Multifunctional CD40L: Pro- and anti-neoplastic activity

Abstract

The CD40 ligand is a type I transmembrane protein that belongs to a tumor necrosis factor (TNF) superfamily. It is present not only on the surface of activated CD4+ T cells, B cells, blood platelets, monocytes, and natural killer (NK) cells but also on cancer cells. The receptor for ligand is constitutively expressed on cells, TNF family protein: CD40. The role of the CD40/CD40L pathway in the induction of body immunity, in inflammation, or in hemostasis has been well documented, whereas its involvement in neoplastic disease is still under investigation. CD40L ligand may potentiate apoptosis of tumor cells by activation of nuclear factor-κB (NF-κB), AP-1, CD95, or caspase-depended pathways and stimulate host immunity to defend against cancer. Although CD40L has a major contribution to anti-cancer activity, many reports point at its ambivalent nature. CD40L enhance release of strongly pro-angiogenic factor, vascular endothelial growth factor (VEGF), and activator of coagulation, TF, the level of which is correlated with tumor metastasis. CD40L involvement in the inhibition of tumor progression has led to the emergence of not only therapy using recombinant forms of the ligand and vaccines in the treatment of cancer but also therapy consisting of inhibiting platelets-main source of CD40L. This article is a review of studies on the ambivalent role of CD40L in neoplastic diseases.

Full text

REVIEW
Multifunctional CD40L: pro- and anti-neoplastic activity
Aleksandra Korniluk &Halina Kemona &
Violetta Dymicka-Piekarska
Received: 6 June 2014 /Accepted: 27 July 2014 /Published online: 13 August 2014
#The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract The CD40 ligand is a type I transmembrane protein
that belongs to a tumor necrosis factor (TNF) superfamily. It is
present not only on the surface of activated CD4+ T cells, B
cells, blood platelets, monocytes, and natural killer (NK) cells
but also on cancer cells. The receptor for ligand is constitu-
tively expressed on cells, TNF family protein: CD40. The role
of the CD40/CD40L pathway in the induction of body immu-
nity, in inflammation, or in hemostasis has been well docu-
mented, whereas its involvement in neoplastic disease is still
under investigation. CD40L ligand may potentiate apoptosis
of tumor cells by activation of nuclear factor-κB(NF-κB),
AP-1, CD95, or caspase-depended pathways and stimulate
host immunity to defend against cancer. Although CD40L
has a major contribution to anti-cancer activity, many reports
point at its ambivalent nature. CD40L enhance release of
strongly pro-angiogenic factor, vascular endothelial growth
factor (VEGF), and activator of coagulation, TF, the level of
which is correlated with tumor metastasis. CD40L involve-
ment in the inhibition of tumor progression has led to the
emergence of not only therapy using recombinant forms of the
ligand and vaccines in the treatment of cancer but also therapy
consisting of inhibiting platelets-main source of CD40L. This
article is a review of studies on the ambivalent role of CD40L
in neoplastic diseases.
Keywords CD40L .CD40 .Inflammation .Apoptosis .
Cancer
Abbreviations
TNF Tumor necrosis factor
TRAF TNF receptor-associated factor
TAP Transporter associated with antigen processing
RANTES Regulated on activation, normal T cell expressed
and secreted
VEGF Vascular endothelial growth factor
FGF-2 Fibroblast growth factor 2
TF Tissue factor
APCs Antigen-presenting cells
DC Dendritic cells
NK cell Natural killer cells
GM-CSF Granulocyte-macrophage colony-stimulating
factor
CLL Chronic lymphocytic leukemia
MM Multiple myeloma
Introduction
The CD40 ligand (CD40L), also known as CD154, T-B
activating molecule (TBAM), tumor necrosis factor (TNF)-
related activation protein (TRAP), and gp39, is a type I
transmembrane protein, a member of the TNF superfamily,
together with lymphotoxin-α(LT-α, TNF-β), LT-β, FasL,
CD30L, CD27L, and 4-1BBL 9 [1]. The CD40L gene is
located on chromosome X (q26.3–q27.1) [2], contains five
exons and four introns, and the protein that is produced
(29 kDa) via translation is built up of 261 amino acids. The
cell membrane presents the 32–33-kDA form, which indicates
the posttranslation modification of the protein [3].
