Editorial
Adenosine pathway and cancer: where do we go from here? 1.
CD73 as viable target in cancer therapy
2.
Adenosine system in cancer microenvironment
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
3.
Expert opinion
Luca Antonioli†, Gy€orgy Hasko´, Matteo Fornai, Rocchina Colucci & Corrado Blandizzi †
University of Pisa, Division of Pharmacology and Chemotherapy, Department of Clinical and Experimental Medicine, Pisa, Italy
Increasing evidence supports the occurrence of an intriguing link between tumor onset and development with the microenvironment in which cancer cells are embedded. In this context, a critical role of CD73, in calibrating the duration, magnitude and composition of adenosine signaling in cancer development and progression, has been identified. Adenosine levels are increased in cancer tissues as the result of genetic alterations that occur during tumor progression. Indeed, a rearrangement of the adenosine metabolic machinery has been described within the neoplastic milieu with the aim of amplifying adenosine generation, thereby creating an immune tolerant microenvironment suitable for tumor onset and development. At the same time, adenosine, through the engagement of receptors expressed on neoplastic cells, finely tunes the growth and dissemination of tumor mass, thus interfering with cancer proliferation, apoptosis and metastasis. Based on current knowledge, an improved understanding of how and to what extent adenosine participates to the molecular mechanisms underlying cancer development and diffusion will pave the way toward new therapeutic advances. The discovery and development of drugs targeted on this system might lead to substantial improvements in the clinical management of various cancers. Keywords: adenosine, adenosine receptors, cancer, CD73, immune system, inflammation, novel therapies Expert Opin. Ther. Targets (2014) 18(9):973-977
The review “Targeting CD73 in triple-negative breast cancer” published by Allard et al. [1] in this issue of Expert Opinion Therapeutic Targets discusses the basic structural and molecular features of CD73 and its involvement in the development of cancer, with a particular attention to the role played by CD73 in the biology of triple negative breast cancer (TNBC). CD73 (designated also as ecto-5¢-nucleotidase or ecto5¢NTase) is a key membrane-bound enzyme, which is deeply involved in calibrating the duration, magnitude and composition of the ‘purinergic halo’ surrounding cells, through the dephosphorylation of extracellular AMP into adenosine (Figure 1). This enzyme is expressed in blood cells, spleen, lymph nodes, bone marrow and endothelium. At the level of macrophages, lymphocytes, regulatory T cells (Treg) and dendritic cells, CD73 plays a critical role in calibrating the activity of immune cells, driving a shift toward an anti-inflammatory environment induced by adenosine [2]. 1.
CD73 as viable target in cancer therapy
The role played by CD73 in cancer development and progression can be better understood looking at this enzyme in the wider context of the hypoxiaadenosinergic pathway. Indeed, CD73 is triggered when the extent of collateral tissue damage by activated immune cells during adverse conditions determines the 10.1517/14728222.2014.925883 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
973
L. Antonioli et al.
↓ NK cell activity
↑ Macrophage M2 polarization
↓ Th 1 cells
↑ Expansion of MDSC
↓ Tumor antigen cross-presentation from
↑ Treg
dendritic cells Immune-tolerant tumor microenvironment
Adenosine
ATP
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
Blood vessel AMP
CD73
CD39
↑ Tumor cell proliferation
Neoplastic cell
↑ Metastatic properties
↑ Angiogenesis
Figure 1. Schematic representation of the main actions mediated by adenosine in tumor microenvironment. ": Increases; #: Decreases; ATP: Adenosine triphosphate; MSDC: Myeloid-derived suppressor cells; NK cells: Natural killer cells; Th1 cells: T helper 1 cells; Treg: regulatory T cells.
