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Pharmacological Research 73 (2013) 8– 19

Contents lists available at SciVerse ScienceDirect

Pharmacological Research

jo ur nal home p age: www.elsev ier .com/ locate /yphrs

eview

ultilevel pharmacological manipulation of adenosine–prostaglandin E2/cAMPexus in the tumor microenvironment: A ‘two hit’ therapeutic opportunity

uzammal Hussaina,1, Aqeel Javeeda,∗,1, Muhammad Ashrafa, Hou Yuzhub,uhammad Mahmood Mukhtarc

Department of Pharmacology & Toxicology, University of Veterinary and Animal Sciences, Lahore, PakistanTransplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Datun Road,eijing 100101, ChinaDana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

a r t i c l e i n f o

rticle history:eceived 30 March 2013ccepted 14 April 2013

eywords:ctonucleotidasesdenosineOX2

a b s t r a c t

Novel trends in cancer treatment research are focused on targeting the tumor microenvironment, therebydeveloping chemo-immunotherapeutic strategies which not only directly kill tumor cells, but also triggerthe anti-tumor immune effector responses. Ectonucleotidases (CD39 and CD73)-generated extracellu-lar adenosine and cyclooxygenase-2 (COX2)-derived prostaglandin E2 (PGE2) are amongst the tumormicroenvironmental factors that have emerged as attractive targets in this regard. Both comprise a piv-otal axis in tumor progression and immune escape via autocrine and paracrine activation of a commonintracellular signaling pathway, the cAMP–protein kinase A (PKA) pathway, in cancer and immune cells.

GE2

hemo-immunotherapyAMP–PKA pathway

In this review, we venture a potential and realistic strategy that this adenosine–PGE2/cAMP nexus istargetable at different levels, thereby pointing out a ‘two hit’ chemo-immunotherapeutic proposition:direct killing of tumor cells on one hand, and the rescuing of endogenous anti-tumor immune responseon the other. The reviewed experimental, preclinical and clinical data provide the proof of concept that‘two hit’ multilevel pharmacological manipulation of adenosine–PGE2/cAMP nexus is achievable withinthe tumor microenvironment.

© 2013 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92. Adenosine–PGE2/cAMP nexus in the tumor microenvironment: double-edge sword effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93. Therapeutic potential of upstream targeting at enzymatic level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.1. COX2: NSAIDs and COXIBs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.2. CD39–CD73 ectoenzymes: ectonucleotidase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4. Therapeutic potential of downstream targeting at receptor level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.1. Selective EP2/EP4 receptor antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.2. Selective A2A/A2B receptor antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

5. Therapeutic potential of downstream targeting in the cAMP–PKA pathw5.1. AC inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2. The cAMP–PKA inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: AC, adenylyl cyclase; ATP, adenosine-5′-triphosphate; ADP, adenosinOX, cyclo-oxygenase; COXIBs, cyclo-oxygenase 2 (COX-2) inhibitors; CINODs, COX-inhi-prostanoid; FOXP3, transcription factor forkhead box protein P3; IFN, interferon; IL, inymphokine-activated killer; MDSC, myeloid-derived suppressor cells; NECA, 5, N-ethylcailler; NSCLC, non-small cell lung cancer; PG, prostaglandin; PKA, protein kinase A; TAM, tndothelial growth factor.∗ Corresponding author at: Department of Pharmacology & Toxicology, University of V

el.: +92 42 99213697.E-mail address: aqeelvet@hotmail.com (A. Javeed).

1 These authors equally contributed to this work.

043-6618/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.phrs.2013.04.006

ay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

e-5′-diphosphate; AMP, adenosine-5′-monophosphate; CADO, 2-chloroadenosine;biting nitric oxide donators; DC, dendritic cells; DDA, 2′ ,5′-dideoxyadenosine; EP,terleukin; IP-10, IFN-induced protein of 10 kDa; 3LL, Lewis lung carcinoma; LAK,

rboxamide adenosine; NSAIDs, non-steroidal anti-inflammatory drugs; NK, naturalumor-associated macrophages; Treg, T regulatory cells; Th, T helper; VEGF, vascular

eterinary and Animal Sciences, Abdul Qadir Jilani Road, Lahore 54600, Pakistan.

M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19 9

6. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Tumors potentiate their own progression and survival by fos-ering a complex and highly dynamic microenvironment thatncompasses a plethora of distinct cellular and molecular compo-ents, which orchestrate a variety of crosstalks, providing essentialues to limitless tumor cell growth, as well as to aid the escapef tumor masses from the host immune system [1–5]. Novelnsights in anti-cancer therapeutics are, therefore, to manipulatehe events and molecules implicated in such crosstalks within theumor microenvironment, thereby developing effective strategieso control tumor progression and immunoescape [2,6–8]. Extra-ellular adenosine and prostaglandin E2 (PGE2) are amongst theumor microenvironmental molecular factors that have emergeds attractive targets in this regard [9–12].

