P2Y Receptors for Extracellular Nucleotides: Contributions to Cancer Progression and Therapeutic Implications

Abstract

Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A₁, A_2A, A_2B and A₃ for extracellular adenosine, P2X1–7 receptors that are ATP-gated ion channels and G protein-coupled P2Y_{1,2,4,6,11,12,13 and 14} receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment.

1. Introduction

In its infancy, the field of purinergic signaling was met with great skepticism. The idea that an intracellular molecule of such immense importance to cellular energy generation and the activation of so many enzymatic reactions could serve as an extracellular signaling molecule was antithetical to the prevailing scientific concepts of the 1970’s. However, the pioneering work of the late Dr. Geoffrey Burnstock, to whom this special issue of Biochemical Pharmacology is dedicated, investigated the responses to direct stimulation of guinea pig smooth muscle nerves during blockade of two classical neurotransmitters, acetylcholine and noradrenaline. This work led to the generation of the ‘purinergic’ hypothesis that extracellular ATP was the neurotransmitter responsible for non-adrenergic, non-cholinergic responses observed in gastrointestinal smooth muscle nerve fibers [1, 2]. In the ensuing decades, the molecular cloning and functional characterization of P1 adenosine receptors, P2 nucleotide receptors and ectonucleoside triphosphate diphosphodihydrolases (ENTPDases) laid to rest any doubts on the importance of extracellular ATP (eATP) as a signaling molecule [3]. In addition to neurotransmission, purinergic receptors contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis.

While P2 receptors are found in all species of vertebrates and some invertebrates, they are not found in Caenorhabditis elegans or Drosophila melanogaster. P2X receptor homologs are found in several single-cell organisms and it seems that P2 receptors appeared evolutionarily on the boundary of single cellular and multicellular organisms, highlighting their important role in cell-cell communication [4]. Intriguingly, P2 receptors have been identified in fungi and plant lineages, as evidenced by P2X receptor homologs in Dictyostelium amoeba and Medicago truncatula (barrel clover), yet they are strikingly absent from Saccharomyces cerevisiae [5, 6]. Also absent is molecular evidence for the existence of prokaryotic P2 receptors, although the effects of extracellular nucleotides on bacterial growth have been described [7], suggesting that as yet undiscovered prokaryotic purinergic signaling mechanisms do exist. More recently, forward genetic screening of Arabadopsis thaliana seedlings identified ATP-insensitive dorn1 (does not respond to nucleotides) mutants lacking a cytoplasmic calcium response to extracellular nucleotides [8]. Further whole-genome sequencing of dorn1 mutants identified kinase-inactivating mutations in a transmembrane lectin receptor (LecRK-I.9; DORN1) that binds extracellular ATP with high affinity [8] and was suggested to be the founding member of the plant-specific P2K (K for kinase) receptor family involved in innate immunity from plant pathogens [9].

In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A₁, A_2A, A_2B and A₃ for extracellular adenosine, P2X1–7 receptors that are ATP-gated ion channels and G protein-coupled P2Y_{1,2,4,6,11,12,13 and 14} receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose [10]. Additionally, cell-surface ectonucleotidases (i.e., CD39/ENTPD1 and CD73) and ecto-ATP synthases play important roles in modulating purinergic receptor signaling by catalyzing the breakdown of extracellular nucleotide triphosphates (NTPs) into nucleotide diphosphate and nucleoside derivatives or the regeneration of NTPs that serve as ligands for P2Y and P1 receptors [11, 12]. The P2Y receptors (P2YRs), whose role in cancer progression and metastasis will be the focus of this review paper, can be further subdivided according to their associated G protein subtypes and their second messenger systems. The P2Y_1,2,4,6 receptors couple to heterotrimeric G_q proteins to activate the Gα_q subunit-dependent PLCβ/IP₃/DAG cascade to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activation of protein kinase C, whereas P2Y_12,13,14 receptors couple to heterotrimeric G_i/o protein to inhibit Gα_i/o-dependent adenylyl cyclase activity, which decreases intracellular cAMP levels (Table 1) [13]. The P2Y₁₁R uniquely couples to both G_q and G_s proteins to increase both Gα_q-dependent [Ca²⁺]_i and Gα_s-dependent cAMP levels, respectively [14]. Additionally, an extracellularly-oriented Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y₂R allows for its interaction with RGD-binding α_vβ_3/5 integrins, which enables ATP to activate integrin-dependent Gα_o/Rac GTPase and Gα₁₂/Rho GTPase signaling [15, 16]. The P2Y₆R has been shown to couple directly to heterotrimeric G₁₃ proteins to activate Gα₁₃-dependent Rho signaling [17]. Through these signaling cascades and downstream activation of protein kinases, including protein kinase A (PKA), protein kinase C (PKC), Src family kinases and mitogen-activated protein kinases (MAPKs), P2Y receptors and their selective extracellular nucleotide ligands modulate numerous cellular responses relevant to cancer tumorigenesis, metastasis and chemotherapeutic drug resistance.

