The effect of chemotherapy on programmed cell death 1/ programmed cell death 1 ligand axis: Some chemotherapeutical drugs may finally work through immune response

Abstract

Most tumors are immunogenic which would trigger some immune response. Chemotherapy also has immune potentiating mechanisms of action. But it is unknown whether the immune response is associated with the efficacy of chemotherapy and the development of chemoresistance. Recently, there is a growing interest in immunotherapy, among which the co-inhibitory molecules, programmed cell death 1/programmed cell death 1 ligand (PD-1/PD-L1) leads to immune evasion. Since some reports showed that conventional chemotherapeutics can induce the expression of PD-L1, we try to summarize the effect of chemotherapy on PD-1/PD-L1 axis and some potential molecules relevant to PD-1/PD-L1 in chemoresistance in this review.

Full text

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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 20
The eect of chemotherapy on programmed cell death 1/
programmed cell death 1 ligand axis: some chemotherapeutical
drugs may nally work through immune response
Min Luo1 and Liwu Fu1
1 State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong
Esophageal Cancer Institute, Sun Yat-Sen University Cancer Center, Guangzhou, China
Correspondence to: Liwu Fu, email: Fulw@mail.sysu.edu.cn
Keywords: programmed cell death 1, programmed cell death 1 ligand, chemotherapy, immunotherapy
Received: October 30, 2015 Accepted: February 15, 2016 Published: February 23, 2016
ABSTRACT
Most tumors are immunogenic which would trigger some immune response.
Chemotherapy also has immune potentiating mechanisms of action. But it is unknown
whether the immune response is associated with the ecacy of chemotherapy
and the development of chemoresistance. Recently, there is a growing interest in
immunotherapy, among which the co-inhibitory molecules, programmed cell death
1/programmed cell death 1 ligand (PD-1/PD-L1) leads to immune evasion. Since
some reports showed that conventional chemotherapeutics can induce the expression
of PD-L1, we try to summarize the eect of chemotherapy on PD-1/PD-L1 axis and
some potential molecules relevant to PD-1/PD-L1 in chemoresistance in this review.
INTRODUCTION
Immunotherapy is a rising hope for cancer patients,
utilizing the immune system to detect and eliminate
foreign tumor antigens. But immune response is such a
complex phenomenon involving clonal T cell selection,
activation, proliferation and trafcking to antigen sites
to deliver immune effector functions, that it’s hard to hit
the target in immunotherapy [1]. The process of T cell
activation requires two major signals (Figure 1): the co-
stimulatory signals and co-inhibitory signals [2]. The
co-inhibitory signals could be a main actor in cancer
progression through the inhibition of anti-cancer immune
response [3]. One of the inhibitory signals is PD-1/PD-L1
axis. PD-1 is a member of the B7 receptor family and is
inducibly expressed on activated T cell subsets including
T follicular helper (Tfh) cells [5] and T regulatory (Treg)
cells. It attenuates immune responses by negatively
regulating T cell proliferation and function [4]. And the
relationship between PD-L1 expression on tumor and/or
immune cells and objective immune response has been
reported [6-9].
Chemotherapy is a conventional treatment for
cancer with different extent of cytotoxicity but has
immune potentiating mechanisms of action [10]. Whether
chemotherapy can stimulate immune response and nally
kill tumors is unknown. Recent introduction of immune
modulators, PD-1/PD-L1 adds much excitement to this
eld. It is reported that the expression rate of PD-L1 in
human malignant tumors varies from 19% to 92% [11]
and the expression of PD-L1 is positively correlated
with tumor progression [12-15]. PD-L1 overexpression
predicted better pathological response to chemotherapy,
independently of histo-clinical variables and predictive
gene expression signatures [16]. Zhang et al. [18]
demonstrated that paclitaxel, etoposide and 5-uorouracil
were able to induce PD-L1 surface expression in human
breast cancer cells and increase PD-L1-mediated
T cell apoptosis, revealing a potential link between
chemotherapy and cancer immunoresistance. PD-L1 is
expressed by cancer cells, the exact mechanism of how the
chemotherapeutic drugs work on tumor microenvironment
especially PD-1/PD-L1 axis and how this PD-1/PD-L1
axis induces chemoresistance is not clear. Herein, in this
review, we try to summarize the relationships between
chemotherapy and immune response through PD-1/PD-
L1 axis.