CD40L associated with cell membrane contains a globular
TNF-like extracellular domain, long extracellular domain
(ECD), short transmembrane domain (TMD), and a small
cytoplasmic intracellular domain (ICD). [3] The extracellular
structure of CD40L composed of beta sheet-alpha helix-beta
sheet that is arranged in the so-called Greek key motif is
specific to the TNF superfamily [4]. If CD40L is produced
A. Korniluk (*):H. Kemona :V. Dymicka-Piekarska
Department of Clinical Laboratory Diagnostics, Medical University
of Bialystok, Bialystok, Poland
e-mail: alex.korniluk@wp.pl
Tumor Biol. (2014) 35:9447–9457
DOI 10.1007/s13277-014-2407-x

as a single polypeptide chain, it can be found on the cell
surface as a homotrimeric complex. The long protein chain
is accompanied by two shorter forms of CD40L, which
through proteolytic hydrolysis are released from the cell mem-
brane to the circulation, where they form soluble trimmers,
sCD40L, having the receptor-binding ability [4,5]. Since both
the surface ligand and its soluble form have structural do-
mains, they show high biological activity. Due to the presence
of the KGD sequence (lysine-glycine-aspartic acid), they can
bind to the GPIIb–IIIa receptor, and therefore, the ligand may
play a role in platelet activation and stimulate further release
of sCD40L [6,7]. The trimeric protein structure facilitates
signal transfer to the cell interior via binding to the receptor,
whereas the TNF-like domain allows ligand binding to its
main receptor-CD40. The presence of the ligand has been
observed, first of all, on the surface of activated CD4+ T cells,
but also on activated B cells and blood platelets, monocytes,
natural killer (NK) cells, adipose cells, and basophils during
inflammation [8]. It is assumed that over 95 % of CD40L
originates from blood platelets [9]. Henn et al. [9]haveshown
that CD40L is stored in platelet granules αand released after
their activation and degranulation.
The CD40 receptor is a transmembrane type I protein
belonging to the TNF family, encoded by the gene located
on chromosome 20 (q12–q13.2). It is present on the cell
surface as a trimeric complex (40–45 kD) [10]. Structurally,
CD40 contains a long ECD, transmembrane region, and short
C-terminal cytoplasmic fragment [11]. Since the cytoplasmic
part does not exhibit kinase activity, signals are transmitted
mainly through the ligand-dependent recruitment of adaptor
proteins of the TNF receptor-associated factor (TRAF) family
[12]. The CD40 receptor has been found on the surface of B
cells and on the membrane of the antigen-presenting cells
(APCs), epithelial cells, endothelial cells, smooth muscle
cells, fibroblasts, basophils, and blood platelets, where it is
constitutively expressed [13,14]. However, expression of the
receptor can be induced by the combination of TNF-αwith
interferon (IFN)-γ. These molecules significantly increased
de novo expression of CD40 on human endothelial and vas-
cular smooth muscle cells, and this process is mediated by the
simultaneous activation of nuclear factor-κB (NF-κB) by
TNF-αand STAT-1αby IFN-γ[15,16]. Activation of
CD40 receptor by CD40 ligand on epithelial cells leads to
the secretion of cytokines and chemokines by these cells,
whereas on fibroblasts and endothelial cells, it contributes to
their proliferation [17]. Interaction betweenCD40 and CD40L
can be inhibited by specific antibodies like 4D11, a novel fully
human anti-CD40 mAb which is produced by genetically
modified mice. Also, lucatumumab and Chi 220 are anti-
CD40 antibodies which inhibit ligation between receptor and
ligand [18].
Neither CD40L nor its receptor CD40 can function inde-
pendently, and only their interaction leads to the production of
intracellular signal that is responsible for enhanced humoral
and cellular response and for the cellular production of cyto-
kines, chemokines, and adhesion proteins [19].
CD40L in the immune response and inflammation
The first studies on the roleof CD40Lin the immune response
were performed when the ligand was detected on the surface
of T and B cells, confirming its involvement in cellular and
humoral immune response. The activation of B cells by
CD40L turned out to be essential in the process of lymphocyte
proliferation, differentiation, and maturation and in isotope
switching [10]. The interaction of CD40L with CD40 exerts
an effect on the production of cytokines (IL-6, IL-10, TNF-α,
LT-α), expression of adhesion molecules and co-stimulatory
receptors (intercellular adhesion molecule (ICAM), CD23,
B7.1/CD80, B7.2/CD86), as well as class I MHC, class II
MHC, and transporter associated with antigen processing
(TAP) present on B cells [20].
The significant role of the CD40L-CD40 system in the
immune response was confirmed with the discovery of
CD40L gene mutation, leading to the hyper-IgM syndrome
(HIGM). In these patients, overproduction of IgM antibodies
and lack of IgG, IgA, and IgE antibodies can be observed [3].