reduction of local blood/oxygen supply, and thereby a hypoxic condition [3]. Inflamed areas of otherwise normal tissues as well as solid tumors are characterized by transient or chronic hypoxia, followed by accumulation of extracellular adenosine [3]. In particular, hypoxic conditions are associated with a massive release of ATP into the extracellular space, which is quickly converted into AMP by CD39 (also designed as ectonucleoside triphosphate diphosphohydrolase 1 [ENTPDase1]) and then into adenosine by CD73 (Figure 1) [4]. The hypoxia-driven extracellular adenosine, through signaling via A2A and A2B receptors, elicits the accumulation of immunosuppressive intracellular cyclic AMP, and thereby the inhibition of activated immune cells [5]. Based on this concept, several studies have revealed a crucial role of the hypoxia/CD39/CD73/adenosine/A2A-A2B receptor axis in protecting hypoxic normal and cancerous tissues from collateral damage during immune responses [3]. In particular, conclusive evidence for the role of extracellular adenosine, and thus for the relevance of CD73 in tumor protection, stems from studies on A2A-receptor deficient mice [6-8]. Under adverse conditions, such as hypoxia, inflammation or neoplastic disorders, levels of extracellular adenosine are increased in these microenvironments due to changes in the activities of enzymes involved in adenosine metabolism [5]. An increased expression and activity of CD73 has been observed in neoplastic tissues, suggesting an involvement of this catabolic enzyme in cancer onset and progression [2,4]. Within the neoplastic milieu, CD73 overexpression can be ascribed to several mechanisms such as: i) hypoxia inducible factor-1a, which is activated under hypoxic conditions [9]; 974
ii) activation of Wnt signaling [10]; iii) counteraction of immune system functions since CD73-positive cancer cells are better equipped to evade antitumor immune responses via the release of immunosuppressive adenosine [11]; and iv) loss of estrogen receptor (ER) expression, which is known to maintain the constitutive expression of CD73, as observed in ER-negative breast cancer cells [10]. Within cancer microenvironments, CD73 plays a critical role in promoting tumor growth through paracrine and autocrine actions on cancer cells, endothelial cells and immune cells [11]. In this regard, it is emerging that the main mechanism underlying CD73-mediated tumor growth is related to the release of adenosine and subsequent immunosuppression of tumor- infiltrating immune cells [12]. Indeed, this catabolic enzyme, widely expressed both in tumor and stromal cells, negatively regulates both the activation and effector phase of CD8+ T cells and natural killer (NK) cells, thus contributing to tumor immune evasion [12]. It is becoming increasingly evident that tumor-mediated immunosuppression frequently occurs in concomitance with tumor angiogenesis, possibly as a result of the cooption of tissue repair programs. In particular, CD73-derived adenosine is being appreciated as a critical factor promoting concurrent immunosuppression and angiogenesis. In support of this view, adenosine produced by tumor-derived CD73 has been shown to elicit the autocrine release of vascular endothelial growth factor, thus promoting neovessel formation in neoplastic microenvironment [13]. Of note, several human solid tumors express high levels of CD73 involved in cancer cell proliferation [2]. In particular, as
Expert Opin. Ther. Targets (2014) 18(9)
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
Adenosine pathway and cancer
described by Allard et al. [1], CD73, via intratumoral adenosine generation, plays a pivotal role in in vitro and in vivo breast cancer tumorigenesis, supporting cell proliferation, adhesion, migration and invasion through EGFR and IL-8 expression [1]. The evaluation of CD73 expression in cancer cells has unraveled its prognostic significance in neoplastic progression. A direct correlation has been observed between the high expression of CD73 in cancer tissues and a poor outcome of the disease [14]. In addition, the involvement of CD73 in cancer resistance to anthracycline treatment has been reported [15]. Indeed, following massive release upon anthracycline-induced tumor cell death, ATP is converted into immunosuppressive adenosine, via CD73, thus promoting cancer cell growth and resistance to chemotherapy [15]. Based on current knowledge, CD73 appears to be a clinically key target in the management of cancer. In line with this concept, several preclinical studies have shown that the pharmacological blockade of CD73 as well as treatment with anti-CD73 monoclonal antibody is effective in restraining both tumor growth and metastatic spread in mice [4]. In addition, anti-CD73 treatment has been found to enhance the antiblastic activity of other anticancer drugs, such as anthracyclines or immunomodulatory monoclonal antibodies, like antiprogrammed cell death 1 or anticytotoxic T-lymphocyte antigen4 [1]. At present, monoclonal antibodies targeting human CD73 have also been generated and tested in a human xenograft model of TNBC with encouraging results [16]. Of note, targeting CD73 is currently supported not only by preclinical data, but also by clinical observations. In particular, a study by Loi et al. [17] pointed out that TNBC patients with high CD73 expression displayed a worse prognosis, associated with a higher risk of distant metastases, thus corroborating the significance of anti-CD73 therapy in this specific pathological setting. When discussing potential clinical applications of CD73 targeting, also worth mentioning that the development of small molecule inhibitors of CD73 would be a preferable strategy, owing to their limited half-life in vivo [18]. By contrast, it would be more difficult to counteract the activity of antiCD73 monoclonal antibodies in case of adverse effects, as a consequence of their prolonged half-life (about 20 days) in humans.
Adenosine system in cancer microenvironment
2.