DIFFERENT CHEMOTHERAPEUTIC
AGENTS HAVE DIFFERENT EFFECTS ON
IMMUNE SYSTEM
Accumulating evidences suggest that conventional
therapeutic regimens as well as targeted anticancer agents,

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originate (at least in part) from their ability to elicit a novel
or reinstate a pre-existing tumor-specic immune response
[19, 20]. One of the mechanisms is that chemotherapy can
provoke the immune system to recognize and destroy
malignant cells called immunogenic cell death (ICD) [21].
Several common chemotherapeutics share the ability to
trigger ICD, (e.g., doxorubicin, epirubicin, idarubicin,
mitoxantrone, bleomycin, bortezomib, cyclophosphamide
and oxaliplatin) [21, 22] as well as some anticancer agents
that are still under preclinical or clinical development (e.g.,
some microtubular inhibitors of the epothilone family) [21,
22]. Among the various chemotherapeutic drugs that have
been tested on mice, anthracyclines are the only agents that
provide enhanced immunity to further battle with tumor
cells [23]. Recent data indicate that cyclophosphamide at
high doses have the immunosuppressive properties, while
metronomic cyclophosphamide regimens exert contrary
immunostimulatory effects [20] by selectively depleting or
inhibiting Tregs [24]. Such immunostimulatory properties
seem to, at least in part, contribute to the therapeutic
success by cyclophosphamide as a conventional
anticancer agent [25]. Importantly, many clinical studies
demonstrated that metronomic cyclophosphamide led
to improved T cell effector functions [19, 26]. Cancer
cells evade immune recognition via down-regulating
human leukocyte antigen (HLA) Class I expression,
allowing their escape from immune surveillance and
destruction [27]. While in ovarian cancer cells, low-dose
epothilone B, taxol and vinblastine greatly increased
expression of HLA Class I and HLA-A2 molecules, and
low-dose epothilone B treatment markedly increased the
expression of interferon-α, IL-1β, IL-12 and IL-6 [27]. In
the inammatory microenvironment, interferon-γ (IFN-γ)
and other inammatory cytokines, secreted by anti-
tumor Th1 cells or macrophages, may upregulate PD-L1
expression in response to immune-mediated attack [6],
to decrease the cytotoxic local immune response. Some
anti-tumor drugs can promote the cytokines (IFN, IL-6)
release to upregulate PD-L1 constitutively or in response
to inammation [6]. PD-L1 is upregulated in cancerous
cells in vitro by immune cytokines that are critical for
T cell functioning, such as IFN-γ [28], which may even
positively feedback to enhance immune tolerance in
vivo (Figure 2). Collectively, these studies indicate that
different chemotherapeutic agents have different effects on
immune system.
CHEMOTHERAPY ALTERS THE
EXPRESSION OF PD-1/PD-L1
Besides inducing ICD, oxaliplatin are reported
to inhibit the expression of programmed death ligand 2
(PD-L2), thereby limiting immunosuppression by both
dendritic cells (DCs) and tumor cells [29]. Treatment with
paclitaxel and etoposide upregulated PD-L1 expression
Figure 1: The regulation of T cell activation. T cell receptor (TCR) recognizes the tumor antigens in the context of major
histocompatibility complex (MHC) expressed on professional antigen presenting cells (APCs). Then APCs deliver a second signal by
positive co-stimulatory molecules CD28/B7-1/B7-2 to fully activate naive T cells. CTLA-4 can bind with B7-1/B7-2 competitively to
inhibit the activation of CD28/B7-1/B7-2, resulting in inactivation of T cells. PD-1 binds with its ligands, PD-L1 and PD-L2 to attenuate
lymphocyte activation.