The binding of CD40L to CD40 present on endothelial
cells and fibroblasts increases the secretion of metalloprotein-
ases and activates the production of chemokines (IL-8, MCP-
1, macrophage inflammatory protein (MIP)-1α,RANTES)
and cytokines (IL-1, IL-6, IL-12, and TNF-α), which attract
lymphocytes to the site of inflammation and increase expres-
sion of adhesion molecules (ICAM-1, VCAM-1, and E-
selectin), responsible for the recruitment of monocytes and
lymphocytes, thus leading to their accumulation in the internal
vascular membrane [21].
Interaction between ligand and receptor causes overexpres-
sion of cycloxygenase 2 (COX-2) and prostaglandin E2
(PGE2) [22]. The soluble form of CD40L is able to activate
blood platelets and stimulate the release of β-thromboglobulin
(β-TG) and 5-hydroxytryptoamine from their granules,
whereas cell membrane CD40L causes the release of regulated
on activation, normal T cell expressed and secreted
(RANTES), the protein of strong pro-inflammatory proper-
ties, from the platelets [23,24].
The evidence for the involvement of the CD40/CD40L
pathway in inflammation includes highly elevated expression
of these molecules in inflammatory conditions of the colon,
such as Lesniewski-Crohn disease or ulcerative colitis [19].
The pro-inflammatory and pro-thrombotic activities of the
CD40/CD40L system have been also shown in diabetes
[25], atherosclerosis [26], and cardiovascular diseases [27].
High level of sCD40L can be a prognostic risk factor of death,
heart infarct, and recurrent angina in acute coronary
9448 Tumor Biol. (2014) 35:9447–9457

syndromes and future cardiovascular episodes in healthy
women [28]. It is also a reliable marker for the population of
patients with acute coronary syndrome at a high risk of heart
incidents [29].
CD40L in neoplastic disease
The role of the CD40/CD40L pathway in the induction of
body immunity, in inflammation, or in hemostasis has been
well documented, whereas its involvement in neoplastic dis-
ease is still controversial.
Studies on CD40L have revealed that it enhances anti-
neoplastic immune response of the body, inhibits tumor
growth, and induces apoptosis of cancer cells [30,31]. Subse-
quent reports have suggested that in many cancers, CD40
activation by its ligand results in a completely reverse situa-
tion, i.e., enhancement of tumor growth and progression
(Fig. 1)[32,33]. The effect induced by CD40L has appeared
to depend not only on the type of cells that show the receptor
expression but also on the strength of the signal transmitted by
the ligand. High signal (the cell has many CD40 molecules)
indicates apoptosis of cancer cells, whereas low signal (a small
number of receptors) CD40L promotes cancer growth [34].
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Fig. 1 This figure illustrates some of the mechanisms by which CD40L
can influence on different cell functions and the processes by which these
altered cells can impact on host immunity in neoplastic diseases. CD40L
is present as a membrane-bound form (mCD40L), present first of all on T
lymphocytes (CD4+,CD8+,iNKTc,andNKc), and as a soluble form
(sCD40L) derived primarily from active platelets. Interaction of the
ligand with the receptor (CD40) may not only potentiate the anti-tumor
immunity but also promote the development of cancer. The binding of
CD40L with a receptor on dendritic cells (DC) leads to the expression of
co-stimulatory molecules necessary for the correct antigen presentation
and protects DC against apoptosis induced by factors derived from tumor
cells (TC). Moreover, CD40L/CD40 interaction enhances the prolifera-
tion and maturation of lymphocytes B (Bcells) and the production of
antibodies against the tumor. mCD40L present on various cells induces
apoptosis of tumor cells via the TRAF-JNK/AP-1-caspase-9/caspase-3
pathway. On the other hand, the activation of endothelial cells (EC)may
result in enhance TF pro-coagulant activity and high expression of VEGF,
main mediator of tumor angiogenesis
Tumor Biol. (2014) 35:9447–9457 9449

CD40L and cancer progression
CD40L has a major contribution to immunological activity;
however, many reports point at its ambivalent nature in neo-
plastic diseases. On one hand, the ligand activatesthe immune
system to combat the cancer, but on the other, it stimulates
tumor progression, growth, and metastasis formation.