Extracellular adenosine and PGE2, though different in theirhemical nature, have three commonalities. First, both are enzy-atic products: PGE2 is a major product of cyclooxygenase 2

COX2), while adenosine is generated through the concertedction of two cell-surface ectonucleotidases (CD39 and CD73) ondenosine-5′-triphosphate (ATP) (Fig. 1). Second, both interactith the respective sub-class of same receptor family, called G-rotein-coupled receptors (GPCR): PGE2 binds to E-prostanoid (EP)ubtypes EP1, EP2, EP3 and EP4 [13], whereas adenosine activates1, A2A, A2B, and A3 receptors [14]. Third and most important, bothomprise a pivotal axis in tumor progression and immunoescapeia autocrine and paracrine activation of respective prostanoidnd adenosinergic transcellular signaling pathways in various cellypes, including cancer and immune cells [9,15–19]. Evidence isccumulating which emphasize the existence of links betweenhe prostanoid and adenosinergic transcellular signaling pathwaysithin the tumor microenvironment [20–24]. In particular, recent

tudies have shown that adenosine and PGE2 cooperate addi-ively in the tumor milieu: stimulate cAMP-elevating A2A/A2B andP2/EP4 receptors and, thereby, lead to convergent activation ofAMP–dependent protein kinase A (PKA) pathway [25–28]. TheAMP amplification via adenosine–PGE2 signaling has been impli-ated not only in the initiation and progression of various tumors,ut also in tumor-driven immune suppression [15,17,18,27]. Theocus of current review, therefore, is to specifically discuss the phar-

acological manipulation of this intriguing and novel aspect ofdenosine–PGE2 signaling, namely adenosine–PGE2/cAMP nexusn the tumor microenvironment. Here, after briefly describing someeatures of adenosine–PGE2/cAMP nexus that are relevant to tumorrogression and immunoescape, we discuss the therapeutic poten-ial of selective pharmacological inhibitors at various levels of thisexus in cancer therapy.

. Adenosine–PGE2/cAMP nexus in the tumoricroenvironment: double-edge sword effect

Tumor cells and the activated immune cells, via simultaneousnvolvement of ectonucleotidases (CD39/CD73) and COX2, con-ribute to extracellular adenosine and PGE2 production in the tumor

icroenvironment. Adenosine and PGE2 signaling via A2A/A2B

nd EP2/EP4 receptors, which are richly expressed on cancer andmmune cells, converges on the intracytoplasmic adenylyl cyclaseAC), leading to increased level of canonical second messengerAMP that potently regulates variety of cellular functions at the

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

level of transcription factors through activation of PKA (Fig. 1).The cAMP–activated PKA phosphorylates broad spectrum of pro-teins in both cytoplasm and nucleus, especially cAMP-responseelement binding protein (CREB), which in turn bind to the cAMP-response elements in the promoter regions of cAMP-responsivegenes responsible for versatile functioning of cAMP–PKA pathwayin different cell types.

Persistent elevation of cAMP levels in the tumor bed may changethe behavior of cells: some proliferate and some die, depend-ing upon cAMP-mediated genetic regulation and the type of cells.Tumor cells often hijack the normal physiological functioning ofthe adenosine–PGE2/cAMP nexus and exploit it for their survival,autonomous proliferation, angiogenesis and metastasis. Mountingevidence implicates adenosine- and/or PGE2-mediated activationof cAMP–PKA pathway as the most common tactic in the aberrantgrowth of highly prevalent cancers [15,17,18,29–35]. On the otherside of the coin, activation of the same signaling pathway in tumor-infiltrating immune cells potently inhibits their functional activity,hampering the development of effector mechanisms of anti-tumorimmune surveillance. Several studies have demonstrated thatcAMP amplification via adenosine and/or PGE2 signaling dimin-ishes the executive functioning of tumor-associated macrophages(TAM) [36], natural killer (NK) and gammadelta T cells [23,37–41],T effector cells [28,38,42–45] and lymphokine activated killer (LAK)cells [27,46]; while enhances suppressor functions by inducing theactivity of T regulatory cells (Treg) [25,26,47–54] and myeloid-derived suppressor cells (MDSC) [55], ultimately tilting the balancefrom an immune-active to immune-suppressive tumor microen-vironment. Collectively, adenosine–PGE2/cAMP nexus executes a‘double-edge sword’ effect in the tumor microenvironment, ableon the one hand to directly enhance tumor progression, and onthe other hand, dampen anti-tumor immunity and, in turn, pro-mote tumor immunoescape (Fig. 1). Therefore, it is reasonableto assume that concomitant disarming of adenosine–PGE2/cAMPnexus, either upstream or downstream, by (a) eliminating enzy-matic activities of ectonucleotidases (CD39/CD73) and COX2 (b)blocking cAMP-elevating adenosine and EP receptors, (c) inhibi-ting AC activity, (d) blocking the common intracellular cAMP–PKAcascade or (e) combining these strategies will have ‘two hit’ chemo-immunotherapeutic opportunity against cancer: direct killing oftumor cells, as well as the triggered activation of immunosup-pressed tumor microenvironment to mount a robust anti-tumorimmune response.

