Báo cáo khoa học: Hyaluronan–CD44 interactions as potential targets for cancer therapy pptx

MINIREVIEW
Hyaluronan–CD44 interactions as potential targets for
cancer therapy
Suniti Misra
1
, Paraskevi Heldin
2
, Vincent C. Hascall
3
, Nikos K. Karamanos
4
, Spyros S. Skandalis
2
,
Roger R. Markwald
1
and Shibnath Ghatak
1
1 Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA
2 Ludwig Institute for Cancer Research, Uppsala University Biomedical Centre, Sweden
3 Department of Biomedical Engineering ⁄ ND20, Cleveland Clinic, Cleveland, OH, USA
4 Department of Chemistry, Laboratory of Biochemistry, University of Patras, Greece
Introduction
Ten years ago, Hanahan and Weinberg [1] proposed
seven hallmarks of cancer shared by most tumor cells,
namely self-sufficiency in growth signals, insensitivity
to anti-growth signals, evasion of apoptosis, limitless
Keywords
cancer; CD44-varient; gene-therapy;
hyaluronan; nanoparticles; stem cells;
shRNA-therapy; tumorigenesis; tumor-

stroma; wound-healing
Correspondence
S. Misra, or S. Ghatak, Regenerative
Medicine and Cell Biology, BSB # 613,
Medical University of South Carolina,
Charleston, SC 29425, USA
Fax: 843 792 0664
Tel: 843 792 8642
E-mail: ;
P. Heldin, Ludwig Institute for Cancer
Research, Uppsala University Biomedical
Centre, Box 595, SE-75124 Uppsala,
Sweden
Tel: 0046 18 160414
Fax: 0046 18 160420
(Received 19 October 2010, revised 18
January 2011, accepted 25 February 2011)
doi:10.1111/j.1742-4658.2011.08071.x
It is becoming increasingly clear that signals generated in tumor microenvi-
ronments are crucial to tumor cell behavior, such as survival, progression
and metastasis. The establishment of these malignant behaviors requires
that tumor cells acquire novel adhesion and migration properties to detach
from their original sites and to localize to distant organs. CD44, an adhe-
sion ⁄ homing molecule, is a major receptor for the glycosaminoglycan hyal-
uronan, which is one of the major components of the tumor extracellular
matrix. CD44, a multistructural and multifunctional molecule, detects
changes in extracellular matrix components, and thus is well positioned to
provide appropriate responses to changes in the microenvironment, i.e.
engagement in cell–cell and cell–extracellular matrix interactions, cell traf-
ficking, lymph node homing and the presentation of growth factors ⁄ cyto-

kines ⁄ chemokines to co-ordinate signaling events that enable the cell
responses that change in the tissue environment. The potential involvement
of CD44 variants (CD44v), especially CD44v4–v7 and CD44v6–v9, in
tumor progression has been confirmed for many tumor types in numerous
clinical studies. The downregulation of the standard CD44 isoform
(CD44s) in colon cancer is postulated to result in increased tumorigenicity.
CD44v-specific functions could be caused by their higher binding affinity
than CD44s for hyaluronan. Alternatively, CD44v-specific functions could
be caused by differences in associating molecules, which may bind selec-
tively to the CD44v exon. This minireview summarizes how the interaction
between hyaluronan and CD44v can serve as a potential target for cancer
therapy, in particular how silencing CD44v can target multiple metastatic
tumors.
Abbreviations
CD44s, standard CD44; CD44v, variant CD44; ECM, extracellular matrix; HA, hyaluronan; HAS, hyaluronan synthase; HGF, hepatocyte
growth factor; HYAL, hyaluronidase; MMP, matrix metalloproteinase; MSCs, mesenchymal stem cells; PEG, polyethylene glycol;
PEI, polyethyleneimine; PI3K, phosphoinositide 3-kinase; shRNA, short hairpin RNA; Tf, transferrin.
FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1429
replicative potential, sustained angiogenesis, tissue
invasion and metastasis. More recently, Kroemer and
Pouyssegur [2] further extended these essential hall-
marks of cancer with altered tumor cell-intrinsic
metabolism, proposing the avoidance of immunosur-
veillance as a result of metabolic reprogramming of
tumor cells as another hallmark of cancer. In addition,
it is now widely recognized that the tumor-associated
stroma contributes to malignant tumor progression
[1,3]. The tumor microenvironment contains many dis-
tinct cell types, including vascular cells, fibroblasts,
immune cells and components of the extracellular

matrix (ECM), i.e. growth factors and cytokines, as
well as structural molecules [4,5]. Tumor cells sense
paracrine signals from the local microenvironment and
communicate these signals with their stromal cells. In
this way, they often alter the cellular and molecular
composition of a particular tumor microenvironment
to promote and maintain tumor progression. Hence,
the notion of the tumor microenvironment as an inte-
grated and essential part of the metastatic phenotype
of carcinoma cells has been the subject of intense
investigation. The disruption of ECM promotes abnor-
mal inter- and ⁄ or intracellular signaling, leading to the
dysregulation of cell proliferation, growth and cyto-
skeleton reorganization [6,7].
The glycosaminoglycan hyaluronan (HA) is a major
component in the ECM of most mammalian tissues,
which accumulates in sites of cell division and rapid
matrix remodeling occurring during embryonic mor-
phogenesis, inflammation and tumorigenesis [8–10]. HA
is found in pericellular matrices attached to HA-synthe-
sizing enzymes or its receptors, and is also present in
intracellular compartments [11–14]. The regulation of
transient interactions of HA with its HA-binding pro-
teins, hyaladherins (both extracellular and cell surface
receptors), is crucial for fundamental physiological pro-
cesses, e.g. embryonic development, but also for patho-
logical conditions in which HA affects cell
proliferation, migration and differentiation [10,15,16].
The adhesion ⁄ homing molecule CD44, which is
implicated in cell–cell and cell–matrix adhesion, is the