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in breast cancer cells, resulting in co-inhibitory signals
activation [18]. Yang et al. [30] observed an increase of
PD-L1 and PD-1 antigen expression in leukemia cells
with decitabine treatment, and both PD-L1 and PD-1
expression were increased in a concentration dependent
manner. QIN et al. [31] demonstrated that when the
cisplatin concentration is less than IC50, cisplatin could
upregulate PD-L1 expression in hepatoma H22 cells.
Meanwhile, cisplatin could activate the phosphorylation
of ERK1/2, and that cisplatin-induced PD-L1 expression
is dependent of ERK1/2 phosphorylation [31]. Oki et al.
[32] observed a suppression of PD-1 expression after
treatment with panobinostat (a histone deacetylase). It
suggests that panobinostat may exert anti-tumor activity
by decreasing PD-1 expression in normal lymphocytes,
stimulating the immune reaction against lymphoma
[32]. PD-L1 and its signaling pathway appear to be a
potential therapeutic target for cancer. Interestingly, a
recent research demonstrated that PD-L1 expression
had the capability to change over time with anti-PD-L1
antibody therapy [33]. Therefore, its expression status
of a specic tumor tissue may not reect the present
immunologic phenotype of tumor. Doxorubicin is reported
to downregulate PD-L1 expression on cell surface, while
upregulate its nuclear expression in breast cancer cells
[34]. A decrease in PD-L1 expression on cell surface is
expected to increase the immunogenicity of the cancer
cells, and its translocation to the nucleus is likely to be
responsible for the anti-apoptotic impact of anthracyclines
on cancer cells and their microenvironment [34]. The
translocation of PD-L1 from the cell surface to the
nucleus induced by doxorubicin occurs concurrently with
AKT phosphorylation, but the PI3K/AKT pathway is not
involved in this process [34], which indicates that the PD-
L1 re-distribution from the cell surface to the nucleus is
regulated by two signaling, including an AKT-dependent
pathway (dominant in the nucleus) and an unknown AKT-
independent pathway (dominant on the cell surface).
Moreover, doxorubicin combined with PD-L1 knockdown
has been shown to enhance apoptosis [34]. This indicates
that its nuclear localization can enhance the anti-apoptotic
function, which may link with the apoptotic machinery
of the cell. As for the molecular mechanism of how to
regulate PD-L1 expression has yet to be understood,
but several researchers described the presence of pro-
inammation and inammation may take part in it [35].
Further studies are needed to explore the mechanism of
chemotherapy-induced PD-L1 expression in cancer cells.
SOME SIGNAL MOLECULES
ASSOCIATED WITH PD-L1 EXPRESSION
Zhang et al. [18] showed that chemotherapeutic
agents potentiated IFN-γ-induced PD-L1 expression
in human breast cancer cells. All the chemotherapeutic
Figure 2: Chemotherapeutic agents inuence cytokines network in antitumor immune system. Different chemical agents
work on immune cells, leading to various cytokines released, which can affect the immune cells populations to enhance/attenuate antitumor
response.

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agents tested in the study had similar effects on PD-L1
surface expression in breast cancer cells [18], suggesting
that they may act through a common pathway (Figure 3).