3. Therapeutic potential of upstream targeting atenzymatic level

COX2 and ectonucleotidases (CD39 and CD73) are the rate-limiting enzymes in the arachidonic acid-COX-PGE2 and theATP–ectonucleotidase–adenosine systems, respectively (Fig. 1).Both types of enzymes are overexpressed in many types of humancancers, and appear to be effective intervention points for chemo-immunotherapeutic manipulation of adenosine–PGE2/cAMP nexus[12,56–60].

3.1. COX2: NSAIDs and COXIBs

It is well documented that COX2, via its product PGE2, playsa central role in tumor invasion, angiogenesis, resistance to

10 M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19

Fig. 1. A brief description of multilevel, ‘two hit’, pharmacological manipulation of adenosine–PGE2/cAMP nexus with respect to tumor progression and immunoescape.The adenosine–PGE2 signaling via cAMP-elevating adenosine (A2A/A2B) and E-prostanoid (EP2/EP4) receptors exerts a ‘double-edge sword’ effect: direct enhancement oftumor growth, and dampening of anti-tumor immunity. Pharmacological inhibitors at different levels of this nexus may provide ‘two hit’ therapeutic opportunity: directkilling of tumor cells, as well as tipping the balance of immune responses from tumor protection toward tumor rejection. ATP, adenosine-5′-triphosphate; ADP, adenosine-5 2, prosm NSAID

amastSdcshwrlar

′-diphosphate; AMP, adenosine-5′-monophosphate; COX2, cyclooxygenase 2; PGEacrophages; NK, natural killer cells; Teff, T effector cells; Treg, T regulatory cells;

poptosis, and suppression of host immunity [9]. Upstream phar-acological targeting of the COX2 with conventional non-steroidal

nti-inflammatory drugs (NSAIDs) and, particularly, with highlyelective COX2 inhibitors (COXIBs) look promising in the con-ext of chemo-immunotherapeutic interventions against cancer.everal experimental, epidemiological and clinical studies vali-ate the significance of NSAIDs and COXIBs as bonafide cancerhemopreventive agents (reviewed in Refs. [61–63]). Briefly, theyuppress PGE2-mediated tumor angiogenesis and metastasis, andave been demonstrated to have, either alone or in combinationith other therapies, an indisputable impact in reducing cancer

isk, as well as in treating several types of human cancers. Simi-arly, an impressive number of experimental and clinical studieslso indicate that NSAIDs and COXIBs can switch the immuneesponse from a tumor-promoting profile to a tumor-destructive

taglandin E2; AC, adenylate cyclase; PKA, protein kinase A; TAM, tumor-associateds, nonsteroidal anti-inflammatory drugs; COXIBs, COX2 inhibitors.

profile within the tumor microenvironment (reviewed by us in Ref.[64]). They can reactivate and maintain appropriate anti-tumorimmune responsiveness by modulating the TAM-mediated pro-and anti-tumor effects in favor of effective anti-tumor immu-nity, augmenting the NK cell-mediated cytotoxicity, increasing thetumor-tissue infiltration of T effector cells, as well as by suppressingthe Treg activity in the tumor microenvironment. Moreover, theyalso exhibit a great additive/synergistic potential to boost the effec-tiveness of active, passive and cytokine-based immunotherapeuticapproaches against cancer [64].

Collectively, NSAIDs and COXIBs, either alone or as adjunct

couple with other chemopreventive/immunological/biologicaltherapies, possess a ‘two hit’ therapeutic propensity thatencompasses direct anti-cancer, as well as the anti-tumor immune-enhancing effects. However, treatment-related toxicities, including

M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19 11

ucleo

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he notorious gastrointestinal, renal and cardiovascular com-lications, associated with this class of drugs make their fateloomy at the thought of clinical implementation in cancer chemo-mmunotherapeutics [59,65]. Efforts are now being made to furthermprove their safety profiles, which have ignited the developmentf novel agents that are not plagued with such problems, includingOX-inhibiting nitric oxide donators (CINODs) [66] and third gener-tion NSAIDs that target both COX2 and thromboxane A2 receptors67,68]. CINODs are the NO-NSAIDs hybrids (depicted in Fig. 8 ofef. [69]) that are making a unique niche in the armamentariumf chemopreventive and chemo-immunotherapeutic anti-cancergents. They provide additive anti-tumor effects by each compoundhile minimizing their respective side effects, and are now crossing

he threshold of clinical development [69–72].