major cell surface receptor for HA. CD44 proteins
exist in three states with respect to HA binding: non-
binding; nonbinding unless activated by physiological
stimuli; and constitutively binding [17–19]. HA induces
signaling when it binds to constitutively activated
CD44 variants (CD44v) [20,21]. CD44 can also react
with other molecules, including collagen, fibronectin,
osteopontin, growth factors and matrix metallopro-
teinases (MMPs), but the functional roles of such
interactions are less well known [22]. CD44 is a trans-
membrane protein encoded by a single gene, but, as a
result of alternate splicing, multiple forms of CD44 are
generated that are further modified by N- and O-linked
glycosylations (Fig. 1). The smallest CD44 isoform
that lacks variant exons, designated standard CD44
(CD44s), is abundantly expressed by both normal and
Fig. 1. Structure, binding domains and interactions of CD44. The ectodomain of CD44 contains hyaluronan-binding motifs and is decorated
with chondroitin ⁄ heparan sulfate that both affect its hyaluronan-binding capacity and enable its interactions with growth factors ⁄ growth fac-
tor receptors and matrix metalloproteinases (MMPs). Transmembrane and cytoplasmic domains undergo multiple post-translational modifica-
tions, including palmitoylation and phosphorylation on cysteine and serine residues, respectively, promoting the binding of proteins with
crucial functions in cytoskeletal organization and signaling. ErbB2, epidermal growth factor receptor-2; ERM, ezrin–radixin–moesin; FGF, fibro-
blast growth factor; HGF, hepatocyte growth factor; IQGAP1, IQ motif containing GTPase activating protein 1; MAPK, mitogen-activated pro-
tein kinase; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; TGFR, transforming growth factor receptor;
VEGF, vascular endothelial growth factor.
Targeting CD44 variants in tumors S. Misra et al.
1430 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS
cancer cells, whereas the CD44v isoforms that contain
a variable number of exon insertions (v1–v10) at the
proximal plasma membrane external region are
expressed mostly by cancer cells. In addition, the

CD44 ectodomain can be decorated with chondroitin
sulfate and ⁄ or heparan sulfate enabling CD44 to bind
growth factors, including fibroblast growth factor, vas-
cular endothelial growth factor or hepatocyte growth
factor (HGF) [22,23]. The rather short cytoplasmic tail
of CD44 binds to ankyrin and ezrin–radixin–moesin
proteins, providing a link to the cytoskeleton, as well
as to merlin, which abrogates this binding. However,
the multiple cellular functions of CD44 rely on its
association with partner proteins that regulate cell
migration, growth, survival and differentiation. CD44
is endogenously expressed at low levels on various cell
types of normal tissues [24,25], but requires activation
before binding to HA [17,18,26–29]. The CD44 struc-
ture of normal cells is distinct from that of cancer cells
because pathological conditions promote alternate
splicing and post-translational modifications to pro-
duce diversified CD44 molecules with enhanced HA
binding which lead to increased tumorigenicity [30–36].
Glycosylation is required for spliced variant formation
of CD44, which has high affinity to bind HA on cer-
tain cell types, whereas glycosylations rich in sialic acid
decrease HA binding [37–39]. For example, circulating
lymphocytes express CD44, but do not bind HA until
CD44 is deglycosylated on lymphocyte activation
[37,40,41] and, to internalize CD44, it must be acylated
[42]. This diversification of CD44v functions allows the
production of specific targeting agents that will be use-
ful for both diagnosis and therapy. Systemic applica-
tion of antibodies directed against the variant 6

epitope and the expression of antisense CD44v6 retard
colon tumor growth and metastasis in vivo [43,44]. The
overexpression of the variant, high-molecular-mass iso-
forms CD44v4–v7 and CD44v6–v9 in various cancers
[45–52], as well as the downregulation of CD44s in
colon cancer, are postulated to result in increased
tumorigenicity [53], emphasizing the potential impor-
tance of CD44 splice variants in cancer.
In this article, we review the tumorigenic actions of
HA and its receptor CD44 that occur extensively in
several malignant conditions. We also discuss potential
therapeutic interventions for the development of tar-
geted therapies based on an understanding of the com-
munication between HA and cell surface CD44. In
particular, we highlight possible roles in HA–CD44v-
induced tumor growth and invasion, together with
fresh insights into the enigmatic nature of CD44 splice
variants, and how the suppression of HA–CD44v
interactions may be a therapeutic target.
HA and CD44 in tumor initiation and
progression
Influence of HA in tumorigenesis
Reactive stroma in cancer is often characterized by an
increase in cancer-associated fibroblasts ⁄ myofibroblasts
that produce an array of growth factors and chemokin-
es, and amplify the synthesis of HA and proteoglycans
such as versican. The interaction of the anti-adhesion
molecule versican with HA and CD44 promotes the
expansion of the pericellular matrix. These complexes
increase the viscoelastic nature of the pericellular