The review by Allard et al. [1] highlights the intriguing link occurring between neoplastic onset and development with the microenvironment in which cancer cells are embedded, pointing out the critical role of CD73, via adenosine generation, in tumor development and progression (Figure 1). A large body of evidence supports the notion that adenosine is not a mere passive product of cancer tissue hypoxia, ischemia and necrosis, but rather that its release is actively increased as a result of specific genetic alterations occurring
throughout tumor progression. Indeed, several tumors put in place altered metabolic systems, such as the increased expression and activity of CD73, to amplify adenosine generation, aimed at creating a ‘protective adenosine halo’, useful to ensure the onset and progression of neoplasia [4]. Extracellular adenosine, massively released in cancer tissue, represents a critical player in the alteration of immune cell activity [19,20], mainly via engagement of A2A and A2B receptors. This is possibly because the tightly regulated adenosine receptor system of immune cells is altered in cancer, thus switching these cells from an immune surveillance activity and host defense toward a neoplastic transformation and growth [4]. Beyond its involvement in the generation of an immune tolerant microenvironment suitable for tumor onset and progression, adenosine, through the engagement of receptors expressed on neoplastic cells, regulates also the growth and dissemination of the tumor mass by direct actions on cancer cell proliferation, apoptosis and metastasis (Figure 1) [4]. In particular, it has been reported that adenosine can promote cell proliferation in some cases, via A1, A2A and A2B receptors, while restraining this process in other contexts, via A3 receptors. Moreover, adenosine has been shown to exert proapoptotic activity, primarily via A3 receptors, but also via A2A and A2B receptors. Of note, a particular tumor can coexpress different adenosine receptor types. For this reason, adenosinergic drug therapies should take into account these competing pro- and antiproliferative/apoptotic actions mediated by the different adenosine receptor subtypes [4]. A number of preclinical studies support the efficacy of the pharmacological blockade of A2A receptors in curbing cancer development and spread, through an enhancement of the antitumor actions of CD8+ T cells as well as via an inhibition of tumor neovascularization, growth and metastatic potential [4]. Likewise, the pharmacological blockade of A2B receptors resulted in a delay of tumor growth and reduction of metastatic dissemination, reversing the immune suppression on tumor, via an inhibition of Treg differentiation [21] and an increased density of tumor-infiltrating CD8+ T cells and NK cells [22]. The stimulation of A3 receptors, via selective agonists, such as IB-MECA (designated also as CF101) or Cl-IB-MECA (designated also as CF102), has been found to modulate cell growth-regulatory downstream signaling pathways as well as to downregulate the cell survival protein AKT, with subsequent inhibition of tumor growth [4]. Recently, data obtained from a Phase I/Phase II open-label study (clinical trial NCT00790218), enrolling patients with advanced hepatocellular carcinoma, demonstrated a favorable safety and efficacy profile of CF102, thus justifying its further clinical development [23]. 3.
Expert opinion
An improved understanding of the molecular mechanisms of adenosine pathways underlying the onset, progression and
Expert Opin. Ther. Targets (2014) 18(9)
975
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
L. Antonioli et al.
diffusion of neoplasia may help to translate their viability as therapeutic targets in cancer. The performance of preclinical investigations has improved our knowledge of the functional roles played by adenosine, via the engagement of its own receptors, in cancer biology, thus paving the way toward the discovery of novel antineoplastic therapies. However, despite in vitro studies and animal models support the concept that targeting the adenosine system has a huge potential for treating tumors, the translation of current data into clinical practice will require a more thorough understanding of how adenosine regulates cancer [4]. In this regard, one major hurdle concerns the fact that it is impossible to provide direct genetic in vivo evidence for the role of extracellular adenosine in cancer onset and development since it is impossible to genetically knock-out animals for this nucleoside. In addition, the intrinsic actions of adenosine on neoplastic cells depend on a variety of factors, which include the type of cancer, the adenosine receptor subtype expressed on cancer cells, and whether proliferation, apoptosis or metastasis is considered. Since there is evidence that a particular tumor can express different adenosine receptors, therapeutic strategies targeted on adenosine pathways should take into account the opposing actions mediated by different adenosine receptors. Accordingly, the performance of preclinical studies, using various methodological approaches, will help to better dissect the roles of adenosine in mediating neoplasia onset and progression as well as to highlight the potential for adenosinergic therapies in the management of patients with cancer. Future efforts should be focused, as suggested by Allard et al. [1], on the development of CD73 inhibitors. Likewise, A2A and A2B receptor antagonists hold a great potential for the treatment of cancer, with particular regard for one of the most aggressive breast cancer such as the TNBC. Targeting A2A or A2B receptors or inhibiting adenosine signaling, via CD73 blockade, can represent a promising adjunct to tumor immunotherapy since several immunotherapeutic approaches to curb neoplasia have failed due to CD73 overexpression in cancer cells or high adenosine levels within tumor microenvironment. In this regard, there is evidence that the genetic deletion of immunosuppressive A2A and A2B
976
adenosine receptors or their pharmacological inactivation can prevent the inhibition of antitumor T cells by the hypoxic tumor microenvironment, thus facilitating a process of full cancer rejection [24]. This approach is based on the in vivo genetic evidence demonstrating that A2A receptors play a pivotal role in the protection of normal tissues from overactive immune cells in acutely inflamed and hypoxic areas. Subsequent observations, showing an enhanced T-cell-mediated rejection of tumors in mice with inactivated A2A receptors, highlighted a protective role of these receptors on hypoxic cancer tissues, thus indicating their inactivation as a useful way to improve tumor rejection via antitumor T cells [5]. Along this way, several studies have shown that A2A receptor antagonists, already tested and shown to be safe in clinical trials on Parkinson disease, might also prevent immunosuppression mediated through the generation of adenosine by Tregs, further strengthening the rationale for implementing antiadenosine strategies by A2A antagonists to enhance the immune responses against neoplastic cells [24]. Of note, an attractive feature of these drugs is that they could be used in combination with other clinically available cytotoxic drugs, in order to magnify their antiblastic activity. Overall, a better characterization of how and to what extent adenosine participates in the molecular mechanisms underlying cancer development and diffusion will drive new therapeutic advances in creating more targeted drug interventions, which might lead to substantial improvements of the clinical management of various cancers.