.2. CD39–CD73 ectoenzymes: ectonucleotidase inhibitors

The concerted action of CD39–CD73 ectoenzymes repre-ents the primary step of extracellular adenosine-mediatedrometastatic and immunosuppressive effects in the tumoricroenvironment [16]. Extracellular adenosine exerts pro-etastatic effects by directly acting on tumor cells via activation of

AMP-elevating A2A/A2B receptors, subsequently enhancing tumorell invasion and chemotaxis. On the other hand, adenosine-ediated activation of cAMP-elevating A2A/A2B receptors in

mmune cells suppresses interferon gamma (IFN-�) production andytotoxic killing by CD8+ T-cells and NK cells, promotes CD4+ cellsifferentiation into Treg, and inhibits phagocytosis and antigenresentation in macrophages and dendritic cells (DCs) [15,18,43].argeted inhibition of both CD39 and CD73 with anti-CD39 andnti-CD73 monoclonal antibodies, respectively, has been shownromising to inhibit tumor growth as well as to enhance anti-tumor

mmunity [56,73,74].CD39 drives the sequential dephosphorylation of extracellular

TP to adenosine-5′-monophosphate (AMP) and, thus, stimulatesumor growth by limiting ATP-triggered anti-tumoral activity. Tar-eted inhibition of CD39 may lead to an accumulation of ATPn the tumor milieu, which itself not only directly inhibits cellroliferation and promotes cancer cell death, but also provokesnti-tumor immune responses by serving as a ‘find-me’ signal forhe recruitment of different immune cells in the tumor microen-ironment [56,75]. Currently, pharmacological inhibitors of CD39

re not well-developed, and include only polyoxometalates (POMs)76] and ARL67156 (Fig. 2). POMs are inorganic anionic com-lexes whose anti-tumoral potential has been reported againstany human cancers, including breast cancer, lung cancer and

tidase (CD39 and CD73) inhibitors.

colon cancer [77,78]. However, more convincing molecular evi-dence has been reported recently by Sun et al. [79], demonstratinganti-tumor activity of POM-1 (polyoxometalate-1) in a mousemodel of hepatic metastatic cancer. They showed that tumor-infiltrating Treg inhibit NK cell-mediated anti-tumor immunityin a CD39-dependent manner, and POM-1 inhibited CD39+ Treg-derived adenosine generation in vitro and significantly abrogatedtumor growth in vivo. Similarly, ARL67156, a structural analog ofATP, significantly decreased CD39+ Treg-mediated suppression ofeffector T cell responses in two clinical studies involving head andneck squamous cell carcinoma (HNSCC) [50] and human follicularlymphoma (FL) patients [80], respectively.

CD73 further reduces CD39-derived AMP to adenosine, and hasa pivotal role in tumor growth and metastasis [12]. CD73 inhibitorsinclude ADP, its more stable analog �,�-methylene-ADP (APCP)(Fig. 2), and anthraquinone- and novel sulfonamide-derived antag-onists [81,82]. None of these compounds has yet been investigatedin tumor settings, except APCP which has shown therapeutic poten-tial in various tumor models. APCP induced apoptosis and cell-cyclearrest in human breast cancer cells MDA-MB-231 [83,84]. It sig-nificantly reduced tumor cell proliferation in T24 human bladdercancer [85], and in human U138MG glioma cell lines [86,87]. APCPalso inhibited abilities of invasion, migration and adhesion to extra-cellular matrix in CD73-overexpressed T-47D and MB-MDA-231human breast cancer cell lines [88,89]. These findings suggest thatpharmacological suppression of CD73 activity on tumor cells maydirectly inhibit tumor growth and metastasis.

APCP may also induce anti-tumor immune responses thatare in large part attributed to enhancement of T cell-mediatedadaptive immunity. Substantial correlative evidence suggests thatAPCP can inhibit tumor growth and metastasis by enhancing theanti-tumor T lymphocytes expansion and effector functions. Itsuppresses CD73 activity on hematopoietic (e.g. endothelial cells)and nonhematopoietic cells (e.g. Treg), and thereby can improvethe efficacy of T-cell-based therapy by significantly increasinganti-tumor T cell homing to draining lymph nodes and tumors[49,50,58,73,74]. More recently, Forte et al. [90] showed that APCPtreatment increased CD8+ T cell- and B cell-mediated immuneresponses in melanoma-bearing mice. They demonstrated thatAPCP administration promoted the release of T helper 1 (Th1)-and Th17-associated cytokines, and also induced immunoglobulinG 2b (IgG2b) production from B cells in the tumor microenviron-

ment, which indicates that CD73 suppression may also improve Bcell-mediated anti-tumor immunity.

Collectively, the data discussed above infer that the pharmaco-logical blockade of CD39–CD73 concerted action may find utility

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s an optimal and rational therapeutic approach in cancer chemo-mmunotherapeutics. Nevertheless, it deems further investigations

hether simultaneous inhibition of the expression of both CD39nd CD73 would far exceed the expectations.

. Therapeutic potential of downstream targeting ateceptor level

The cAMP-elevating EP and A2A/A2B receptors provide an excel-ent example of two different receptor subtypes behaving in aomplementary manner to converge on a common intracellularignaling pathway in order to initiate and maintain tumor develop-ent and progression, as well as to aid tumor immunoescape. As

target further downstream of enzymatic level, pharmacologicalntagonists of EP2/EP4 and A2A/A2B receptors may enable a morepecific therapeutic modality with improved selectivity of actions compared to COX2 and ectonucleotidase inhibitors.