matrix, creating a highly malleable extracellular envi-
ronment that supports the cell shape change necessary
for cancer cell proliferation and migration. Further-
more, versican, via its chondroitin ⁄ dermatan sulfate
side-chains, is highly polyanionic, which amplifies the
hydration of the environment caused by HA [54]. A
large number of studies performed during the last three
decades have demonstrated a close correlation between
malignancy and HA-rich ECM, as well as with CD44s
and CD44v expression. CD44 in cancer cells interacts
with HA-rich microenvironments, which affects cell sig-
naling pathways that trigger the ability of malignant
cells to migrate, to invade basement membranes ⁄ ECM
and to lodge at distant sites of the tumor [14,22,23,55–
58] (see also the interesting series of reviews on the
Web Science of Hyaluronan Today at http://www.
glycoforum.gr.jp). However, the underlying molecular
mechanism whereby HA–CD44 cooperation influences
the malignant phenotype and contributes to tumor
progression is not yet clear.
Divergent mechanisms control the expression of
hyaluronan synthase (HAS) genes in response to stim-
uli, and each HAS synthesizes HA molecules of differ-
ent size and amount in a cell-type and context-specific
manner. The study of HAS2-knockout mice [9,59]
clearly demonstrated that HA deposition in the ECM
was required for embryonic heart valve morphogenesis.
In HAS2-null embryos, the endocardial cushion cells
failed to undergo epithelial-to-mesenchymal transition
and did not migrate to the cardiac jelly. This is partly

a result of the lack of HA–CD44-induced Ras signal-
ing. Importantly, this phenotype was seen only for the
HAS2 isoform, indicating functional differences among
the three HASs. The HA-synthesizing capacity of
HASs and, specifically, HAS2 can be regulated by
dimerization and ubiquitination [60]. In this study, the
mutation of the HAS2 lysine residue 190, which is one
major acceptor site for ubiquitin, led to total inactiva-
tion of its enzymatic activity. The different roles of the
three HAS isoforms are also likely to be related to
S. Misra et al. Targeting CD44 variants in tumors
FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1431
different expression patterns [61]. Studies of nonmalig-
nant cells overexpressing different HASs revealed that
the high levels of HA induced by HAS3 were inversely
correlated with cell motility and CD44 expression [62].
Importantly, the overproduction of HA in cancer cells,
such as fibrosarcomas, breast cancer, mesotheliomas
and prostate cancer, transfected with HAS1, HAS2
and HAS3 genes, triggered intracellular signaling path-
ways that promoted anchorage-independent growth
and invasiveness, which correlated with increased
expression of CD44 [63–66]. HAS1 and its splice vari-
ants were detected in multiple myeloma patients, but
not in healthy donors, and were associated with poor
survival of the patients [67]. Invasive and ⁄ or metastatic
breast cancer cells deprived of HAS2 lost their aggres-
sive phenotype [68]. These and many other studies, not
referred to here because of space limitations, suggest
that HA contributes in several ways to the hallmark

properties of malignancy, especially anchorage-inde-
pendent growth and invasiveness.
Although many studies have shown the importance
of HASs in tumor growth and malignant progression,
other studies have suggested a more complex role of
HA. For example, HAS2 overexpression was found to
suppress the tumorigenesis of glioma cells lacking hyal-
uronidase (HYAL) activity [69], and HYAL1 expres-
sion promoted the HAS-mediated growth suppression
and metastatic ability of prostate cancer cells [70].
Notably, the overexpression of HAS2 in colon carcino-
mas that possessed HYAL1 activity promoted, whereas
the overexpression of HYAL1 suppressed, tumorigene-
sis in an experimental model of colon carcinoma [71].
In addition, the HA content in tissues was well corre-
lated with the tumor growth rate. Additional observa-
tions support the notion that HYAL1 can have both
tumor-promoting and tumor-suppressing functions
[72]. It is possible that excess HA synthesis and degra-
dation in concert promote the metastatic phenotype of
certain tumor types. However, the HA content in clini-
cal samples is not always statistically correlated with
tumor grade, suggesting that the transformation-
induced HA overproduction may be a result of differ-
ential upregulation of HAS isoforms and ⁄ or HYALs
at different stages of malignant transformation. Recent
work utilizing the mouse mammary tumor virus-Neu
transgenic model conditionally expressing HAS2 high-
lighted the role of HA in the promotion of the malig-
nant phenotype. The growth rate of mammary tumors