Declaration of interest L Antonioli was supported by National Institutes of Health Grant R01GM66189 (G.H.), Nexus award ‘Marcello Tonini’ (L.A.) and by IBD Research Foundation (mini grant 2012) (L.A.). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Expert Opin. Ther. Targets (2014) 18(9)
Adenosine pathway and cancer
Bibliography
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
1.
Allard B, Turcotte M, Stagg J. Targeting CD73 and downstream adenosine receptor signaling in triple-negative breast cancer. Expert Opin Ther Targets 2014;18(8):863-81
ecto-5’-nucleotidase and increase extracellular adenosine generation. Exp Cell Res 2004;296(2):99-108
20.
Hasko´ G, Szabo´ C, Ne´meth ZH, et al. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Immunol 1996;157(10):4634-40
21.
Nakatsukasa H, Tsukimoto M, Harada H, et al. Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells. Biochem Biophys Res Commun 2011;409(1):114-19
22.
Ghiringhelli F, Bruchard M, Chalmin F, et al. Production of adenosine by ectonucleotidases: a key factor in tumor immunoescape. J Biomed Biotechnol 2012;2012:473712
Iannone R, Miele L, Maiolino P, et al. Blockade of A2b adenosine receptor reduces tumor growth and immune suppression mediated by myeloid-derived suppressor cells in a mouse model of melanoma. Neoplasia 2013;15(12):1400-9
23.
Stemmer SM, Benjaminov O, Medalia G, et al. CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label,dose-escalation study. Oncologist 2013;18(1):25-6
24.
Ohta A, Gorelik E, Prasad SJ, et al. A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 2006;103(35):13132-7
11.
Allard B, Turcotte M, Stagg J. CD73-generated adenosine: orchestrating the tumor-stroma interplay to promote cancer growth. J Biomed Biotechnol 2012;2012:485156
12.
Stagg J, Divisekera U, Duret H, et al. CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis. Cancer Res 2011;71(8):2892-900 Allard B, Turcotte M, Spring K, et al. Anti-CD73 therapy impairs tumor angiogenesis. Int J Cancer 2014;134(6):1466-73
2.
Antonioli L, Blandizzi C, Pacher P, et al. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer 2013;13(12):842-57
3.
Sitkovsky MV. T regulatory cells: hypoxia-adenosinergic suppression and re-direction of the immune response. Trends Immunol 2009;30(3):102-8
4.
Antonioli L, Pacher P, Vizi ES, et al. CD39 and CD73 in immunity and inflammation. Trends Mol Med 2013;19(6):355-67
13.
Lukashev D, Ohta A, Sitkovsky M. Hypoxia-dependent anti-inflammatory pathways in protection of cancerous tissues. Cancer Metastasis Rev 2007;26(2):273-9
14.
6.
Jin D, Fan J, Wang L, et al. CD73 on tumor cells impairs antitumor T-cell responses: a novel mechanism of tumor-induced immune suppression. Cancer Res 2010;70(6):2245-55
15.
Stagg J, Divisekera U, McLaughlin N, et al. Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis. Proc Natl Acad Sci USA 2010;107(4):1547-52
7.
Waickman AT, Alme A, Senaldi L, et al. Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor. Cancer Immunol Immunother 2012;61(6):917-26
16.
Rust S, Guillard S, Sachsenmeier K, et al. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type. Mol Cancer 2013;12:11
5.
8.
9.
10.
Beavis PA, Divisekera U, Paget C, et al. Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors. Proc Natl Acad Sci USA 2013;110(36):14711-16 Synnestvedt K, Furuta GT, Comerford KM, et al. Ecto-5’-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 2002;110(7):993-1002 Spychala J, Kitajewski J. Wnt and betacatenin signaling target the expression of
mechanisms. FASEB J 2000;14(13):2065-74
17.
Loi S, Pommey S, Haibe-Kains B, et al. CD73 promotes anthracycline resistance and poor prognosis in triple negative breast cancer. Proc Natl Acad Sci USA 2013;110(27):11091-6
18.
Zhang B. CD73: a novel target for cancer immunotherapy. Cancer Res 2010;70(16):6407-11
19.
Hasko´ G, Kuhel DG, Chen JF, et al. Adenosine inhibits IL-12 and TNF[alpha] production via adenosine A2a receptor-dependent and independent
Expert Opin. Ther. Targets (2014) 18(9)
Affiliation
Luca Antonioli†1,2 PhD, Gy€orgy Hasko´2, Matteo Fornai1, Rocchina Colucci1 & Corrado Blandizzi1 † Author for correspondence 1 University of Pisa, Department of Clinical and Experimental Medicine, Via Roma 55, 56126 -- Pisa, Italy Tel: +39 050 2218762; Fax: +39 050 2218758; E-mail: [email protected] 2 Rutgers-New Jersey Medical School, Department of Surgery and Center for Immunity and Inflammation, Newark, NJ 07103, USA
977
Adenosine pathway and cancer: where do we go from here? 1.
CD73 as viable target in cancer therapy
2.