.1. Selective EP2/EP4 receptor antagonism

What is our present appreciation of the potential of selectiveP2/EP4 receptor antagonism in the context of cancer chemo-mmunotherapeutics? Selective antagonists for EP2 (PF-04418948,H-6809) and EP4 (AH-23848, BGC-20-1531, CJ-023423, CJ-42794, GW-627368X, L-161982, ONO-AE2-227, ONO-AE3-208)ave been developed as shown in Fig. 3. Purely specific EP2 antag-nism was not possible until 2011, when af-Forselles et al. [91]eported for the first time a novel, potent and selective EP2 antag-nist, PF-04418948. To the best of our knowledge, PF-04418948as not yet been tested in tumor settings. However, there is goodvidence for the widely used EP2 antagonist, AH-6809 that alsoinds to EP1 and PGD2 receptors. Therefore, almost all of supportingata regarding EP2/EP4 antagonism with respect to cancer chemo-

mmunotherapeutics relates to AH-6809 and some of the EP4ntagonists. In terms of tumor growth and progression (angiogen-sis and metastasis), considerable experimental/pre-clinical datahows the therapeutic utility of EP2/EP4 antagonists, which can beummarized as follows:

1- Inhibition of tumor cell proliferation in non-small cell lungcancer (NSCLC) NCI-H1299 cell lines by AH-6809 [92];

2- Induction of apoptosis via inhibition of ubiquitin-proteasome-mediated degradation of Bax protein in human C-33A cervicalcancer cells by AH-6809 and AH-23848 [93];

3- Suppression of PGE2-mediated induction of S100A8 mRNAexpression and subsequent tumorigenesis in prostate cancercells by AH-6809 and AH-23848 [94];

4- Inhibition of aromatase-induced tumorigenesis in MCF-7 andMDA-MB-231 human breast cancer cells by AH-6809 and AH-23848 [95];

5- Reduction of intestinal carcinogenesis in APC 1309 andC57BL/6Cr mice by ONO-AE2-227 [96,97];

6- Inhibition of colorectal tumorigenesis via G0/G1cell cyclearrest and blockade of cell proliferation in HT-29 and HCA7colorectal cancer cells by L-161982 [31,98];

7- Inhibition of castration-resistant progression of tumors inKUCaP-2 mouse model of prostate cancer by ONO-AE3-208[99];

8- Decrease in cell growth and viability of neuroblastoma cells byL-161982 [100];

9- Inhibition of the vascular endothelial growth factor (VEGF)

expression in cervical adenocarcinoma cells by ONO-AE2-227[101], and suppression of VEGF-C and -D production by ahighly metastatic cancer cell line C3L5 in a recent pre-clinicalstudy involving a syngeneic murine breast cancer model by

cal Research 73 (2013) 8– 19

ONO-AE3-208, which markedly reduced tumor growth, lym-phangiogenesis, angiogenesis, and metastasis to lymph nodesand lungs [11]; and

10- Reduction in the metastatic potential of B16 melanoma cellsin an experimental murine model of bone metastasis by ONO-AE3-208 [102].

Cellular migration and invasion are important functions ofmetastatic tumors. EP2/EP4 antagonism with AH-6809 and AH-23848, respectively, inhibited migration of metastatic breast cancercells. In particular, AH-23848 effectively inhibited migration ofmurine (C3L5) and human (MDA-MB-231) breast cancer cells [34].In another study, AH-23848 and ONO-AE3-208 blocked the migra-tory response in vitro and lung metastasis in vivo of murinebreast cancer cells [103]. Treatment with ONO-AE3-208 alsosuppressed tumor metastasis to lung and colon in Lewis lung car-cinoma (3LL) and MC26 colon cancer cells. It significantly inhibitedthe in vitro adhesion, motility, invasion and colony formationin 3LL cells [104]. Moreover, EP4 antagonism with GW627368Xdiminished invasion of human inflammatory breast cancer cells[105,106], whereas EP2 antagonism with AH-6809 was proposedto inhibit angiogenesis and tumor invasion via suppression of theurokinase-type plasminogen activator system in the gastric can-cer cells [107]. AH-6809 also reduced COX2 expression in thehighly metastatic L3.6pl human pancreatic cells [108], which indi-cates its potential to inhibit tumor-shed COX2-derived tumorinvasion, angiogenesis and metastasis. Furthermore, two recentstudies by Ma and colleagues [109,110] have demonstrated thattwo novel EP4 antagonists, frondoside A and RQ-00015986 (CJ-042794), inhibited tumor metastasis to lungs in a syngeneic murinemodel of metastatic breast cancer. Both, frondoside A and RQ-00015986, inhibited the migration of tumor cells in vitro, andprevented their lung colonization in vivo.