increased and an HA-rich intratumoral stroma was
formed, which most probably established interactions
between tumor and stromal cells that promoted angio-
genesis and lymphangiogenesis [73,74]. This and other
mouse models will be useful to further study the mech-
anisms regulating tumor–stroma interplay and stromal
targeting therapy. It should also be mentioned that
there is a connection between HA catabolism and
energy generation, most probably allowing HA to
function as an alternative energy source to glucose for
malignant cells [75]. Such metabolic reprogramming of
tumor cells could add a further dimension to the
importance of HA in cancer progression.
Function of CD44 in tumor initiation and
metastatic behavior
The increased deposition of HA in tumors is not a pas-
sive process during malignancy; rather, it triggers sig-
naling events and promotes the association between
CD44 and other cell surface receptors that become acti-
vated or inhibited either directly or indirectly through
HA-activated CD44 [14,16,57,76]. Early studies by us
and other laboratories revealed that aggressive breast
carcinomas expressed high levels of CD44s and CD44v,
as well as increased synthesis of HA [77,78]. More
recent studies have highlighted the importance of CD44
molecules in the onset of malignant transformation.
There is now increasing evidence that a small popula-
tion of tumor cells (less than 0.1%), referred to as can-
cer stem cells or cancer-initiating cells, exhibit stem cell
properties, i.e. are responsible for maintaining the

tumor and, possibly, for the formation of new tumors
at metastatic loci. CD44 has been identified as an
important marker of such a population of cancer stem
cells in breast, pancreas and colorectal cancers [79–81].
Together, these findings suggest that CD44 plays an
important role in the initiation and ⁄ or maintenance of
cancer stem cells in some tumors.
Specifically, CD44s interacts with growth factor
receptors, such as epidermal growth factor receptor-2
and platelet-derived growth factor receptor. Most
importantly, the binding of HA to CD44s either stimu-
lates [82,83] or inhibits [84] tyrosine phosphorylation
by the associated tyrosine kinase receptors. Most prob-
ably, the binding of HA to CD44 causes clustering,
which triggers differential downstream events depen-
dent on cell type and tissue context. Such clustering
appears to be important for the trapping of MMP9
and the subsequent activation of transforming growth
factor-b, which affects oncogenic functions including
invasion and angiogenesis [85]. Moreover, the cluster-
ing of CD44 also occurs on extensive N- and O-gly-
cosylations of the variant ectodomain of CD44 that
can affect the binding of HA to CD44 [22,23]. How-
ever, there are also indications of clustering-indepen-
dent signaling via CD44. Thus, HA dodecasaccharides,
which most probably are unable to induce CD44
Targeting CD44 variants in tumors S. Misra et al.
1432 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS
clustering, induce chemokine CXCL1 secretion, result-
ing in endothelial cell sprouting in a CD44-dependent

manner [86]. During tumor progression, HAS and
HYAL activities give rise to HA molecules of high or
low molecular mass, with the capacity to bind differen-
tially to CD44 and thereby modulate its function. This
complexity may explain why CD44 expression is not
correlated with tumor aggressiveness in neuroblasto-
mas and prostate cancer [23].
As the detailed description of the expression of
CD44v isoforms from less malignant to more advanced
stages is beyond the scope of this minireview, we high-
light the relevance of CD44v isoforms in cancer which
seem to be suitable targets for anti-cancer therapy. In
several primary and cancer cells, CD44v6 forms a ter-
nary complex with HGF and its receptor c-Met. Most
probably, CD44v6 presents HGF to its receptor, trig-
gering receptor kinase activity and signaling pathways
involving the binding of ezrin to ezrin–radixin–moesin
proteins, and thus actin cytoskeleton binding and Ras
activation. HGF elicits metastatic behavior in various
types of cells, mostly in a paracrine fashion. In a
recent study, we found that insulin-like growth fac-
tor 1, transforming growth factor, prostaglandin E2
and tumor necrosis factor-a, secreted by prostate can-
cer cells, stimulated the synthesis of HGF by myofi-
broblasts. HGF, in turn, stimulated the production of
splice variant 9 of CD44. The interaction of stromal-
derived HA with the upregulated CD44v9 initiated sig-
naling pathways that stabilized androgen receptor
functions and induced anti-apoptotic signaling [87].
Colon cancer cells exhibit the same mechanism, but

utilize CD44v6 (S. Misra et al., unpublished results).
Silencing the appropriate CD44v inhibits tumor cell
adhesion to the tumor cell matrix and in vitro tumor
cell invasion [87]. Cross-talk between the increased HA
synthesized by the stromal cells, which interacts with
colon tumor cell CD44v6, sustains HA–CD44v6–phos-
phoinositide 3-kinase (PI3K) signaling through a posi-
tive feedback loop between CD44v6 and PI3K that
induces invasiveness. In addition, we have demon-
strated that stromal-derived HGF stimulates the syn-
thesis of metalloproteinase (MT1MMP), which induces
shedding of CD44v, and promotes colon ⁄ prostate can-
cer cell invasiveness (S. Misra et al., unpublished
results; depicted in the model in Fig. 2). Thus, thera-
peutic approaches using HA–CD44v interaction with
CD44v short hairpin RNA (shRNA) can target tumors
at one or more of these levels: the microenvironment
(stromal factors such as HGF and its inducers); recep-
tor-based signals (select CD44v, Met ⁄ RTK); and signal
transducers, such as PI3K ⁄ AKT or mitogen-activated
protein kinase (Fig. 2).
CD44 and HA in tumors: wounds that
do not heal
The tumor microenvironment contains many distinct
cell types, including endothelial cells and their
precursors, pericytes, smooth muscle cells, fibroblasts,
carcinoma-associated fibroblasts, myofibroblasts, neu-
trophils ⁄ eosinophils ⁄ basophils ⁄ mast cells, T ⁄ B lympho-
cytes, natural killer cells and antigen-presenting cells,
such as macrophages and dendritic cells [4]. The micro-