Adenosine system in cancer microenvironment
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
3.
Expert opinion
Luca Antonioli†, Gy€orgy Hasko´, Matteo Fornai, Rocchina Colucci & Corrado Blandizzi †
University of Pisa, Division of Pharmacology and Chemotherapy, Department of Clinical and Experimental Medicine, Pisa, Italy
Increasing evidence supports the occurrence of an intriguing link between tumor onset and development with the microenvironment in which cancer cells are embedded. In this context, a critical role of CD73, in calibrating the duration, magnitude and composition of adenosine signaling in cancer development and progression, has been identified. Adenosine levels are increased in cancer tissues as the result of genetic alterations that occur during tumor progression. Indeed, a rearrangement of the adenosine metabolic machinery has been described within the neoplastic milieu with the aim of amplifying adenosine generation, thereby creating an immune tolerant microenvironment suitable for tumor onset and development. At the same time, adenosine, through the engagement of receptors expressed on neoplastic cells, finely tunes the growth and dissemination of tumor mass, thus interfering with cancer proliferation, apoptosis and metastasis. Based on current knowledge, an improved understanding of how and to what extent adenosine participates to the molecular mechanisms underlying cancer development and diffusion will pave the way toward new therapeutic advances. The discovery and development of drugs targeted on this system might lead to substantial improvements in the clinical management of various cancers. Keywords: adenosine, adenosine receptors, cancer, CD73, immune system, inflammation, novel therapies Expert Opin. Ther. Targets (2014) 18(9):973-977
The review “Targeting CD73 in triple-negative breast cancer” published by Allard et al. [1] in this issue of Expert Opinion Therapeutic Targets discusses the basic structural and molecular features of CD73 and its involvement in the development of cancer, with a particular attention to the role played by CD73 in the biology of triple negative breast cancer (TNBC). CD73 (designated also as ecto-5¢-nucleotidase or ecto5¢NTase) is a key membrane-bound enzyme, which is deeply involved in calibrating the duration, magnitude and composition of the ‘purinergic halo’ surrounding cells, through the dephosphorylation of extracellular AMP into adenosine (Figure 1). This enzyme is expressed in blood cells, spleen, lymph nodes, bone marrow and endothelium. At the level of macrophages, lymphocytes, regulatory T cells (Treg) and dendritic cells, CD73 plays a critical role in calibrating the activity of immune cells, driving a shift toward an anti-inflammatory environment induced by adenosine [2]. 1.
CD73 as viable target in cancer therapy
The role played by CD73 in cancer development and progression can be better understood looking at this enzyme in the wider context of the hypoxiaadenosinergic pathway. Indeed, CD73 is triggered when the extent of collateral tissue damage by activated immune cells during adverse conditions determines the 10.1517/14728222.2014.925883 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
973
L. Antonioli et al.
↓ NK cell activity
↑ Macrophage M2 polarization
↓ Th 1 cells
↑ Expansion of MDSC
↓ Tumor antigen cross-presentation from
↑ Treg
dendritic cells Immune-tolerant tumor microenvironment
Adenosine
ATP
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
Blood vessel AMP
CD73
CD39
↑ Tumor cell proliferation
Neoplastic cell
↑ Metastatic properties
↑ Angiogenesis
Figure 1. Schematic representation of the main actions mediated by adenosine in tumor microenvironment. ": Increases; #: Decreases; ATP: Adenosine triphosphate; MSDC: Myeloid-derived suppressor cells; NK cells: Natural killer cells; Th1 cells: T helper 1 cells; Treg: regulatory T cells.
reduction of local blood/oxygen supply, and thereby a hypoxic condition [3]. Inflamed areas of otherwise normal tissues as well as solid tumors are characterized by transient or chronic hypoxia, followed by accumulation of extracellular adenosine [3]. In particular, hypoxic conditions are associated with a massive release of ATP into the extracellular space, which is quickly converted into AMP by CD39 (also designed as ectonucleoside triphosphate diphosphohydrolase 1 [ENTPDase1]) and then into adenosine by CD73 (Figure 1) [4]. The hypoxia-driven extracellular adenosine, through signaling via A2A and A2B receptors, elicits the accumulation of immunosuppressive intracellular cyclic AMP, and thereby the inhibition of activated immune cells [5]. Based on this concept, several studies have revealed a crucial role of the hypoxia/CD39/CD73/adenosine/A2A-A2B receptor axis in protecting hypoxic normal and cancerous tissues from collateral damage during immune responses [3]. In particular, conclusive evidence for the role of extracellular adenosine, and thus for the relevance of CD73 in tumor protection, stems from studies on A2A-receptor deficient mice [6-8]. Under adverse conditions, such as hypoxia, inflammation or neoplastic disorders, levels of extracellular adenosine are increased in these microenvironments due to changes in the activities of enzymes involved in adenosine metabolism [5]. An increased expression and activity of CD73 has been observed in neoplastic tissues, suggesting an involvement of this catabolic enzyme in cancer onset and progression [2,4]. Within the neoplastic milieu, CD73 overexpression can be ascribed to several mechanisms such as: i) hypoxia inducible factor-1a, which is activated under hypoxic conditions [9]; 974