Induction of angiogenesis by CD40L induces VEGF
production
Tumor growth and metastasis depend on angiogenesis and
lymphangiogenesis caused by different factors released from
host and tumor cells [35]. Binding of CD40L to CD40 present
on endothelial cells leads to the expression of strongly pro-
angiogenic factors, such as vascular endothelial growth factor
(VEGF) or fibroblast growth factor 2 (FGF-2) that can pro-
mote in vivo angiogenesis. [36–38]. VEGF participates in
mobilization of endothelial stem cells, which take part in
formation of new blood vessels in tumor microenvironment
[35]. In addition, VEGF increases expression of tissue factor
(TF), which also promotes blood coagulation. Moreover,
VEGF induces monocyte chemotaxis and activation and also
impairs the immune system functions through the inhibition of
dendritic cell maturation [39]. In gastric cancer, ligation of
CD40 by CD40L causes up-expression of VEGF by PI3K
pathway [40]. CD40L/CD40 interaction also has been shown
to induce COX-2 expression in cells and subsequent VEGF
production, what was confirmed by Miura et al. [41]. Tai YT
et al. [42] suggested that in human multiple myeloma (MM)
cells, CD40 activation by CD40L can induce secretion of
VEGF by p53 pathway. In this case, VEGF stimulates IL-6
secretion in bone marrow stromal cells and thereby augments
paracrine IL-6-mediated MM cell growth. What is more,
Farahani et al. [43] shows that in cells from patients with
chronic lymphocytic leukemia (CLL), CD40L also can up-
regulate production of VEGF, which leads to CLL cell sur-
vival. This process depends, in part, on NF-κB activation.
CD40L-induced survival of malignant cells depends on com-
bined signaling by CD40 and VEGF receptor (VEGFR).
Inhibition of CD40L-induced production of VEGF and cyto-
kines (IL-6) and activation of signaling pathways, prolifera-
tion, and survival of CLL [44] and MM [42] cells is caused by
lucatumumab (HCD122). HCD122 binds to CD40 and block-
ade CD40/CD40L interactions that induce apoptosis and me-
diate antibody-depended cellular toxicity on lucatumumab-
bound CD40-expressed malignant cells. This antibody is cur-
rently in phase I/II clinical trials in CLL. VEGF can also
enhance cleavage of membrane-bound CD40L and cause
increased level of sCD40L. Inhibition of this process is pos-
sible through the use of bevacizumab-anti-VEGF antibody
[18].
Hematological malignancy
The activation of CD40 contributes to the increased survival
and resistance to chemotherapy of follicular lymphoma, hairy
cell leukemia, and CLL cells [45–47]. It is suggested that the
co-stimulation of IL-4 and CD40L causes long-term prolifer-
ation of B cells and short-term proliferation and increased
percentage of coat cells in hairy cell leukemia [48]. Kato
et al. [49] showed that such interaction in diffuse non-
Hodgkin’s lymphoma depending on the type of cancer cell
line enhanced short- or long-term proliferation of cancer cells.
Research conducted on the established cell lines (GDLBGCB-
1 and GDLBGCB-2) and cells taken from the patients has
proved that lack of IL-4 or introduction of antibodies against
CD40L causes inhibition of cell proliferation. In some types
of CLL, cell proliferation does not depend on endogenous
CD40L, but on the stimulation with the ligand from tumor
microenvironment. Pham et al. [50] suggest that endogenous
CD40L which is present on aggressive B cells of lymphoma
binds to CD40 and the signal transferred into the cell activates
the NF-κB pathway.
The effect of CD40L on the action of many drugs has been
reported. The CD40L induces resistance to a number of anti-
cancer drugs, including doxorubicin or vinblastin. In non-
Hodgkin’
s lymphoma, lack of reaction to drugs is caused by
caspase-independent and independent pathways [51], whereas
in breast cancer, only by the caspase-dependent route [52]. A
study conducted on transgenic mice showed that the admin-
istration of clopidogrel, a drug inhibiting platelet aggregation
and CD40L release, decreased tumor size [53]. This may
suggest that tumor growth inhibition by the drug is associated
with an indirect decrease in CD40L expression [51]. The
sCD40L/CD40 pathway activation in gastric cancer inhibited
Fas- and drug-dependent apoptosis and also increased the
motility of CD40-positive cancer cells, thus facilitating the
formation of metastases, what was confirmed in humans [54].
Rui Li et al. [55], who investigated the expression of CD40
and CD40L on gastric cancer cells from patients, suggest that
a simultaneous expression of the two molecules and perma-
nent activation of the CD40/CD40L pathway have a major
significance in neoplastic progression. CD40 activation in-
hibits apoptosis and promotes spread of transformed cells.
Likewise, in cancer of the urinary bladder, the activity of the
CD40/CD40L system protects cells against apoptosis and
increases their survival, which is associated with the inhibition
of CD95-dependent apoptosis [56]. The assessment of CD40
expression on the surface of pancreatic cancer cells revealed
that at stage I of the disease, the cells were CD40-negative,
whereas in patients with lymph node involvement and distant
metastases, the cells exhibited high expression of CD40. Also,
the serum level of sCD40L in these patients was significantly
elevated. The two molecules correlated with cancer stage,
which may indicate their involvement in the disease
9450 Tumor Biol. (2014) 35:9447–9457

development. The use of shrCD40L on pancreatic cancer cell
lines significantly inhibited their proliferation and enhanced
apoptosis, although the effect was most pronounced in the
cells showing high CD40 expression [57].