IFN-γ enhances cancer immunoresistance by upregulating
the expression of PD-L1 and PKD2 (Polycystic Kidney
Disease Gene 2) in human oral squamous carcinoma cells
in both time and dose dependent manner [36]. PKD2
knockdown with shRNA / siRNA or PKD chemical
inhibitor resulted in IFN-γ production, then downregulated
the expression of PD-L1 [36]. And the activation of PKD2
can stimulate the expression of P glycoprotein (P-gp)
[37]. Inhibition of PKD2 activation could signicantly
inhibit the expression of P-gp and decrease multiple drug
resistance (MDR) in human breast cancer cells [38],
indicating that PKD2 may be an important target for
tumor biotherapy and MDR reversal. Signaling through
key proliferative pathways, like MEK/ERK and PI3K/
AKT can also increase PD-L1 expression in malignant
glioma, prostate and breast carcinoma [39, 40]. Berthon
et al. [41] conrmed that blocking MEK inhibited PD-
L1 transcription in the AML cell lines THP-1 and U937,
suggesting that MEK is an important regulator of PD-L1
expression in leukemic cells. Non-small-cell lung cancer
(NSCLC) cell lines bearing EGFR, KRAS, BRAF, ALK
or RET mutations were found with high level of PD-L1
expression, and this may be correlated with high levels of
PI3K/AKT/mTOR pathway activation. PD-L1 expression
markedly increased in a subset of patients after acquiring
resistance to getinib in EGFR-mutant NSCLC [42].
Loss of the phosphatase and tensin homolog (PTEN)
through genetic deletions or mutations accelerates PD-L1
expression in tumors [43]. Crane et al. [40] demonstrated
that PI3K activation caused by loss of PTEN function
enhanced PD-L1 protein level expression in breast cancer
cell lines. Parsa et al. [43] found an increase of the post-
transcriptional PD-L1 expression in other types of PTEN
loss cancers with the activation of the PI3K pathway. The
transformed cells can also utilize PI3K pathway to evade
the immune system by mimicking immune cells [44],
developing resistance to T cell induced apoptosis [45],
secreting immunosuppressive cytokines [46], enhancing
the immunosuppressive potential of Treg cells [47] or
emulating immune cell chemotactic responses [48].
Inhibition of PI3K or its downstream signaling molecule
AKT decreased PD-L1 expression in tumor cells and
increased cytotoxic T cells-induced killing [43]. Therefore,
Figure 3: Chemotherapeutic agents promote PD-1/PD-L1 expression through various signals. Chemotherapeutic agents
via IFN-γ-dependent and IFN-γ-independent way to upregulate PD-L1 expression by activating different signals, like RAS/RAF, PI3K/
AKT, JAK/STAT3 and release some immune suppression cytokines to attenuate antitumor immune response.

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targeting PD-1/PD-L1 interaction may be enhanced when
combined with PI3K inhibitors. The complex interaction
between PI3K signal and anti-tumor immune response
needs further studies. It also represents a promising
avenue to best exploit the anti-tumor effects of clinical
PI3K inhibitors. Toso et al. [49] showed that in PTEN-null
senescent tumors, activation of the JAK2/STAT3 pathway
induced an immunosuppressive tumor microenvironment
that contributed to tumor growth and chemoresistance.
Inhibition of the JAK2/STAT3 pathway in PTEN-decient
prostate tumors led to senescence-associated cytokine
network reprogrammed, and improved the efcacy of
docetaxel-induced senescence by triggering a strong anti-
tumor immune response. Soliman et al. [37] reported
that high PD-L1 basal cell lines had lower expression of
IRF2BP2 (interferon regulatory factor 2 binding protein 2)
and higher STAT1 levels compared to those with low PD-
L1 expression. All ndings above suggest that regulation
of PD-L1 expression varies widely among cell types and
drugs targeting signal transduction pathways might have
different immunological effects in different tumors.
PD-1/PD-L1 IN CHEMORESISTANCE
A research [41] showed that in ve out of nine
patients with AML, spontaneous PD-L1 expression
increased when they relapsed. Lower expression of PD-L1
was positive correlated with a tendency to longer survival
[30]. Jennifer et al. [50] found that in progressing prostate
cancer patients, more PD-L1/2+ DCs led to poorer response
to Enzalutamide (ENZ) treatment and shorter treatment
duration. They also observed that circulating PD-L1/2+
DCs signicantly increased in mice bearing Enzalutamide
resistant (ENZR) tumors compared to castration resistant
Figure 4: Some strategies for blocking PD-1/PD-L1 axis. Targeting PD-1/PD-L1 and its downstream molecules can block the
axis to activate. And siRNA can downregulate PD-L1 expression from gene level. Changing the construct of PD-1 can convert it into a
co-stimulatory molecule to enhance immune response. Target IFN-γ can decrease IFN-γ-induced PD-L1 expression. Administration of
molecules bind with PD-1/PD-L1 can interfere the binding between PD-1 and PD-L1, thus inactive the PD-1/PD-L1 axis.