environment of a solid tumor closely resembles the
environment of wound healing and tissue repair sites
of an injured tissue. On tissue injury, platelets are acti-
vated. These activated platelets release vasoactive
mediators for vascular permeability, serum fibrinogen
for fibrin clot formation and growth factors ⁄ cyto-
kines ⁄ matricellular proteins to initiate granulation tis-
sue formation, activate fibroblasts, and induce and
activate MMPs necessary for ECM remodeling. Epi-
thelial and stromal cell types engage in a reciprocal sig-
naling cross-talk to assist healing. The reciprocal
signaling collapses after the wound is healed. In the
case of tumorigenesis, the invasive inflammatory tumor
cells produce an array of cytokines ⁄ chemokines
that are mitogenic for granulocytes ⁄ monocytes ⁄ macro-
phages ⁄ fibroblasts ⁄ endothelial cells. These factors
Fig. 2. Proposed model for the cross-talk between tumor cells (epi-
thelial cells) and tumor-associated stromal myofibroblasts. Cancer
cells and stroma-derived fibroblasts influence each other’s develop-
ment. The extracellular domain of CD44 variants, which contains
the sequence encoded for variants of CD44 and their interaction
with HA, is required for the stromal factor-dependent activation of
receptor tyrosine kinases (RTK, such as hepatocyte growth fac-
tor ⁄ Met) and its downstream anti-apoptotic signaling involving
phosphoinositide 3-kinase (PI3K) ⁄ AKT and mitogen-activated pro-
tein kinase (MAPK) pathways. Tumor-associated stromal myofibro-
blast-derived hyaluronan, synthesized in response to stromal
factors (such as hepatocyte growth factor) and cancer cell-derived
CD44 variant, and RTK are involved in tumorigenesis.
S. Misra et al. Targeting CD44 variants in tumors

FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1433
(cytokines ⁄ chemokines) potentiate tumor growth, stim-
ulate angiogenesis, induce fibroblast migration and
enable metastatic spread. During this process, non-
hematopoietic mesenchymal stem cells (MSCs) origi-
nating from bone marrow localize to the sites of
hematopoiesis, sites of inflammation and sites of
injury, as well as to solid tumors [88–90]. Inactivated
MSCs have been shown to inhibit tumor growth by
inhibiting a PI3K ⁄ AKT pathway in an E-cadherin-
dependent manner, prompting the use of these cells as
tumor inhibitory cells in vivo [91], whereas activated
MSCs within the solid tumors are the source of carci-
noma-associated fibroblasts that contribute to tumor
growth in several ways [92,93]. Tissue injury and
inflammation are accompanied by increased production
of stromal HA, which, in addition to cell–cell and cell–
matrix adhesion [94,95], and cell proliferation and sur-
vival [10,83,87,96], helps to create highly hydrated
ECM that may facilitate local cellular trafficking
[97,98]. In the bone marrow, HA is also abundantly
produced by both stromal and hematopoietic cells.
CD44, in addition to its function to regulate cell prolif-
eration ⁄ differentiation ⁄ survival ⁄ migration into tissues,
is implicated in hematopoietic progenitor trafficking to
the bone marrow and spleen [99–101]. The concept of
the use of MSCs as delivery vehicles originates from the
fact that tumors, similar to wounds, produce chemo-
attractants, such as cytokines ⁄ chemokines (e.g. vascular
endothelial growth factor, transforming growth factor-

b), to recruit MSCs to form the supporting stroma of
the tumor, and also pericytes for angiogenesis. MSCs
transfected with the interferon- b gene can increase the
production of interferon-b at the local site [102,103].
Likewise, Herrera et al. [104] presented a convincing
case indicating that interactions between CD44 and HA
influence the homing of exogenous MSCs that localize
to the kidneys during acute renal failure, i.e. CD44 on
exogenous cells is important in helping MSCs to local-
ize to the damaged renal tissue in vivo. However, this
in vivo function of MSCs depends partly on signals
from the target tissue microenvironment, i.e. endothe-
lial progenitor cells were used as gene delivery vehicles
to the site of angiogenesis rather than to the quiescent
vasculature [105]. On the basis of these observations, it
is possible to deliver immune-activating cytokines ⁄
secreted proteins to the site of tumors through MSCs
[103]. As human MSCs can be easily expanded in vitro
and retain an extensive multipotent capacity for differ-
entiation [106,107], in a recent study, we found that
genetically engineered human MSCs which secrete solu-
ble CD44v that acts as an antagonist to HA–CD44v
signaling inhibit malignant properties in cancer cells
(S. Misra et al., unpublished results). These studies and
co-implantation models combining tumor cells and
MSCs [102,103,108] hold great promise for therapeutic
strategies [106], in which the interaction between tumor
and stroma can be manipulated and studied (the
concept of using MSCs for tumor therapy is depicted in
the model in Fig. 3).