Solid tumors
Rosseli et al. [58] showed a high level of sCD40L in patients
with adenocarcinoma and squamous cell carcinoma of the
lungs, with the highest level of the ligand observed in patients
with distant metastases of the latter. Moreover, sCD40L
showed a positive correlation with the pro-thrombotic system
components, i.e., fragments of pro-thrombin 1 and 2 (F1+ 2),
and the thrombin/anti-thrombin complex (TATc). The re-
searchers forwarded the hypothesis that the pro-thrombotic
agents released from lung cancer cells stimulate platelet acti-
vation and release of sCD40L, which in turn interacts with
other types of CD40-positive cells, including tumor-
associated macrophage (TAM) cells and cancer cells of the
lungs. Also, Amirkhosravi et al. [59] suggested that the inter-
action of CD40L present on blood platelets with CD40 on
tumor cells may potentially promote activation of coagulation
on human melanoma cells A375 through the effect of in-
creased expression of TF.
Anti-neoplastic stimulation of immune response
Research on CD40L indicates that the inhibition of cancer
progression can be associated with the activation of two
mechanisms by the ligand, namely, stimulation of anti-
neoplastic immune response and induction of apoptosis of
transformed cells.
The enhancement of immune response is inextricably as-
sociated with the activation of antigen-presenting cells
(APCs), especially dendritic cells, monocytes, and B cells,
showing CD40 expression. Cancer cells release a number of
cytokines and soluble agents that have an immunosuppressive
effect and cause APC dysfunction [60]. Most data seem to
confirm a beneficial effect of CD40L/CD40 interaction on the
function of dendritic cells (DC). It has been proved that
CD40L/CD40 binding protects DC against apoptosis, by in-
ducing the expression of the anti-apoptotic molecules Bcl-2 or
serpin serine protease inhibitor 6 (SPI-6) [61,62]. The appli-
cation of recombinant CD154 therapy leads to the formation
of mature dendritic cells, increased expression of adhesion
and co-stimulatory molecules (ICAM-1, CD83, CD80/86),
and secretion of pro-inflammatory cytokines (TNF-α,IL-6,
IL-12) and chemokines (IL-8, MIP-1α), which confirms an
exceptionally important role of the ligand in the activation of
these cells [63]. CD40L, together with pro-inflammatory cy-
tokines and INF-γ, is indispensable for the presentation of
foreign antigens absorbed by dendritic cells (cross-priming) to
T cells. It has been suggested that IL-12 and INF-γregulate
the expression of CD154 on effector cells, whereas the ele-
vated level of the ligand may modulate NK cell cytotoxicity
[64]. The ligand binding to the receptor present on monocytes
and macrophages is associated with the activation of numer-
ous immune processes involving these cells [65].
The activation of CD40 by CD40L enhances the expres-
sion of TLR-9 on macrophages, whereas its stimulation by
sCD40L increases the activity of monocytes, especially in
cancer of the uterine cervix, which is related to the activation
of the NF-κB and MARK pathways [66]. Studies on the
stimulation of CD40 present on cancer cell lines in the blad-
der, pancreas, or breast through the recombinant CD40L have
shown increased expression of ICAM and Fas by cancer cells
and the production of IL-6, IL-8, GROα,GM-CSF,and
TNF-α. The incubation of transformed cells with the expres-
sion of CD40 and CD40L has been found to considerably
inhibit their proliferation, disturbances in the cell cycle, and
decreased cell viability [66]. Moreover, the administration of
anti-CD40 antibodies has been observed to activate macro-
phages, thus enabling them to inhibit proliferation of melano-
ma B-16 cells and to stimulate them to interferon production
[67]. CD40 binding to CD40L not only leads to the increased
production of cytokines by immune cells, but also stimulates
many types of cancers to release pro-inflammatory proteins.
Cells of Hodgkin’s lymphoma due to CD40 activation release
IL-8, IL-6, or TNF, which in consequence can increase Tcell-
dependent anti-neoplastic immune response [68]. Also, in
acute lymphoblastic leukemia, the activation of CD40 present
on cancer cells by CD40L enhances the secretion of MDC and
TARC; i.e., the proteins that play a role of chemoattractans for
CCR4+ T cells [69]. These lymphocytes possess CCR4 re-
ceptor on such chemokines as MPC-1, MIP-1, or RANTES.