EP2/EP4 antagonists may counteract the PGE2-mediatedimmunosuppression, thereby enhancing anti-tumor immunesurveillance to optimal level. NK cells orchestrate innate immu-nity against tumors, both directly by exerting cytolytic effects andindirectly by secreting immunostimulatory cytokines like IFN-�.Frondoside A promoted NK cell functions in tumor-bearing mice.It inhibited breast tumor metastasis in NK-dependent manner byprotecting PGE2-mediated suppression of IFN-� production fromNK cells [37]. Similarly, RQ-00015986 protected tumor-mediatedNK-cell immunosuppression in a syngeneic murine model ofmetastatic breast cancer [109]. EP2/EP4 antagonists may also inhibitthe expression of tumor-derived chemokine signals and, in turn,may modulate immune cell functions to inhibit cancer progres-sion. For example, RQ-00015986 inhibited the recruitment oftumor-promoting phenotype of TAM in a mouse model of gastriccancer by suppressing the expression of tumor-derived chemokineCCL2, which attracts the macrophages in the tumor milieu [111].Moreover, AH-6809 and AH-23848 reduced the accumulation ofhuman MDSC in ovarian cancer environment [112]. Both inhibitedthe tumor-derived PGE2-induced production of CXCL12 (stromal-derived factor 1, SDF-1) in ovarian cancer cells, as well as reducedthe expression of its receptor CXCR4 in MDSC. Finally, AH-23848counteracted the PGE2-mediated suppression of IFN-induced pro-tein of 10 kDa (IP-10), which not only induces anti-tumor T cellimmunity by regulating lymphocyte chemotaxis, but also acts asan angiostatic agent, thus, inhibiting tumor growth [113–115].

The data described above clearly suggest that EP2/EP4 antago-nists may have chemopreventive and chemo-immunotherapeutic

utility against cancer. Interestingly, the extraintestinal safetyof some of the EP antagonists has already been shown inrodent models [96,97], which indicates that selectively target-ing the PGE2–EP2/EP4 arm of adenosine–PGE2/cAMP nexus will

M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19 13

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Fig. 3. Chemical struct

ikely avoid the cardiovascular and gastrointestinal complicationsttributed to selective targeting of COX2 alone.

.2. Selective A2A/A2B receptor antagonism

Fig. 4 shows the commonly used A2A/A2B receptor antagonists.imilar to that of EP2/EP4 antagonists, pharmacologic inhibitors

f EP2/EP4 antagonists.

of A2A/A2B receptors may also have chemo-immunotherapeuticutility against cancer. Several preclinical and early clinical stud-ies have shown that they can inhibit tumor growth, as well

as enhance anti-tumor immune functioning in an immune sup-pressed tumor microenvironment. In terms of tumor growth andmetastasis, SCH 58261 (a specific A2A antagonist) significantlydecreased cell growth and viability of two gefitinib-resistant NSCLC

14 M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19

Fig. 4. Chemical structures of A2A/A2B antagonists.

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ell lines, H1975 and HCC827GR, in a concentration-dependentanner [116]. Similarly, MRS 1754 (a selective A2B antagonist)

ignificantly inhibited cell growth in human colon carcinoma cellines in a dose-dependent manner [117]. PSB 1115 (a highlyelective A2B antagonist) decreased chemotaxis in vitro and lungetastasis in vivo of mouse breast cancer cell line 4T1.2 [118].

heobromine inhibited angiogenesis in human ovarian cancer cells119]. Moreover, A2B antagonists have been shown to inhibit tumoreovascularization by down-regulating the production of angio-enic factors. For instance, A2B blockade by MRE 2029F20 andaffeine reduced proangiogenic cytokine interleukin (IL)-8 releasen the human A375 melanoma, and in the human HT29 colon can-er cell lines [120,121]. IPDX (a selective A2B antagonist) inhibited-N-ethylcarboxamide adenosine (NECA)-stimulated IL-8 release

n the U87MG tumor cells [122].A2A/A2B antagonists may counteract tumor microenvironmen-

al adenosine-induced immunosuppression by improving theffector functioning of tumor-infiltrating lymphoid cells. ZM41385, a nonselective A2A/A2B antagonist, increased LAK cyto-oxicity by significantly blocking the inhibitory effects of stabledenosine analog 2-chloroadenosine (CADO) and NECA (an A2A/A2Bgonist) on their cytokine production and the cytotoxic activ-ty against 3LL tumor cells [23,46]. A2A/A2B antagonists may also

eaken inhibition of anti-tumor T cells by adenosine and, thereby,mprove rejection of established tumors by cytotoxic T lympho-ytes. The majority of supporting data relates to ZM 241385,hich: restored the cytotoxic activity of human anti-melanoma