Therapeutic strategy involving
perturbation of HA–CD44 interactions
Importance of targeting CD44v in vivo
CD44v interaction with HA is known for its role in
the metastatic cascade, as this interaction regulates
the ability of malignant cells to activate receptor tyro-
sine kinases, and to stimulate migration, invasion of
basement membranes ⁄ ECM and migration to distant
sites [22,57,109–115]. HA induces intracellular signal-
ing when it binds to constitutively activated CD44v
during cell dynamic processes, but does not do so
under conditions of adult tissue homeostasis, which
generally involves CD44s. The CD44 structure on
normal cells is distinct from that on cancer cells
because, under various physiological and pathological
conditions, the local environmental pressure (stromal
factors) influences alternate splicing and post-transla-
tional modification to produce diversified CD44
molecules [35,36]. This diversification allows the pro-
duction of specific targeting agents that will be useful
for both diagnosis and therapy. Pathological condi-
tions that stimulate alternate splicing and post-transla-
tional modifications produce diversified CD44
molecules with enhanced HA binding that leads to
increased tumorigenicity [30–36]. The systemic applica-
tion of antibodies directed against a CD44v epitope
[43] reduced the metastasizing activity of a pancreatic
adenocarcinoma. The overexpression of variant, high-
molecular-mass isoforms CD44v4–v7 and CD44v6–v9
in various cancers [45–52], as well as the downregula-

tion of the CD44s isoform in colon cancer, has been
postulated to result in increased tumorigenicity [53],
emphasizing the potential importance of CD44 splice
variants in cancer.
Inhibition of HA–CD44 interactions
To explore the mechanism of constitutive HA–CD44
interactions and the consequent outcomes in cancer
cells, four different methods were used. The first
method uses small HA oligosaccharides (2.5 kDa)
that compete with the endogenous HA polymer
[83,96,110,116–118]. The second method overexpresses
soluble HA-binding proteins (e.g. soluble CD44) that
Targeting CD44 variants in tumors S. Misra et al.
1434 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS
act as competitive decoys for CD44 and thus bind to
endogenous HA [83,96,110,116,117]. The third method
blocks the HA–CD44 interaction specifically by treat-
ing the cells with a blocking antibody against the
HA-binding site of CD44 [96,104,117,119]. The fourth
method inhibits the post-transcriptional expression of
CD44v with CD44 siRNAs [83,87,110,112,119,120].
Although these methods yield valuable information on
how epithelial cell-derived HA and its interaction with
CD44v can influence malignant properties in vitro, they
do not address the tumor cell responses to cell-specific
perturbation of the HA–CD44v interaction at the
genetic level in vitro and in vivo. In addition, by using
the CD44 siRNA to interrupt HA–CD44v6 signaling
processes at a cellular level [110,112], it has been
observed that the phenotypic changes induced by siR-

NAs only persist for 1 week because of a lack of trans-
fer of siRNA or the dilution of siRNA concentration
after each cell division, or a lack of stability of siRNA,
which limits their use in the inhibition of tumor pro-
gression in vivo. Moreover, the dose of siRNA remains
undefined, and the induction of innate immune
responses is another obstacle that will obscure the use
of siRNAs as therapeutics.
Strategies that target CD44 to perturb HA–CD44
interactions in tumors [121]
HA-conjugated drugs
CD44 can internalize HA [122]. Thus, HA-carrying
drugs alone or encapsulated drugs in liposomes have
the potential to be used as targeted drugs, as well as
drug transport vehicles. Chemical groups of HA, such
as the carboxylate on glucuronic acid, the N-acetylglu-
cosamine hydroxyl and the reducing end, can poten-
tially be used to conjugate a drug [123]. HA–drug
conjugates are internalized via CD44, and the drug is
released and activated mainly by intracellular enzy-
matic hydrolysis [124–126]. Activated CD44 is overex-
pressed on solid tumors, but not on their
nontumorigenic counterparts. Several preclinical stud-
ies have shown that HA chemically conjugated to cyto-
toxic agents improves the anticancer properties of the
agent in vitro [125,127,128]. Drugs with low solubility
can be successfully applied when conjugated with HA.
For instance, the antimitotic chemotherapeutic agent
paclitaxel has low water solubility. On conjugation to
HA, its solubility and CD44-dependent cellular uptake

