
cancer [##REF##12731885##215##, ##REF##14979731##216##]. Overall, it seems that the predominant prostaglandin within tumors
is PGE<sub>2</sub>, and 15dPGJ<sub>2</sub> may not be present in high levels at
all. Thus, 15dPGJ<sub>2</sub>-dependent suppression of CXCR4 seems to be a
restraint mechanism that is not operative in a cancer situation.</p> <title>19. SYNTHETIC PPAR<italic>γ</italic> AGONISTS DOWNREGULATE
CXCR4 ON CANCER CELLS</title> <p>As indicated
above, the PPAR<italic>γ</italic> agonist rosiglitazone also decreased CXCR4 expression on human
colorectal cancer cells, congruent with an effect of 15dPGJ<sub>2</sub> through
PPAR<italic>γ</italic>. This effect was seen at both the mRNA and protein level, and was more
durable than the effect of 15dPGJ<sub>2</sub>, as it would be expected for a
more chemically stable ligand [##REF##17390024##101##, ##REF##17707368##204##]. Moreover, we found that other
glitazone agents also downregulate CXCR4, with a rank order of potency
(rosiglitazone &gt; pioglitazone &gt; ciglitazone &gt; troglitazone) consistent
with their potencies for interaction with PPAR<italic>γ</italic> [##REF##7768881##206##, ##REF##9830009##217##, ##REF##10871190##218##]. Further confirming that these agents were
acting through their expected target, PPAR<italic>γ</italic>, and that this target is linked to
elimination or reduction of CXCR4 at the cell surface, we showed that the
ability of rosiglitazone to decrease CXCR4 was blocked by the PPAR<italic>γ</italic> antagonists
GW9662 and T0070907 (##TAB##1##Table 2##), or by shRNA knockdown of PPAR<italic>γ</italic> expression in the
cancer cells [##REF##17390024##101##].</p> <p>Therefore,
rosiglitazone and its analogues act through PPAR<italic>γ</italic> to cause substantial and
persistent suppression of CXCR4 on cancer cells. Since these agents are the
same chemicals as the thiazolidinedione (TZD) class of drugs that have been
used clinically for the treatment of diabetes (although recent concerns
regarding side effects have limited their utility), it opens up the possibility
that we may already have a means to manipulate CXCR4 levels in cancer. Given
that CXCR4 expression is linked to metastasis, judicious use of TZDs may allow
us an opportunity to influence the metastatic process (##FIG##2##Figure 3##). Recent studies
have shown that a unique population of CXCR4+ stem cells may be crucial for
expansion of tumor cell populations [##REF##18371365##130##]. We suggest that TZD therapy, by
stimulating PPAR<italic>γ</italic>-dependent downregulation of CXCR4 on cancer cells, may slow
the rate of metastasis and may impact beneficially on disease progression.</p>
<title>ACKNOWLEDGMENTS</title> <p>This work was supported by grants
to Jonathan Blay from the Natural Sciences and Engineering Research Council of
Canada (NSERC), and the Canadian Institutes for Health Research (CIHR); and by
studentship awards to Cynthia Lee Richard from NSERC, the Killam
Foundation, and Cancer Care Nova Scotia.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Production of PGD<sub>2</sub> and conversion
to its metabolites. Prostanoids follow an initial common pathway in which
arachidonic acid is released from membrane phospholipids by phospholipase A<sub>2</sub> and then converted to the short-term intermediates PGG<sub>2</sub> and PGH<sub>2</sub> by cyclooxygenases. Prostaglandin D synthase forms PGD<sub>2</sub> itself, but
subsequent nonenzymatic reactions in aqueous media lead to the sequential
production of prostaglandin J<sub>2</sub> (PGJ<sub>2</sub>), 9-deoxy-Δ<sup>9</sup>,Δ<sup>12‐13,14</sup>-dihydro-PGD<sub>2</sub> (Δ<sup>12</sup>‐PGJ<sub>2</sub>),
and 15-deoxy-Δ<sup>12,14</sup>‐PGJ<sub>2</sub> (15dPGJ<sub>2</sub>).</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Time course of changes in cell-surface CXCR4
protein expression on HT-29 cells by PGD<sub>2</sub> and its metabolites. HT-29 cells were treated with vehicle or
with 10 <italic>μ</italic>M PGD<sub>2</sub> (light gray bars), 10 <italic>μ</italic>M PGJ<sub>2</sub> (dark gray
bars), or 3 <italic>μ</italic>M 15dPGJ<sub>2</sub> (hatched bars), and cell-surface CXCR4
protein expression was measured at the indicated time points. The data shown
are expressed relative to the level of CXCR4 receptor on cells treated with
vehicle alone at that time point. Values have also been corrected for any
possible changes in cell number. The data are mean values ± SE 
(<italic>n</italic> = 4). Significant decrease due to prostaglandin, **<italic>P</italic> &lt; .01;*<italic>P</italic> &lt; .05. The figure is taken from
[##REF##17707368##204##] with permission.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>How PPAR<italic>γ</italic>
downregulation of CXCR4 may act to decrease metastasis. Tumor cells typically have high levels of