pecific CD8+ T cells by blocking the inhibitory effects of CADO123]; significantly blocked the adenosine-induced suppression ofytokine release by CD8+ and CD4+ Th1 cells [123]; abrogatedhe T cell hyporesponsiveness in human FL patients by increas-ng their cytokine production [80], and significantly decreasedreg-mediated suppression of effector T cell responses in HNSCCatients [25,49,50]. Moreover, treatment with ZM 241385 or caf-eine (1,3,7-trimethyxanthine) rendered endogenously developedr adoptively transferred anti-tumor CD8+ T cells much moreesistant to inhibition in the tumor microenvironment and, inurn, delayed the onset of rapid growth of CL8-1 melanoma inumor-bearing mice [44,124]. In addition, ZM 241385 and caffeinemproved the rejection of LL-LCMW lung carcinoma, destructionf lung metastasis of CMS4 sarcoma, and prevention of neo-ascularization by anti-tumor CD8+ T cells [44,124]. Similarly,ombination of SCH58261 or caffeine with adoptive T cell therapyemarkably improved survival in tumor-bearing mice by abrogat-ng the adenosine-induced suppression of tumor-specific T-cellroliferation [58]. A2B receptor blockade by a nonselective antag-nist, aminophylline (AMO), and a selective antagonist, ATL801,lowed the growth of bladder and breast tumors by increasingXCR3-dependent anti-tumor T cell responses. AMO and ATL801ignificantly increased tumor levels of IFN-� and IFN-induciblehemokine CXCL10, which is a ligand for CXCR3 [125]. CVT-6883a selective A2B antagonist) inhibited NECA-stimulated VEGF pro-uction from host tumor-associated immune cells in a mouse LLC

sograft model, thus, inhibiting tumor growth and increasing hosturvival [126].

. Therapeutic potential of downstream targeting in theAMP–PKA pathway

The convergent activation of cAMP-dependent PKA type 1athway via EP2/EP4 and A2A/A2B receptors represents the

utually shared facet of the ‘double-edge sword’ execution of

denosine–PGE2 signaling in the tumor microenvironment. Thisathway is tightly regulated at several levels to maintain speci-city in the extracellular signal inputs [127]. As a target even further

cal Research 73 (2013) 8– 19 15

downstream of receptor level, pharmacological blockade at differ-ent levels of this pathway may further increase the specificity ofaction in the context of chemo-immunotherapeutic manipulationof adenosine–PGE2/cAMP nexus.

5.1. AC inhibition

AC is a central regulator of cAMP-elevating adenosine–PGE2pathway in the tumor microenvironment. The adenosine–PGE2signaling via the A2A/A2B and EP2/EP4 receptors converge on AC,stimulating its activity and leading to increased production ofcytosolic cAMP. Pharmacological inhibitors of AC may exert clin-ically relevant and beneficial effects that might not be achievedwhen just either arm, PGE2-EP2/EP4 or adenosine-A2A/A2B, ofthe adenosine–PGE2/cAMP nexus is antagonized alone. Cur-rently, non-receptor dependent pharmacological inhibitors of ACinclude MDL-12,330A, 2′,5′-dideoxyadenosine (DDA), and SQ22536(Fig. 5).

At least nine transmembrane isoforms of AC have been reportedin mammalian cells, which are differentially expressed in var-ious tissues [128]. Nevertheless, detailed data regarding theparticipation of specific AC isoforms in tumorigenesis and tumorimmunoescape is sparse. AC-7 is the major isoform selectivelyexpressed in hematopoietic cells, regulating cAMP synthesis inimmune cells in order to shape the immune responses in a con-text dependent manner [129]. It may be a fruitful starting point forpharmacologic interventions aimed at disarming adenosine–PGE2pathway in the tumor microenvironment [130]. Interestingly, evi-dence is available which implies that AC inhibitors may inhibitPGE2-mediated enhancement of tumor growth and progression.For instance, 2′,5′, DDA significantly blocked PGE2-induced VEGFsecretion by inhibiting EP2-dependent cAMP signaling pathway inPC-3 cells, a prostatic small cell carcinoma cell line [35]. Moreover,2′,5′, DDA inhibited PGE2-promoted cell proliferation and VEGFproduction in human colon cancer cell lines LS174 T and HCT15,while MDL-12330A reversed PGE2-derived up-regulation of cellviability [131]. More importantly, SQ22536, which has shown anti-tumor potential in various tumor models [132–135], counteractedPGE2-mediated suppression of IP-10 production in epidermoidcarcinoma A431 [113], tempting to speculate that AC inhibitorsmay not only inhibit tumor growth and angiogenesis, but alsoinduce anti-tumor immunity [115]. However, neither in vitro norin vivo pharmacological data are yet available regarding anti-tumorimmune enhancing potential of AC inhibitors. Given the existenceof pharmacological studies supporting the anti-neoplastic utilityof AC inhibitors, further testing and development of such com-pounds in the context of chemo-immunotherapeutic manipulationof adenosine–PGE2/cAMP nexus is anticipated.

5.2. The cAMP–PKA inhibitors

Looking from the perspective of cAMP–PKA cascade, extensiveliterature survey indicates a good in vitro and in vivo pharmaco-logical evidence for the chemo-immunotherapeutic potential oftwo specific and competent inhibitors (Fig. 5): Rp-8-Br-cAMPSthat antagonize binding of cAMP to the regulatory 1 subunit ofPKA, and H89 that selectively inhibits PKA catalytic subunit. Rp-8-Br-cAMPS reduced tumor development in Apc (Min/+) micemodel of intestinal cancer by inhibiting PGE2-induced phosphory-lation of beta-catenin by PKA [136]. Immunohistochemical analysisrevealed that Rp-8-Br-cAMPS significantly down-regulated theexpression and nuclear translocation of beta-catenin, and then,

subsequent expression of its target genes c-Myc and COX2. This wasalso confirmed in parallel experiments by the same research groupinvolving human colon cancer cell line (HCT116) in which Rp-8-Br-cAMPS blocked PGE2-induced beta-catenin phosphorylation