ii) activation of Wnt signaling [10]; iii) counteraction of immune system functions since CD73-positive cancer cells are better equipped to evade antitumor immune responses via the release of immunosuppressive adenosine [11]; and iv) loss of estrogen receptor (ER) expression, which is known to maintain the constitutive expression of CD73, as observed in ER-negative breast cancer cells [10]. Within cancer microenvironments, CD73 plays a critical role in promoting tumor growth through paracrine and autocrine actions on cancer cells, endothelial cells and immune cells [11]. In this regard, it is emerging that the main mechanism underlying CD73-mediated tumor growth is related to the release of adenosine and subsequent immunosuppression of tumor- infiltrating immune cells [12]. Indeed, this catabolic enzyme, widely expressed both in tumor and stromal cells, negatively regulates both the activation and effector phase of CD8+ T cells and natural killer (NK) cells, thus contributing to tumor immune evasion [12]. It is becoming increasingly evident that tumor-mediated immunosuppression frequently occurs in concomitance with tumor angiogenesis, possibly as a result of the cooption of tissue repair programs. In particular, CD73-derived adenosine is being appreciated as a critical factor promoting concurrent immunosuppression and angiogenesis. In support of this view, adenosine produced by tumor-derived CD73 has been shown to elicit the autocrine release of vascular endothelial growth factor, thus promoting neovessel formation in neoplastic microenvironment [13]. Of note, several human solid tumors express high levels of CD73 involved in cancer cell proliferation [2]. In particular, as
Expert Opin. Ther. Targets (2014) 18(9)
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
Adenosine pathway and cancer
described by Allard et al. [1], CD73, via intratumoral adenosine generation, plays a pivotal role in in vitro and in vivo breast cancer tumorigenesis, supporting cell proliferation, adhesion, migration and invasion through EGFR and IL-8 expression [1]. The evaluation of CD73 expression in cancer cells has unraveled its prognostic significance in neoplastic progression. A direct correlation has been observed between the high expression of CD73 in cancer tissues and a poor outcome of the disease [14]. In addition, the involvement of CD73 in cancer resistance to anthracycline treatment has been reported [15]. Indeed, following massive release upon anthracycline-induced tumor cell death, ATP is converted into immunosuppressive adenosine, via CD73, thus promoting cancer cell growth and resistance to chemotherapy [15]. Based on current knowledge, CD73 appears to be a clinically key target in the management of cancer. In line with this concept, several preclinical studies have shown that the pharmacological blockade of CD73 as well as treatment with anti-CD73 monoclonal antibody is effective in restraining both tumor growth and metastatic spread in mice [4]. In addition, anti-CD73 treatment has been found to enhance the antiblastic activity of other anticancer drugs, such as anthracyclines or immunomodulatory monoclonal antibodies, like antiprogrammed cell death 1 or anticytotoxic T-lymphocyte antigen4 [1]. At present, monoclonal antibodies targeting human CD73 have also been generated and tested in a human xenograft model of TNBC with encouraging results [16]. Of note, targeting CD73 is currently supported not only by preclinical data, but also by clinical observations. In particular, a study by Loi et al. [17] pointed out that TNBC patients with high CD73 expression displayed a worse prognosis, associated with a higher risk of distant metastases, thus corroborating the significance of anti-CD73 therapy in this specific pathological setting. When discussing potential clinical applications of CD73 targeting, also worth mentioning that the development of small molecule inhibitors of CD73 would be a preferable strategy, owing to their limited half-life in vivo [18]. By contrast, it would be more difficult to counteract the activity of antiCD73 monoclonal antibodies in case of adverse effects, as a consequence of their prolonged half-life (about 20 days) in humans.
Adenosine system in cancer microenvironment
2.