increase in vitro [126].
Fig. 3. Bone marrow-derived nonhematopoietic human mesenchymal stem cells (hMSCs) are pluripotent cells that are capable of differenti-
ating into various tissue lineages, including osteoblasts, adipocytes, chondrocytes, myoblasts, hepatocytes and possibly even neural cells
[107]. After systemic injection, hMSCs can selectively migrate to solid tumors, where they proliferate and become cancer-associated stromal
myofibroblasts [103]. As hMSCs can be easily expanded in vitro and possess an extensive multipotent capacity for differentiation, they have
been explored as vehicles for tissue repair and gene therapy [106], when they are appropriately engineered for therapy. We established that
tissue-specific floxed plasmid ⁄ nanoparticle delivery is efficient for the activation of a gene of interest [120]. In a pilot study (S. Misra et al.,
unpublished results) using genetically modified hMSCs in nanoparticles, the tropism was altered, because the secreted proteins from trans-
duced hMSCs interacted with stromal hyaluronan, and thus inhibited the malignant properties of cancer cells by more than 20-fold by per-
turbing hyaluronan–CD44v interaction.
S. Misra et al. Targeting CD44 variants in tumors
FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1435
HA-conjugated nanocarriers
HA, when conjugated to a nanocarrier, acts as a pro-
tective structural component and a targeting coating.
The circulation time and biodistribution (pharmacoki-
netic properties) are influenced by incorporation of the
targeting and cell-specific uptake properties of HA
onto large carriers. Cargo liposomes or nanoparticles
delivered to CD44-overexpressing cells include anti-
cancer drugs (epirubicin [127], doxorubicin [129–139],
paclitaxel [125,126] and mitomycin c [127,133]) as well
as siRNA [140,141]. Results from the above studies
using HA-targeted nanocarriers do not differ from
many of the studies performed with HA–drug conju-
gates.
Targeting with anti-CD44 antibodies
Anti-CD44 antibodies against highly expressed vari-
ants can actively target drugs to CD44, inhibit and dis-
rupt CD44–matrix interactions, occupy CD44 and

induce CD44 signaling, which can cause apoptosis
[142]. Anti-CD44 antibodies targeting ligands for either
radiolabels or anti-cancer chemotherapeutics partially
stabilize some patients [143,144]. CD44v6 is expressed
in breast, cervical and colon cancers, and in squamous
cell carcinomas. Thus CD44v was chosen as a model
for therapy. A Phase 1 clinical trial was performed
with an immunotoxin (humanized antibody coupled
with a cytotoxic drug mertansine) against CD44v6 in
30 patients with incurable squamous cell carcinoma
[76].Three patients showed a partial response and it
was thought that the trial was successful. Unfortu-
nately, one of the patients died, and the trial was
abruptly withdrawn.
Tissue-specific deletion of CD44v signalling
The technique of using shRNA in an expression vector
is an alternative strategy to stably suppress selected
gene expression, which suggests that the use of shRNA
expression vectors holds potential promise for thera-
peutic approaches for silencing disease-causing genes
[145]. There are two ways to deliver shRNA in cancer
cells: using either a viral vector or a nonviral vector.
Viral vectors have been used to achieve this proof of
principle in animal models and, in selected cases, in
human clinical trials [146]. Systemic targeting by viral
vectors to the desired tissue is difficult because the host
immune responses activate viral clearance. Systemic
administration of a large amount of adenovirus (e.g.
into the liver) can be a serious health hazard, which
even caused the death of one patient [146]. Neverthe-

less, there has been considerable interest in developing
nonviral vectors for gene therapy. In this regard, non-
viral vectors, such as positively charged polyethylenei-
mine (PEI) complexes shielded with polyethylene
glycol (PEG), can be used safely to avoid the nonspe-
cific interactions with nontarget cells and blood com-
ponents [147]. Nonviral vectors were once limited
because of their low gene transfer efficiency. However,
the incorporation of various ligands, such as peptides,
growth factors and proteins, or antibodies for targets
highly expressed on cancer cells, has circumvented this
obstacle [148]. In addition, enhanced permeability
caused by the aberrant vasculature in solid tumors,
and retention (known as the enhanced permeability
and retention effect) of ligand-coated vectors around
the receptors of tumor cells, can increase the chances
for a high probability of interaction with the cells
[120]. Thus, nonviral vectors can acquire high gene
transfer efficiency [120]. This concept has been tested
by preparing nonviral vector nanoparticles with plas-
mids packed inside an outer PEG–PEI layer coated
with transferrin (Tf), an iron-transporting protein
[120,148], which binds with Tf receptors (Tf-R) with
high affinity. Tf-R is present at much higher levels on
tumor cells [120] than on phenotypically normal epi-
thelial cells. The association of Tf with nanoparticles
significantly enhances the transfection efficiency of
shRNA generator plasmids by promoting the internali-
zation of nanoparticles in dividing and nondividing
cells through receptor-mediated endocytosis [148].

Finally, the uptake of nanoparticles carrying multi-
ple functional domains (surface-shielding particles Tf–
PEG–PEI, shRNA generator plasmids, tissue-specific
promoter-driven Cre recombinase and conditionally
silenced plasmid) can overcome the intracellular barri-
ers for the successful delivery of the shRNA gene.
The newly developed cell-specific shRNA delivery
approach by Misra et al. [120] confirmed that the tar-
geting of the HA–CD44v6-induced signaling pathway
inhibited distant colon tumor growth in Apc Min ⁄ +
mice. Tissue-specific shRNA delivery was made possi-
ble by the use of Cre recombinase produced in
response to a colon tissue-specific promoter, which
deletes the interruption between the U6 promoter and
the CD44v6shRNA oligonucleotide. This approach,
depicted in the model in Fig. 4, has successfully dem-
onstrated that CD44v6shRNA is localized to the colon
tumor cells by an endpoint assay of CD44v6 expres-
sion, and by perturbation of HA–CD44v6 interaction,
as reflected in the reduction in the number of tumors
[120]. In our recent in vivo studies with C57Bl ⁄ 6 mice,
we are optimistic that the systemic delivery of a
mixture of two plasmids in Tf ⁄ nanoparticles (pARR
2
-
Targeting CD44 variants in tumors S. Misra et al.
1436 FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS
probasin-Cre ⁄ nanoparticles and floxed pSico-CD44v
9shRNA ⁄ nanoparticles) will target both localized and
metastatic prostate cancer cells (S. Ghatak et al. ,