Table 1.

P2Y receptor agonists and coupling to G protein subunits - second messenger systems.

P2Y Receptor Subtype	Preferred Agonist	Gα Protein Subunit	Second Messenger System/Effector
P2Y1R	ADP	Gαq	PLCβ activation, IP3/DAG generation, increased [Ca2+]i/PKC
P2Y2R	ATP, UTP	Gαq	PLCβ activation, IP3/DAG generation, increased [Ca2+]i/PKC
		Gαo	Rac activation
		Gα12	Rho activation
P2Y4R	UTP	Gαq	PLCβ activation, IP3/DAG generation, increased [Ca2+]i/PKC
P2Y6R	UDP	Gαq	PLCβ activation, IP3/DAG generation, increased [Ca2+]i/PKC
		Gα12/13	Rho activation
P2Y11R	ATP	Gαq	PLCβ activation, IP3/DAG generation, increased [Ca2+]i/PKC
		Gαs	Adenylyl cyclase activation, increased [cAMP]i
P2Y12R	ADP	Gαi/o	Adenylyl cyclase inhibition, decreased [cAMP]i
P2Y13R	ADP	Gαi/o	Adenylyl cyclase inhibition, decreased [cAMP]i
P2Y14R	UDP-glucose	Gαi/o	Adenylyl cyclase inhibition, decreased [cAMP]i

Abbreviations: PLCβ - phospholipase C β, DAG - diacylglycerol, IP₃ - 1,4,5 inositol trisphosphate, PKC - protein kinase C, cAMP - cyclic adenosine monophosphate.

Through the activity of cell-surface ectonucleotidases, the concentration of extracellular nucleotides is tightly regulated within the physiological nM to low μM range [18, 19]. In fact, in vitro studies with ovarian and endometrial cancer cells demonstrate that eATP is quickly broken down by ectonucleotidases so that only 20% of eATP remains in the cell culture media after 3 hours [20, 21]. However, during periods of cellular stress, such as inflammation, hypoxia in the tumor microenvironment or in response to γ-radiation therapy for cancer, extracellular ATP can accumulate to levels of 100 μM or more [18, 22], where subsequent activation P2X and P2Y receptors in cancer cells has been shown to directly stimulate cancer cell proliferation, apoptosis, migration and extracellular matrix remodeling. Additionally, activation of P2 receptors in host endothelial cells, immune cells and platelets can modulate cancer progression indirectly by promoting tumor cell extravasation and metastasis and enhancing the host anti-tumor response [23–25]. Here, we review the role of extracellular nucleotides in cancer cell proliferation, examine the cell-specific contributions of P2Y receptor subtypes to oncogenic processes and discuss the therapeutic implications of P2Y receptor-targeting agonists and antagonists to the regulation of cancer progression.

2. Does extracellular ATP enhance or inhibit cancer cell proliferation?

Early in vitro experiments demonstrated that application of eADP or eATP arrested tumor cell growth [26], although P1 and P2 receptors had not yet been cloned and functionally characterized. Further studies showed that the inhibitory effects of eATP, eADP or extracellular adenosine monophosphate (AMP) on cell growth were dependent on nucleotide hydrolysis and the slow generation of extracellular adenosine, whose uptake through cell-surface nucleoside transporters increased intracellular adenine nucleotide levels and led to pyrimidine starvation [27]. In vivo studies of exogenous nucleotides for cancer treatment found that daily intraperitoneal administration of ATP inhibited the growth of adenocarcinoma xenografts, prevented weight loss and increased red blood cell and blood plasma levels of ATP in tumor-bearing mice [28, 29]. Subsequent clinical trials of intravenous ATP administration in human lung cancer patients failed to demonstrate efficacy in slowing tumor progression or increasing survival [30]; however, beneficial effects of intravenous ATP on weight loss, quality of life and muscle strength in non-small cell lung cancer patients have been reported [31], leading to the hypothesis that the effects of intravenous ATP may result from increased nutritional (intracellular) ATP levels rather than the action of extracellular ATP on purinergic receptors [32].