The review by Allard et al. [1] highlights the intriguing link occurring between neoplastic onset and development with the microenvironment in which cancer cells are embedded, pointing out the critical role of CD73, via adenosine generation, in tumor development and progression (Figure 1). A large body of evidence supports the notion that adenosine is not a mere passive product of cancer tissue hypoxia, ischemia and necrosis, but rather that its release is actively increased as a result of specific genetic alterations occurring
throughout tumor progression. Indeed, several tumors put in place altered metabolic systems, such as the increased expression and activity of CD73, to amplify adenosine generation, aimed at creating a ‘protective adenosine halo’, useful to ensure the onset and progression of neoplasia [4]. Extracellular adenosine, massively released in cancer tissue, represents a critical player in the alteration of immune cell activity [19,20], mainly via engagement of A2A and A2B receptors. This is possibly because the tightly regulated adenosine receptor system of immune cells is altered in cancer, thus switching these cells from an immune surveillance activity and host defense toward a neoplastic transformation and growth [4]. Beyond its involvement in the generation of an immune tolerant microenvironment suitable for tumor onset and progression, adenosine, through the engagement of receptors expressed on neoplastic cells, regulates also the growth and dissemination of the tumor mass by direct actions on cancer cell proliferation, apoptosis and metastasis (Figure 1) [4]. In particular, it has been reported that adenosine can promote cell proliferation in some cases, via A1, A2A and A2B receptors, while restraining this process in other contexts, via A3 receptors. Moreover, adenosine has been shown to exert proapoptotic activity, primarily via A3 receptors, but also via A2A and A2B receptors. Of note, a particular tumor can coexpress different adenosine receptor types. For this reason, adenosinergic drug therapies should take into account these competing pro- and antiproliferative/apoptotic actions mediated by the different adenosine receptor subtypes [4]. A number of preclinical studies support the efficacy of the pharmacological blockade of A2A receptors in curbing cancer development and spread, through an enhancement of the antitumor actions of CD8+ T cells as well as via an inhibition of tumor neovascularization, growth and metastatic potential [4]. Likewise, the pharmacological blockade of A2B receptors resulted in a delay of tumor growth and reduction of metastatic dissemination, reversing the immune suppression on tumor, via an inhibition of Treg differentiation [21] and an increased density of tumor-infiltrating CD8+ T cells and NK cells [22]. The stimulation of A3 receptors, via selective agonists, such as IB-MECA (designated also as CF101) or Cl-IB-MECA (designated also as CF102), has been found to modulate cell growth-regulatory downstream signaling pathways as well as to downregulate the cell survival protein AKT, with subsequent inhibition of tumor growth [4]. Recently, data obtained from a Phase I/Phase II open-label study (clinical trial NCT00790218), enrolling patients with advanced hepatocellular carcinoma, demonstrated a favorable safety and efficacy profile of CF102, thus justifying its further clinical development [23]. 3.
Expert opinion
An improved understanding of the molecular mechanisms of adenosine pathways underlying the onset, progression and
Expert Opin. Ther. Targets (2014) 18(9)
975
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
L. Antonioli et al.
diffusion of neoplasia may help to translate their viability as therapeutic targets in cancer. The performance of preclinical investigations has improved our knowledge of the functional roles played by adenosine, via the engagement of its own receptors, in cancer biology, thus paving the way toward the discovery of novel antineoplastic therapies. However, despite in vitro studies and animal models support the concept that targeting the adenosine system has a huge potential for treating tumors, the translation of current data into clinical practice will require a more thorough understanding of how adenosine regulates cancer [4]. In this regard, one major hurdle concerns the fact that it is impossible to provide direct genetic in vivo evidence for the role of extracellular adenosine in cancer onset and development since it is impossible to genetically knock-out animals for this nucleoside. In addition, the intrinsic actions of adenosine on neoplastic cells depend on a variety of factors, which include the type of cancer, the adenosine receptor subtype expressed on cancer cells, and whether proliferation, apoptosis or metastasis is considered. Since there is evidence that a particular tumor can express different adenosine receptors, therapeutic strategies targeted on adenosine pathways should take into account the opposing actions mediated by different adenosine receptors. Accordingly, the performance of preclinical studies, using various methodological approaches, will help to better dissect the roles of adenosine in mediating neoplasia onset and progression as well as to highlight the potential for adenosinergic therapies in the management of patients with cancer. Future efforts should be focused, as suggested by Allard et al. [1], on the development of CD73 inhibitors. Likewise, A2A and A2B receptor antagonists hold a great potential for the treatment of cancer, with particular regard for one of the most aggressive breast cancer such as the TNBC. Targeting A2A or A2B receptors or inhibiting adenosine signaling, via CD73 blockade, can represent a promising adjunct to tumor immunotherapy since several immunotherapeutic approaches to curb neoplasia have failed due to CD73 overexpression in cancer cells or high adenosine levels within tumor microenvironment. In this regard, there is evidence that the genetic deletion of immunosuppressive A2A and A2B
976
adenosine receptors or their pharmacological inactivation can prevent the inhibition of antitumor T cells by the hypoxic tumor microenvironment, thus facilitating a process of full cancer rejection [24]. This approach is based on the in vivo genetic evidence demonstrating that A2A receptors play a pivotal role in the protection of normal tissues from overactive immune cells in acutely inflamed and hypoxic areas. Subsequent observations, showing an enhanced T-cell-mediated rejection of tumors in mice with inactivated A2A receptors, highlighted a protective role of these receptors on hypoxic cancer tissues, thus indicating their inactivation as a useful way to improve tumor rejection via antitumor T cells [5]. Along this way, several studies have shown that A2A receptor antagonists, already tested and shown to be safe in clinical trials on Parkinson disease, might also prevent immunosuppression mediated through the generation of adenosine by Tregs, further strengthening the rationale for implementing antiadenosine strategies by A2A antagonists to enhance the immune responses against neoplastic cells [24]. Of note, an attractive feature of these drugs is that they could be used in combination with other clinically available cytotoxic drugs, in order to magnify their antiblastic activity. Overall, a better characterization of how and to what extent adenosine participates in the molecular mechanisms underlying cancer development and diffusion will drive new therapeutic advances in creating more targeted drug interventions, which might lead to substantial improvements of the clinical management of various cancers.