CXCR4 at their cell surface. During metastasis, cancer cells that find their
way into the bloodstream lodge in tissues that have high concentrations of CXCL12
(e.g., lungs, liver, and bone marrow). CXCL12 both encourages the entry of
cells into the tissue and promotes growth of the cell population. Downregulation
of CXCR4 by PPAR<italic>γ</italic> activation (endogenous 15dPGJ<sub>2</sub> or
thiazolidinedione drugs, TZDs) will interfere with this process and may impede
metastasis.</p></caption></fig>
<table-wrap id="tab1" position="float"><label>Table 1</label><caption><p>Involvement of CXCL12/CXCR4 in different
cancers.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Cancer</th><th align="left" rowspan="1" colspan="1">Comments</th><th align="center" rowspan="1" colspan="1">References</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Acute lymphoblastic leukemia</td><td align="left" rowspan="1" colspan="1">Levels of CXCR4 are elevated on
lymphoblasts. Elevated levels of CXCR4 are associated with increased
infiltration in liver and spleen</td><td align="center" rowspan="1" colspan="1">[##REF##11736934##58##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Acute myelogenous leukemia</td><td align="left" rowspan="1" colspan="1">High CXCR4 expression is associated with relapse and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##16888090##59##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Brain cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 expression is demonstrated
in tissues and cell lines derived from glioblastoma, medulloblastoma, and
astrocytoma. Cell lines respond to CXCL12 with increased proliferation,
survival and migration. Gliomas expressing CXCR4 are associated with
increased tumor size and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##11591806##41##, ##REF##9808513##60##–##REF##17881971##64##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Breast cancer</td><td align="left" rowspan="1" colspan="1">High CXCR4 expression is noted in breast cancer tissues compared to normal tissues and cell lines with invasive characteristics. CXCR4 expression is associated with more extensive lymph node metastasis and with liver metastasis, although CXCR4 expression in lymph node metastases may be lower than primary cancers. CXCR4 co-expression with HER2/neu is an indicator of more extensive lymph node involvement</td><td align="center" rowspan="1" colspan="1">[##REF##11242036##25##, ##REF##12927045##28##, ##REF##16061624##65##–##REF##16756955##67##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Cervical cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 expression is associated with increased tumor size, stromal invasion, lymph node metastasis, and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##17032700##68##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Chronic lymphocytic leukemia</td><td align="left" rowspan="1" colspan="1">Malignant B cells express 3- to 4-fold higher cell-surface CXCR4 levels than normal B cells. High CXCR4 expression on B cells is associated with reduced survival in patients with familial chronic lymphocytic leukemia</td><td align="center" rowspan="1" colspan="1">[##REF##10602415##69##, ##REF##12150154##70##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Colorectal cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 is over-expressed in colorectal carcinoma tissues compared to normal tissues, and on certain established cell lines. In patients with liver metastasis, higher CXCR4 expression is found on liver metastases compared to the primary tumor. In patients with stage I/II disease, high CXCR4 mRNA expression in tumor samples is associated with increased disease recurrence. In patients with stage IV disease, patients with high CXCR4 have decreased overall survival. High CXCR4 expression is associated with increased lymph node involvement and distant metastasis, as well as reduced 3-year
survival</td><td align="center" rowspan="1" colspan="1">[##REF##15592929##40##, ##REF##12839981##71##–##REF##16675573##75##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Endometrial cancer</td><td align="left" rowspan="1" colspan="1">Endometrial adenocarcinoma
tissues and human cell lines express CXCR4 protein. CXCL12 induces proliferation of endometrial
carcinoma cells</td><td align="center" rowspan="1" colspan="1">[##REF##15146553##76##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Esophageal cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 expression is associated
with reduced survival and increased lymph node/bone marrow metastasis</td><td align="center" rowspan="1" colspan="1">[##REF##16368946##77##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Gastric cancer</td><td align="left" rowspan="1" colspan="1">A majority of primary gastric tumors