16 M. Hussain et al. / Pharmacological Research 73 (2013) 8– 19

ate cy

am[msitprmbo

tccocCaRaccitr8aecpmPc

c

adenosine–PGE2/cAMP nexus. It may offer a new avenue in can-

Fig. 5. Chemical structures of adenyl

nd c-Myc up-regulation [136]. Moreover, Rp-8-Br-cAMPS reducedigration of highly metastatic murine (C3L5) breast cancer cells

34], which shows its inhibitory potential against invasive andetastatic tumors. Likewise, H89 may also inhibit tumorigene-

is and tumor progression [108]. It inhibited PGE2/PKA-mediatedncreases in S100A8 mRNA and aromatase protein expressions andheir subsequent tumorigenesis promoter activities in human PC-3rostate [94], and MCF-7 and MDA-MB-231 breast cancer cell lines,espectively [95]. H89 also induced apoptosis in human papillo-avirus type 16 E5 protein-expressing C-33A cervical cancer cells

y recovering E5-induced, COX2/PGE2/PKA-mediated degradationf Bax protein expression [93].

The cAMP–PKA inhibitors may enhance immune-mediatedumor destruction by down-regulating adenosine–PGE2-mediatedAMP up-regulation to optimal levels in tumor-infiltrating immuneells. For instance, Rp-8-Br-cAMPS blocked the suppressive effectsf adenosine–PGE2/cAMP nexus on LAK cell cytotoxic activity andytokine production [27]. It also blocked the inhibitory effects ofADO (adenosine analog) on cytokine production and cytotoxicctivity of LAK cells against 3LL tumor cells [46]. Furthermore,askovalova et al. [123] showed that Rp-8-Br-cAMPS inhibited thedenosine/cAMP-induced impairment of cytokine production andytotoxic activity of tumor-infiltrating human anti-melanoma spe-ific CD4+ and CD8+ T lymphocytes. Tumor-infiltrating Treg impedemmune surveillance and hamper the development of an effec-ive anti-tumor immunity by expressing COX2 and suppressingesponder T cells in PGE2-cAMP-dependent manner [54,137]. Rp--Br-cAMPS significantly improved the anti-tumor T-cell immunectivity that was suppressed by Treg-derived PGE2 in the periph-ral blood of colorectal cancer patients [53,54]. Similarly, H89ompletely reversed the tumor-shed PGE2-cAMP-dependent sup-ression of CCL5 secretion in LPS-activated macrophages of 4T1ammary gland tumor-bearing mice [36], and also counteracted

GE2-mediated suppression of IP-10 in human epidermoid A431ancer [113].

In summary, the dual activity findings related to Rp-8-Br-AMPS and H89 in the tumor experimental models provide the

clase (AC) and cAMP–PKA inhibitors.

proof of concept that concomitant disarming of adenosine–PGE2signaling, via single pathway blockade, is achievable in the tumormicroenvironment, and might have profound implications in thecontext of ‘two hit’ proposition. Nevertheless, the research in thisfield is still in its infancy, and a lot more basic investigationsare required before translating these findings into phases I andII clinical studies/trials. First, extensive pre-clinical documenta-tion regarding anti-neoplastic and anti-tumor immune-enhancingeffects of selective AC and cAMP–PKA inhibitors is needed. Sec-ond, reasonable pharmacological data about dual activity findingsof such compounds in various types of human cancers is stillpending. Third, our in-depth understanding of the pharmacody-namics of respective antagonists/inhibitors for cAMP–PKA pathwayis far from complete, and a critical evaluation of potential toxi-cities is required. Hopefully, future research efforts in appropriatemouse models, as well as intelligently conducted clinical evalua-tions will fine tune the spectrum of their efficacy in relevance toconcomitant inhibition of adenosine–PGE2 signaling in the tumormicroenvironment.

6. Conclusion

This is an attention-grabbing exercise based on a provoca-tive speculation that multilevel pharmacologic blockade/inhibitionof adenosine–PGE2/cAMP nexus may produce ‘two kills’ effectagainst cancer, able on the one hand to inhibit tumor growth ormetastatic potential, and on the other hand, may restore/activatethe endogenous anti-tumor immune responses. The experimen-tal and clinical information reviewed in this article clearly supportour thesis, and deem to be self-evident for the ‘two hit’ exploita-tion of selective inhibitors/antagonists at different levels of the

cer chemo-immunotherapeutics, in which these agents may begiven either alone as ‘two-in-one’ chemo-immunotherapy or asadjunctive couple with other forms of anti-tumor immunotherapyto induce improved anti-cancer effects.

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onflict of interest

The authors have no conflict of interest.

cknowledgment

We thank the many researchers who have contributed to oururrent understanding of adenosine–PGE2/cAMP nexus.

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