Declaration of interest L Antonioli was supported by National Institutes of Health Grant R01GM66189 (G.H.), Nexus award ‘Marcello Tonini’ (L.A.) and by IBD Research Foundation (mini grant 2012) (L.A.). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Expert Opin. Ther. Targets (2014) 18(9)
Adenosine pathway and cancer
Bibliography
Expert Opin. Ther. Targets Downloaded from informahealthcare.com by The University of Manchester on 11/12/14 For personal use only.
1.
Allard B, Turcotte M, Stagg J. Targeting CD73 and downstream adenosine receptor signaling in triple-negative breast cancer. Expert Opin Ther Targets 2014;18(8):863-81
ecto-5’-nucleotidase and increase extracellular adenosine generation. Exp Cell Res 2004;296(2):99-108
20.
Hasko´ G, Szabo´ C, Ne´meth ZH, et al. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Immunol 1996;157(10):4634-40
21.
Nakatsukasa H, Tsukimoto M, Harada H, et al. Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells. Biochem Biophys Res Commun 2011;409(1):114-19
22.
Ghiringhelli F, Bruchard M, Chalmin F, et al. Production of adenosine by ectonucleotidases: a key factor in tumor immunoescape. J Biomed Biotechnol 2012;2012:473712
Iannone R, Miele L, Maiolino P, et al. Blockade of A2b adenosine receptor reduces tumor growth and immune suppression mediated by myeloid-derived suppressor cells in a mouse model of melanoma. Neoplasia 2013;15(12):1400-9
23.
Stemmer SM, Benjaminov O, Medalia G, et al. CF102 for the treatment of hepatocellular carcinoma: a phase I/II, open-label,dose-escalation study. Oncologist 2013;18(1):25-6
24.
Ohta A, Gorelik E, Prasad SJ, et al. A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 2006;103(35):13132-7
11.
Allard B, Turcotte M, Stagg J. CD73-generated adenosine: orchestrating the tumor-stroma interplay to promote cancer growth. J Biomed Biotechnol 2012;2012:485156
12.
Stagg J, Divisekera U, Duret H, et al. CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis. Cancer Res 2011;71(8):2892-900 Allard B, Turcotte M, Spring K, et al. Anti-CD73 therapy impairs tumor angiogenesis. Int J Cancer 2014;134(6):1466-73
2.
Antonioli L, Blandizzi C, Pacher P, et al. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer 2013;13(12):842-57
3.
Sitkovsky MV. T regulatory cells: hypoxia-adenosinergic suppression and re-direction of the immune response. Trends Immunol 2009;30(3):102-8
4.
Antonioli L, Pacher P, Vizi ES, et al. CD39 and CD73 in immunity and inflammation. Trends Mol Med 2013;19(6):355-67
13.
Lukashev D, Ohta A, Sitkovsky M. Hypoxia-dependent anti-inflammatory pathways in protection of cancerous tissues. Cancer Metastasis Rev 2007;26(2):273-9
14.
6.
Jin D, Fan J, Wang L, et al. CD73 on tumor cells impairs antitumor T-cell responses: a novel mechanism of tumor-induced immune suppression. Cancer Res 2010;70(6):2245-55
15.
Stagg J, Divisekera U, McLaughlin N, et al. Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis. Proc Natl Acad Sci USA 2010;107(4):1547-52
7.
Waickman AT, Alme A, Senaldi L, et al. Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor. Cancer Immunol Immunother 2012;61(6):917-26
16.
Rust S, Guillard S, Sachsenmeier K, et al. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type. Mol Cancer 2013;12:11
5.
8.
9.
10.
Beavis PA, Divisekera U, Paget C, et al. Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors. Proc Natl Acad Sci USA 2013;110(36):14711-16 Synnestvedt K, Furuta GT, Comerford KM, et al. Ecto-5’-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 2002;110(7):993-1002 Spychala J, Kitajewski J. Wnt and betacatenin signaling target the expression of
mechanisms. FASEB J 2000;14(13):2065-74
17.
Loi S, Pommey S, Haibe-Kains B, et al. CD73 promotes anthracycline resistance and poor prognosis in triple negative breast cancer. Proc Natl Acad Sci USA 2013;110(27):11091-6
18.
Zhang B. CD73: a novel target for cancer immunotherapy. Cancer Res 2010;70(16):6407-11
19.
Hasko´ G, Kuhel DG, Chen JF, et al. Adenosine inhibits IL-12 and TNF[alpha] production via adenosine A2a receptor-dependent and independent
Expert Opin. Ther. Targets (2014) 18(9)
Affiliation
Luca Antonioli†1,2 PhD, Gy€orgy Hasko´2, Matteo Fornai1, Rocchina Colucci1 & Corrado Blandizzi1 † Author for correspondence 1 University of Pisa, Department of Clinical and Experimental Medicine, Via Roma 55, 56126 -- Pisa, Italy Tel: +39 050 2218762; Fax: +39 050 2218758; E-mail: [email protected] 2 Rutgers-New Jersey Medical School, Department of Surgery and Center for Immunity and Inflammation, Newark, NJ 07103, USA
977