unpublished results). This novel approach opens up
new ways to combat cancer, and to understand tumor-
igenesis in vivo, for the following reasons: (a) the
cell-specific release of shRNA by the application of a
tissue-specific promoter-driven Cre-lox mechanism; (b)
silencing of the expression of the selected CD44v in
target tissue cancer cells; (c) no effect on normal target
tissue cells, which do not express targeted CD44v and
rely on the CD44s form, which is not affected by the
plasmids; (d) the target CD44vshRNA is not expressed
in other types of cells because the tissue-specific pro-
moter only unlocks the Cre recombinase in the tar-
geted tissue cells, thereby reducing potential side-
effects [120]; (e) the nanoparticles that carry plasmids
are biodegradable and cleared from the system; (f) it
addresses the pathophysiological role of HA–CD44v
interactions in cancer; (g) it can establish diagnostic
markers for the targeted cancer, including CD44v, sol-
uble CD44 and HA; and (h) it can establish CD44v–
HA interactions as an innovative novel therapeutic tar-
get against cancer progression. Thus, the conditional
suppression of gene expression by the use of a
CD44vshRNA-expressing plasmid holds potential
promise for therapeutic approaches for silencing HA–
CD44v signaling, and hence the downstream signaling
that promotes disease-causing genes [145] (Fig. 4).
Advantages of the tumor-specific delivery of
CD44vshRNA versus other therapeutic strategies
First, this technique avoids the multiple chemical steps
needed to prepare HA-conjugated cytotoxic drugs and

conjugation to nanocarriers. Second, it abolishes
CD44v in cancer cells only. Third, a number of cell
types in normal tissues that express CD44 are not
affected because they are not activated. Fourth, inflam-
mation-associated cancers accumulate activated
immune cells having upregulated Tf receptors and
CD44v. However, they may take up the nanoparticles,
but no deletion of CD44v will take place because the
promoter is not lymphocyte specific (S. Misra et al.,
unpublished results). To target activated lymphocytes,
specific promoter-driven Cre plasmids should be used.
Fifth, the accumulation of antibody in nontumor areas
is a major limitation of anti-CD44 antibody therapy.
Experiments so far have not produced any such effect
in shRNA delivery.
Fig. 4. Model for delivery of short hairpin RNA (shRNA). This illustration depicts the cellular uptake of plasmid transferrin–polyethylene gly-
col–polyethyleneimine (Tf–PEG–PEI) nanoparticles and the mechanism of action of shRNA. First, a pSico vector containing a U6 promoter-
loxP-CMV-GFP-STOP signal-loxP-CD44vshRNA (gene of interest) is made. Second, an expression vector containing the Cre recombinase
gene controlled by the tissue-specific promoter is created. Third, the two vectors are packaged in Tf-coated PEG–PEI nanoparticles that bind
with Tf receptors (Tf-R) present at high levels in the targeted tumor cells. The delivery of the vectors in normal and malignant cells from the
targeted tissue results in the deletion of the STOP signal and the transcription of Cre recombinase driven by the tissue-specific promoter.
The target gene (CD44vshRNA) is then unlocked and transcribed through the strong U6 promoter for high expression. The normal tissue
cells are not affected because they do not make the targeted CD44 variant.
S. Misra et al. Targeting CD44 variants in tumors
FEBS Journal 278 (2011) 1429–1443 ª 2011 The Authors Journal compilation ª 2011 FEBS 1437
Concluding remarks
Despite the increasing number of studies conducted so
far, a complete understanding of HA–CD44-induced
signaling still remains elusive. However, both HA and
CD44 appear to be vitally important from embryogene-

sis to morphogenesis, in inflammation and in cancer,
which accompanies the overexpression of CD44 and its
splice variants and the aberrant synthesis ⁄ turnover of
HA. On the basis of the above-mentioned functions of
HA and its interaction with CD44, it seems likely that
the impact of HA–CD44 and its variant-induced tumor
growth is multifactorial. Importantly, CD44v-induced
proteolysis [24,149] of the matrix facilitates the detach-
ment of malignant tumor cells from their confined
tumor area, and therefore promotes the spread of malig-
nant tumor cells to distant sites. Moreover, partial deg-
radation of HA molecules promotes angiogenesis, a
vital requirement for tumor growth. Furthermore, by
providing increased tissue hydration, HA molecules
provide a suitable environment to support malignant
cell migration, similar to cardiac cushion cell movement
[9,59,150–153]. In summary, CD44 and, more specifi-
cally, CD44v are promising target molecules for therapy
and diagnosis, at least in some tumors.
Acknowledgements
This work was supported, as a whole or in part,
by the National Institutes of Health Grants
P20RR021949 (to SG) and P20RR016434 (to SM, SG
and RRM), HL RO1 33756 and 1 P30AR050953 (to
VCH). This work was also supported by Mitral-07
CVD 04 (to RRM), Medical University of South
Carolina University Research Council Project
2204000-24330 (to SM) and 2204000-24329 (to SG).
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