The presence of this receptor also allows migration of lym-
phocytes to the skin. High percentage of CCR4+ T cells have
been observed in malignant skin lymphomas, such as mycosis
fungoides or Sezary’ssyndrome[70].
CD40L activation-dependent apoptosis of cancer cells
Apart from its effect on the body immunity, the CD40L/CD40
system can inhibit cancer growth via enhanced apoptosis of
cancer cells. Interestingly, binding of CD40L by CD40 in-
duces the apoptosis of cancer cells, but not of the healthy ones.
CD40L prevents death of fibroblasts, dendritic cells, mono-
cytes or B cells, and enhances the death of transformed mes-
enchymal cells, epithelial cells, neurons, or hepatocytes. This
phenomenon is referred to as “activation-induced cell death”
(AICD) [31,71,72]. The ligand induces the apoptosis of
CD40-positive cancer cells. The presence of CD40 molecule
was first identified on cancer cells of urinary bladder.
Tumor Biol. (2014) 35:9447–9457 9451

Subsequent research showed CD40 expression on cancer cells
of the breast [73], ovary, intestines [74], liver [75], glioma
[17], nasopharynx [76], melanoma [77], or lymphoma.
As shown by literature data, cells with CD40 on their
surface very seldom express CD40L. Moreover, various can-
cer cell lines exhibit a varied level of CD40, which is associ-
ated with cancer stage and usually decreases with disease
progression [30]. The receptor present on the transformed
cells can bind to the cell membrane-bound ligand (mCD40L),
soluble CD40L (sCD40L), recombinant CS40L (srhCD40L),
and anti-CD40 antibody. The effect induced by the ligand-
receptor interaction depends on the type of cells on which the
molecules are located and on the form of the ligand that binds
to the receptor.
The CD40L/CD40 pathway may activate CD95-dependent
apoptosis, which has been proved in the research on neuro-
blastoma, and via the activation of caspase-8 that is induced
by recombinant CD40L administration [78]. Investigations of
normal and transformed epithelial cells of the urinary tract
indicate that the activation of cancer cells by mCD40L, but not
by sCD40L, leads to the growth and stabilization of TRAF-3
and causes activation of caspase-9 and caspase-3. In healthy
cells, CD40L does not induce apoptosis but leads to a decrease
in the activity of TRAF-3 and TRAF-2 [79]. The TRAF-2
factor exerts a positive effect on differentiation of B cells,
whereas TRAF-3 inhibits the growth and blockade of NF-κB
factor in cancer cells [79]. Other authors have investigated the
significance of CD40 expression on cancer cells of the urinary
tract. The incubation with CD40L leads to the apoptosis of
transformed cells, whereas decreased expression of CD40 on
these cells is associated with enhanced cancer progression
[80].
It has been shown that CD40L may enhance apoptosis of
solid tumor cells and transformed cells of blood cancers [17,
79].
Hematological malignancy
The CD40/CD40L interaction on human and murine B cell
lymphoma resulted in the arrest of cell cycle, which had a
major significance for the induction and maintenance of tumor
quiescent state. Also, in the case of Burkitt lymphoma, CD40
activation caused tumor growth inhibition, increased activity
of Fas, and enhanced apoptosis of tumor cells [46]. Tong et al.
[31] have demonstrated the expression of CD40 by MM cells
and blockage of their progression by the use of srCD40L
therapy. In turn, Young et al. [81] have suggested that the loss
of CD154 expression on the myeloma cells leads to the
ligand-dependent loss of epithelial growth regulation and
uncontrolled inflammations and infections, which may con-
tribute to the increased and uncontrolled susceptibility of
patients to the development of cancer. It has been also ob-
served that despite preserved expression of CD40 by oral
basal epithelial cells, the loss of CD154 expression may
contribute to epithelial metastasis [76]. Also, in patients with
CD40L gene mutation (HIMN), an increase was noted in the
incidence of cancer of the liver, pancreas, or bile ducts, which
seems to confirm the Young et al. theory [82].
Solid tumors
It is assumed that the level and type of the cytokines secreted
by cancer cells depend on whether the ligand is membrane-
bound or soluble. Numerous studies have shown that binding
of both mCD40L and sCD40L to the receptor CD40 leads to
the activation of NF-κB, although in the case of the transcrip-
tion factor AP-1, the pro-apoptotic cascade is induced only by
mCD40L [74]. The activation of NF-κBfactorstimulatesthe
release of IL-8, whereas AP-1 enhances the production of
GM-CSF [75].