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prostate cancer, and ENZR tumors expressed signicantly
increasing levels of tumor-intrinsic PD-L1. Altogether,
it suggests that there are high expressions of PD-1/PD-
L1 pathway molecules in peripheral blood immune
cells in patients with ENZR castration resistant prostate
cancer (CRPC). Another research [51] showed that drug-
resistant osteosarcoma cell line KHOSR2 and virally-
derived osteoblast cell line hFOB had high (3-log) PD-
L1 gene expression, and osteosarcoma cell line SaOS
and breast cancer cell line MCF-7 had low (< 1-log)
expression. Each osteosarcoma cell line generally has
various PD-L1 expression, ranged from low to high
PD-L1 expression, with slightly higher expression from
drug-resistant variants (KHOSR2 and U-2OSTR) than
their parental cell lines (KHOS and U-2OS) [51]. Thus,
PD-1/PD-L1 blockade has the potential to overcome
resistance, and the combination therapy of chemotherapy
and PD-1/PD-L1 blockade has the potent synergistic
effects to enhance antitumor immunity. Schatton et al.
[52] identied tumorigenic human ABCB5+ MMICs (a
novel type of cancer stem cells, malignant melanoma-
initiating cells), expressing chemoresistance determinant
ABCB5 preferentially expressed PD-1 and B7-2, but
with downregulated expression of PD-L1 compared to
ABCB5
-
cells [53]. The relationship between PD-L1 and
chemoresistance determinant ABCB5 is worthy to further
study. The PD-L1
high
cells were demonstrated signicantly
resistant to CDDP (compound Danshen dripping pills)
and TXL (Tongxinluo) compared with the PD-L1low cells
[54]. As for the mechanism of how PD-L1 increases the
chemoresistance, Yu Fujita et al. [54] found that a miR-
197 mimic can sensitize PD-L1high drug-resistant cells to
chemotherapy, indicating that the biological interaction
between PD-L1 and chemoresistance occurs through
the microRNA regulatory cascade. The overexpression
of miR-197 induced decreased expression of PD-L1 in
NSCLC cells [54]. The miR-197/CKS1B/STAT3 axis
can drive tumor PD-L1 expression as a biomarker of
this cascade, and miR-197 replacement therapy may be
a potential treatment strategy for chemoresistant NSCLC
[54].
STRATEGIES FOR BLOCKING PD-1/PD-L1
AXIS IN CHEMORESISTANCE
With its profound immunosuppressive effect, PD-1/
PD-L1 axis has been the focus of several recent studies
aiming at neutralizing its detrimental effects on T cell
anti-tumor response (Figure 4). There are now multiple
agents targeting the PD-1/PD-L1 at different stages
of clinical development [9, 55-57] (Table 1). PD-1 and
PD-L1 antibodies have shown considerable clinical
efcacy and durability across a range of malignancy
subtypes, including melanoma and lung cancer [9, 58]
and most recently in refractory Hodgkin’s disease [59],
and quite a lot of phase II studies are ongoing in prospect
(NCT02572167, NCT02181738, NCT02327078). It
decreases the metastatic risk and improves the therapeutic
response when associated with immunogenic anti-
cancer chemotherapy such as doxorubicin [60, 61]. As
some signal molecules are reported to upregulate PD-
L1 expression, the target inhibitors may be a potential
treatment (Figure 4). Other immune modulatory
agents, like IFN-α-2b, are going on clinical trials
combined with different anti-PD-1 and anti-PD-L1
antibodies (NCT01943422, NCT01608594). Nivolumab
(NCT02464657, NCT01658878), pembrolizumab
(NCT02551432), MEDI4736 (NCT02027961),
and MPDL3280A (NCT01633970, NCT02525757,