and many human gastric carcinoma cell lines express CXCR4. Primary tumors
that express CXCR4 protein are associated with peritoneal carcinomatosis</td><td align="center" rowspan="1" colspan="1">[##REF##16489019##78##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Head and neck squamous cell
cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 expression is found in
tissues and cell lines. High CXCR4 expression is associated with increased
occurrence of distant metastases and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##15837745##79##, ##REF##18357387##80##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Hepatocellular carcinoma</td><td align="left" rowspan="1" colspan="1">CXCR4 is correlated with tumor
progression, metastasis, and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##16819541##81##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Melanoma</td><td align="left" rowspan="1" colspan="1">CXCR4 protein is expressed on
human melanoma cell lines, as well as on cells isolated from melanoma surgical
specimens. CXCL12 enhances cell
adhesion to fibronectin, the binding of murine melanoma cells to endothelial
cells, and invasion of human melanoma cells across basement membranes. CXCR4
expression is associated with reduced disease-free survival and overall
survival</td><td align="center" rowspan="1" colspan="1">[##REF##15059909##35##, ##REF##14583470##43##, ##REF##11571298##82##, ##REF##15756007##83##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Multiple myeloma</td><td align="left" rowspan="1" colspan="1">Multiple myeloma cells isolated
from bone marrow and multiple myeloma cell lines express cell-surface CXCR4
protein. CXCL12 enhances adhesion to fibronectin and stimulates cell
migration</td><td align="center" rowspan="1" colspan="1">[##REF##11154207##84##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Nasopharyngeal cancer</td><td align="left" rowspan="1" colspan="1">Most primary human nasopharyngeal
carcinoma biopsy samples and metastatic lymph nodes stain positively for
CXCR4 protein. Nasopharyngeal carcinoma cell lines also express CXCR4 mRNA</td><td align="center" rowspan="1" colspan="1">[##REF##16000558##85##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Non-Hodgkin's lymphoma</td><td align="left" rowspan="1" colspan="1">Most tissue samples and cell
lines express high levels of CXCR4 mRNA and cell-surface protein. CXCR4 is
implicated in transendothelial migration and proliferation of non-Hodgkin's
lymphoma cells</td><td align="center" rowspan="1" colspan="1">[##REF##12036921##86##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Nonmelanoma skin cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 is expressed on invasive
squamous cell carcinoma and basal cell carcinoma tissues. Expression on invasive squamous cell
carcinoma is increased compared to normal skin</td><td align="center" rowspan="1" colspan="1">[##REF##18312436##87##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Non-small cell lung cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 mRNA is upregulated in
NSCLC tissues compared to normal tissues, and levels are higher in tissue
samples taken from patients with metastasis than from those without
metastasis. Overexpression of CXCR4 in
NSCLC cells leads to enhanced migratory, invasive, and adhesive responses to
CXCL12. Nuclear CXCR4 staining is
associated with longer survival and reduced incidence of metastasis</td><td align="center" rowspan="1" colspan="1">[##REF##16322285##88##, ##REF##15033669##89##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Osteosarcoma</td><td align="left" rowspan="1" colspan="1">CXCR4 mRNA is expressed in most
human osteosarcoma samples, and two of three osteosarcoma cell lines. CXCR4
expression is higher at metastatic sites than in the primary tumor</td><td align="center" rowspan="1" colspan="1">[##REF##15814634##90##, ##REF##16528367##91##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Ovarian cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 mRNA is expressed in
ovarian cancer cell lines, as well as in biopsies from primary tumors and
ovarian cancer ascites. High levels of CXCL12 are present in ascitic fluid
taken from patients with ovarian cancer. CXCL12 stimulates the growth of
ovarian cancer cells. CXCR4 expression is associated with increased
recurrence and reduced survival</td><td align="center" rowspan="1" colspan="1">[##REF##11431324##26##, ##REF##12384559##33##, ##REF##16631235##92##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Pancreatic cancer</td><td align="left" rowspan="1" colspan="1">Most human pancreatic cancer
tissues stain positively for CXCR4 expression, and more than half of
pancreatic cancer cell lines express CXCR4 mRNA and cell-surface protein.