The results obtained by Georgopoulos et al. [74] indicate
that the interaction between mCD40L and CD40 on colon
cancer cells leads to the formation of a strong pro-apoptotic
signal. Interestingly, the authors have revealed that this effect
is induced only by mCD40L binding. Apoptosis of colon
cancer cells was not observed when they were CD40-
negative and subject to the action of sCD40L [79,80].
mCD40L interaction with CD40-positive cancer cells was
accompanied by increased production of IL-8 by transformed
cells. It has also been shown that in the course of colon cancer,
mCD40L affects the expression of GM-CSF. Growth inhibi-
tion or death in carcinoma cells mediated by CD40 can be
accompanied also by induction of IL-6 production [74]. These
cytokines have an ambivalent character. On one hand, up-
regulated production of cytokines in response to CD40 acti-
vation can enhance the immunogenicity through augmented
presentation of tumor-associated antigens by the tumors and
increasing recruitment of immune cells into tumor sites, which
was observed after CD40L stimulation [83]. Enhance produc-
tion of IL-6, IL-8, TNF-α, and GM-CSF and corresponding
caspase activation can contribute to CD40-dependent tumor
growth inhibition [31]. On the other hand, prolonged produc-
tion of IL-6 and GM-CSF and activation NF-κB pathway can
exacerbate the inflammation and induce cancer progression
through intensifying angiogenesis and inhibition of trans-
formed cells apoptosis.
CD40L inhibits in vitro melanoma cell proliferation by
inducing their apoptosis or stimulating the production of
cytokines that activate the immune system (IL-6, IL-8,
TNF-α) by cancer cells [77]. In another study, two indepen-
dent teams assessed the role of the ligand and CD40 receptor
activation in breast cancer. The results reported by Hirano
et al. [84] suggest that the action of the CD40/CD40L pathway
may inhibit the growth and enhancement of apoptosis of
transformed cells. The use of genetically modified CD40L-
srhCD40L by these authors caused death of cancer cells via
9452 Tumor Biol. (2014) 35:9447–9457

Fas activation. In turn, Tong et al. [73]haverevealedthat
breast cancer cell apoptosis does not require the presence of
INF-γ, whichwas earlier suggested by Wingett et al. [85]. The
authors showed that modified sCD40L by binding on cancer
cells inhibits tumor growth, whereas the receptor-free trans-
formed cells do not undergo apoptosis.
Some authors believe that the CD40L/CD40 pathway may
inhibit cancer progression through only one mechanism, i.e.,
enhanced immune response or apoptosis induction. However,
most researchers favor the theory that anti-neoplastic activity
is only possible when both mechanisms by CD40L are in-
volved in anti-cancer therapy.
Since CD40L and CD40 molecules are strong
immunostimulators, they are considered useful in anti-
cancer therapy. Studies on novel methods of treatment
have been conducted on murine models, using anti-CD40
and anti-CD40L antibodies, recombinant CD40L, and
transformed viral vectors, carrying encoding sequences/
ligand genes or vaccines, to the production of which
dendritic cells containing tumor peptides and CD40L
gene are used [86,87].
The activation of antigen-presenting cells (APCs, i.e.,
CD4+ T cells and dendritic cells) by CD40L present on
CD4+T cells enhances the specific anti-cancer immune re-
sponse. Therefore, the major therapeutic purpose was target
activation of CD40, which leads to the intensified CD-
dependent T cell activation and the resulting effective anti-
cancer immune response. This assumption is accomplished
with the use of various methods, including agonistic antibod-
ies and recombinant forms of sCD40L. Currently, researchers
are widely interested in the transfer of the CD40L encoding
sequences to various cells, including cancer cells, fibroblasts,
or dendritic cells. The introduction of the CD40L encoding
sequence by means of viral vector (adenoviruses, retroviruses)
to cancer cells leads to the enhancement of the ligand expres-
sion and increased activity of dendritic cells that stay in
contact with cancer cells, which accelerates their maturation
and induces anti-cancer activity [88].
One of the cancer treatment strategies is to use CD40-
ligand/interleukin-2 vaccines. Chronic lymphocytic leukemia
(B-CLL) cells express many of tumor-associated and tumor-
specific antigens but lack co-stimulatory molecules, which are
required for effective antigen presentation. Up-regulating of
CD80, CD86, and CD54 can be caused by CD40L interaction
with CD40, and further immunostimulatory effect can be
enhance by IL-2 [89]. Biagi et al. [90] prepared autologous
B-CLL cells expressing human CD40 ligand (hCD40L) and
human interleukin-2 (hIL-2). Vaccine leads to CD4+ and
CD8+ effector cells reactive with autologous B-CLL cells
and consequently to immunomodulatory effect. In non-
Hodgkin’s lymphoma, cells after cultured with embryonic
lung fibroblasts were transducted by adenoviral vector with
Adh CD40L and Adh IL2. This combination caused enhanced
initial T cell activation and generation autologous T cells
against B-NHL cells [91].