NCT02409355) are being evaluated in combination
with chemotherapies, tyrosine kinase inhibitors, or other
targeted therapies. Recently, Tang et al. [62] converted
PD-1 to a T cell co-stimulatory receptor by exchanging
its trans-membrane and cytoplasmic tail with CD28 and
4-1BB signaling domains (PD-1-CD28-4-1BB, PD-1-
ACR), which retained the ability to bind PD-L1, but
resulting in T cell activation as evidenced by the elevated
activity of PI3K/AKT, the augmentation of cytokine
Table 1: Some anti-PD-1/PD-L1 antibodies in clinical trials
Agents Alias Target Clinical trial Application Details
Nilvolumab BMS-936559,
MDX-1106 PD-1
Approved by
FDA
Melanoma, renal cell
carcinoma and non-
small cell lung cancer
/
Pembrolizumab MK-3475,
Lambrolizumab PD-1
Approved by
FDA
Advanced melanoma
/
Pidilizumab [66] CT-011 PD-1 Phase II Hematological
malignant tumor
72 patients enrolled, PFS: 0.72 (90%
CI, 0.60 to 0.82), toxicity: 23/72
(31.94%) blood and lymphatic system
disorders
MPDL3280A Atezolizumab PD-L1 Phase III
Non-small cell lung
cancer, Ongoing (NCT01846416)
MEDI4736 Durvalumab PD-L1 Phase II
Non-small cell lung
cancer Ongoing (NCT02087423)
Avelumab MSB0010718C PD-L1 Phase II
M-Merkel cell cancer
Ongoing (NCT02155647)

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secretion and the increased proliferative capacity. Samuel
et al. [63] reported that CD80-Fc (the fragment of CD80
IgG) is more effective in preventing PD-1/PD-L1-induced
suppression and restoring T cell activation compared to
treatment with mAb to either PD-1 or PD-L1. Soluble
PD-1 (sPD-1) is an efcient way to bind PD-L1 and to
block PD-1/PD-L1 interactions, in conjunction with a
two-domain molecule of bronectin (CH50) in inhibiting
tumor invasion and growth in hepatoma [64]. A study [65]
showed that using short-hairpin double-stranded silencing
RNA (siRNA) to restrain the expression of PD-1 on the
cell surface of tumor-specic T cells, improved immune
responses. In addition, it remains to be seen whether
ICD inducers (e.g., doxorubicin, epirubicin, etc.) may
be advantageously combined with non-immunogenic
conventional chemotherapeutics, targeted anticancer
agents and/or immunostimulatory strategies. It is crucial
for the discovery of next-generation chemotherapeutics,
i.e., molecules that simultaneously hit cancer cells while
exerting potent immunostimulatory effect. These agents
may be particularly relevant for the development of
combinatorial chemotherapeutic regimens that actively
engage the host immune system against malignant cells.
SUMMARY
A better understanding of how chemotherapy affects
the anti-tumor immunity and causes chemoresistance is
crucial. But it is still unknown how the signaling events
regulate the expression of these molecules in resistant
cancer cells. Blockade of the PD-1/PD-L1 pathway is
a new, promising immunotherapy for cancer. Strategic
combination of immunotherapy and chemotherapy can
effectively change the overall tumor microenvironment, as
well as immune tolerance and immune suppression, which
can maintain effective and durable anti-tumor immune
response.
ACKNOWLEDGMENTS
We thank Xiao-Na Fang and De-Lan Li for revising
the manuscript for better reading.
CONFLICTS OF INTEREST
Authors have no relevant, potential conicts of
interest to declare.
FUNDING
The research was supported by Major State Basic
Research Development Program of China (973 Program,
No.2012CB967000) and Natural scientic Foundation of
China (No. 81473233).
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