CXCL12 induces chemotaxis of human pancreatic carcinoma cells, as well as
stimulates proliferation and promoted survival</td><td align="center" rowspan="1" colspan="1">[##REF##15548713##42##, ##REF##10999740##93##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Prostate cancer</td><td align="left" rowspan="1" colspan="1">Prostate cancer cell lines
express CXCR4 mRNA and protein, and approximately half of prostate cancer
tissues stain positively for CXCR4. Treatment of cells with CXCL12 increases
their adherence to osteosarcoma cells and bone marrow endothelial cells,
transendothelial migration, and invasion into Matrigel. CXCR4 expression is a
positive predictor of bone metastasis, particularly in patients with elevated
prostate specific antigen (PSA) levels. High CXCR4 expression is associated
with increased cancer-specific mortality</td><td align="center" rowspan="1" colspan="1">[##REF##11912162##29##, ##REF##15467730##36##, ##REF##15240098##94##, ##REF##18201276##95##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Renal cell cancer</td><td align="left" rowspan="1" colspan="1">One of four human renal cell
cancer lines express CXCR4 mRNA, which is upregulated in renal cell cancer tumor
samples compared to normal tissue. High CXCR4 expression is associated with
poor tumor-specific survival, independent of tumour stage and differentiation
grade</td><td align="center" rowspan="1" colspan="1">[##REF##11953881##96##, ##REF##13679920##97##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Rhabdomyo sarcoma</td><td align="left" rowspan="1" colspan="1">Several rhabdomyosarcoma cell
lines express cell-surface CXCR4 protein. CXCL12 increases cell motility,
induces chemotaxis, increases adhesion to extracellular matrix, and
stimulates secretion of MMP-2</td><td align="center" rowspan="1" colspan="1">[##REF##12239174##37##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Small cell lung cancer</td><td align="left" rowspan="1" colspan="1">CXCR4 mRNA and cell-surface protein
are detected in cell lines. CXCL12 induces proliferation, increases adherence
and motility, and induces morphological changes such as filopodia formation</td><td align="center" rowspan="1" colspan="1">[##REF##12414661##98##]</td></tr><tr><td align="center" colspan="3" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Thyroid cancer</td><td align="left" rowspan="1" colspan="1">Human thyroid carcinoma cell
lines express CXCR4 protein, and CXCR4 is upregulated in primary papillary
thyroid carcinomas compared to normal thyroid tissue. CXCL12 increases
proliferation, inhibits apoptosis, and increases migration and invasion of
human thyroid cancer cells</td><td align="center" rowspan="1" colspan="1">[##REF##12519884##99##, ##REF##15184868##100##]</td></tr></tbody></table></table-wrap> <table-wrap id="tab2" position="float"><label>Table 2</label><caption><p>Rosiglitazone
downregulation of CXCR4 on HT-29 cells and suppression by PPAR<italic>γ</italic> antagonists. HT-29 cells were treated with
the PPAR<italic>γ</italic> antagonists (I) GW9662 at 1 <italic>μ</italic>M or (II) T0070907 at 100 nM for 30 minutes before exposure to
rosiglitazone (10 nM). Cell-surface CXCR4 protein expression was measured after
48 hours. The data are mean values ± SE
(<italic>n</italic> = 4). The table is taken from [##REF##17390024##101##] with permission.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1">Experiment </th><th align="center" rowspan="2" colspan="1">PPAR<italic>γ</italic> antagonist</th><th align="center" colspan="2" rowspan="1">Treatment </th><th align="center" rowspan="2" colspan="1">Decrease due to rosiglitazone (%)</th></tr><tr><th align="center" rowspan="1" colspan="1">Control</th><th align="center" rowspan="1" colspan="1">Rosiglitazone</th></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">I</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">2.53 ± 0.14</td><td align="center" rowspan="1" colspan="1">0.95 ± 0.09***
</td><td align="center" rowspan="1" colspan="1">63</td></tr><tr><td align="center" rowspan="1" colspan="1">GW9662</td><td align="center" rowspan="1" colspan="1">2.47 ± 0.22</td><td align="center" rowspan="1" colspan="1">2.43 ± 0.27 n.s.</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="center" colspan="5" rowspan="1">
<hr/>
</td></tr><tr><td align="left" rowspan="2" colspan="1">II</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">1.90 ± 0.17</td><td align="center" rowspan="1" colspan="1">0.81 ± 0.11**
</td><td align="center" rowspan="1" colspan="1">57</td></tr><tr><td align="center" rowspan="1" colspan="1">T0070907</td><td align="center" rowspan="1" colspan="1">2.74 ± 0.17</td><td align="center" rowspan="1" colspan="1">3.07 ± 0.18 n.s.</td><td align="center" rowspan="1" colspan="1">0</td></tr></tbody></table></table-wrap>
<table-wrap-foot><fn id="TF1"><p>Significant change due to
rosiglitazone, ***<italic>P</italic> &lt; .001; **<italic>P</italic> &lt; .01; n.s.: not significant.</p></fn></table-wrap-foot>
<graphic xlink:href="PPAR2008-769413.001"/> <graphic xlink:href="PPAR2008-769413.002"/> <graphic xlink:href="PPAR2008-769413.003"/>
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PPAR Res. 2008 Sep 2; 2008:769413