Murine model research on urinary bladder cancer has
shown that gene therapy with the use of vectors that carry
the CD40L encoding sequence (AdCD40L) has high potential
in the inhibition of tumor growth. The introduction of
AdCD40L has led to enhanced expression of IL-12 and gen-
eration of specific all-systemic defense response against tu-
mor. At the same time, the development and function of
regulator T cells were suppressed in the lymph nodes [92].
Kipps et al. [93] proposed gene therapy for patients suffering
from chronic lymphoblastic leukemia. Leukemia cells were
transduced by Ad-CD154, which promoted antigen presenta-
tion and generation of autologic T cell response. This caused
the formation of cytotoxic T cells, specific to leukemia cells
[65]. Similar results have been reported by researchers who
studied the effect of gene therapy with the use of CD40L on
gastric cancers [94], non-small-cell lung cancer [95], breast
cancer [85], cancers of the uterine cervix, and the prostate
[96]. Studies on the new more effective and safer forms of
vectors that carry genes into the cells are still performed. For
instance, conditionally replicative oncolytic adenovirus
(AdEHCD40L) containing hybrid promoter ERE/HRE that
regulates transgenic gene CD40L has been proposed for the
treatment of breast cancer. The application of gene therapy has
led to the inhibition of tumor growth, enhanced apoptosis of
cancer cells, and turned out to be completely safe for the body
[97,98]. Vardouli et al. [99] assessed the effect of therapy
using recombinant non-replicating adenovirus, showing the
expression of CD40L, replication-defective recombinant ade-
novirus (RAd)-hCD40L (RAd vector expressing hCD40L) on
cancer cells. Their findings indicate that permanent activation
of CD40 by CD40L inhibits proliferation and enhances apo-
ptosis of cancer cells. Transduction of Rad-hCD40L to CD40-
positive cancer cells of the urinary bladder, uterine cervix, and
ovary inhibits strong proliferation of these cells [99].
CD40L genes can be transduced not only to cancer cells,
but also to the healthy ones in order to enhance their activity.
Kikuchi et al. [100] have forwarded the hypothesis that ge-
netically modified CD40L-positive dendritic cells will be able
to activate each other and enhance anti-cancer response after
being introduced to tumor microenvironment. Research
in vivo has confirmed the theory. The use of modified den-
dritic cells (AdmCD40L-modified syngenic DCs) increased
the immune response and suppressed tumor growth. In order
to increase the activity of antigen-presenting cells, therapy is
used with anti-CD40 antibodies (SGN-40). In MM, the use of
these antibodies significantly enhanced cell apoptosis and
decreased the expression of the receptor for IL-6 [101].
CD40L on cell membrane is known as a homotrimeric
molecule, which allows its binding to the receptor and activa-
tion of the signaling path. At present, an increasing number of
literaturedata suggest that such a structure is indispensable for
Tumor Biol. (2014) 35:9447–9457 9453

the ligand functioning, although the transmitted signal is too
weak to exert the maximum effect [102]. The modified
multimeric forms of CD40L considerably more strongly acti-
vate the receptor than the homotrimeric form does [103]. This
correlation was applied to CD40L therapies. Naito et al. [108]
have suggested an innovative preparation of human recombi-
nant CD40L (CD40L Tri), in which the ECD of CD40L is
connected to the “trimeric motif”of CD40L (CD40L-Tri) by a
long elastic peptide bond. CD40L-Tri considerably increases
the population of CD19 B cells and induces their transition
into antigen-presenting cells. The B cells activated by CD40L-
Tri can effectively stimulate the T cell-dependent response.
Also, the multimeric molecule, SP-D-CD154, being the fusion
of CD40L and lung surfactant protein D (SP-D), activates
proliferation of B cells more strongly than the CD40L-Tri
does [104].
Conclusions
The knowledge of the role of CD40L in normal functioning of
the immune system has initiated a numberof studies that have
revealed not only its involvement in the functioning of T and
B cells, but also its key role in the pathogenesis of such
diseases as atherosclerosis, cardiovascular disorders, and can-
cer. The latest research on the ligand has concentrated around
its practical therapeutic use. At present, the recombinant li-
gand is effectively used in the treatment of renal failure and
has very promising implications in the therapy for breast
cancer and pancreatic cancer. Moreover, new forms of the
modified ligand, vaccines that contain its gene, and
multimetric forms of the protein are produced. In a few years,
therapies that use CD40L are likely to be part of combined
therapy.
Conflicts of interest None.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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