Prior to the widespread use of specific gene-targeting techniques, such as siRNA and CRISPR-Cas9, numerous pharmacodynamic studies investigated whether extracellular nucleotides promote or inhibit cell proliferation in cancer cells. Extracellular ATP stimulates cell proliferation in the OVCAR-3 ovarian cancer cell line that is sensitive to pertussis toxin, suggesting that eATP promotes cell growth via P2Y receptor-mediated G protein signaling cascades [33], yet the slowly hydrolyzable ATP analogue ATP-γ-S inhibited proliferation of ovarian and endometrial cancer cells at higher concentrations (100 μM) [20, 21]. In Kyse-140 esophageal carcinoma cells, high eATP concentrations (100–500 μM) had anti-proliferative effects and induced caspase-3 dependent apoptosis. Additionally, eATP was equipotent to eUTP at increasing [Ca²⁺]_i, a response that was blocked by the PLC inhibitor U73122, suggesting that activation of the G_q-coupled P2Y₂R was responsible [34]. However, these cells also express numerous P2X receptors that may have been preferentially activated by the high concentration of eATP being investigated. The differential effects of low vs. high [eATP] on cell growth have been further demonstrated in liver [35], prostate [36–38], skin [39] and breast [40, 41] cancer cell lines where eATP at low concentrations (10 μM) induced cell proliferation, likely through P2Y₂ receptor activation, and eATP at higher concentrations (> 100 μM) inhibited cell proliferation, likely through activation of P2X7 receptor subtypes that have previously been shown to induce apoptotic responses [42, 43]. Similarly, high eATP concentrations (e.g., 1 mM) induced anti-proliferative and apoptotic responses in normal BEAS-2B lung epithelial cells, but not in A549 lung cancer cells, where lower P2X7R expression was detected [44]. The anti-proliferative effects of high eATP have also been demonstrated in human leukemia cells [45, 46]. In contrast, eATP stimulated the proliferation of lung cancer cells that was attenuated by inhibition of PLC, store-operated Ca²⁺ channels or the epidermal growth factor receptor (EGFR) [47], which has been demonstrated to be transactivated by P2Y₂Rs [48–50].

Following the widespread use of gene targeting techniques and the development of selective pharmacological antagonists, it has now become clear that indivdual P2Y receptor subtypes have differential effects on cell growth, where P2Y₁ receptors generally inhibit cancer cell proliferation [51], whereas P2Y₂ receptors promote proliferation [48]. While it is enticing to link the anti-proliferative responses to high [eATP] based solely on the prolonged activation of P2X7Rs, which induces inflammatory and apoptotic responses in numerous cell types [42, 43, 52, 53], studies have demonstrated that P2X7Rs also mediate trophic responses in cancer cells [54, 55]. Taken together, these studies highlight the importance of extracellular nucleotide ligand concentration and the cell-specific expression of P2X and P2Y receptor subtypes in determining the overall cellular response to P2 receptor activation.

3. Purinergic receptors and ectonucleotidases comprise the extracellular “purinome”

Current research on intercellular purinergic signaling has further highlighted the concept of receptomes, the hierarchical networks of complex interactions between transmitters, hormones and growth factors and their cognate receptors and unique cellular responses [56]. Receptomes conceptualize how heterogenous cell responses result from common signaling components by taking into consideration receptor cooperativity, magnitude and directionality of signal propagation and amplification and regulatory mechanisms [57]. The extracellular purinome is the cell- and tissue-specific receptome where extracellular nucleotides and their degradation products can elicit divergent cellular responses in both autocrine and paracrine fashions (Figure 1) [58]. During instances where high extracellular nucleotide levels are present, such as tissue inflammation or in the tumor microenvironment [59, 60], the purinergic response to extracellular nucleotides represents the integrated signals from each activated P2 and P1 receptor. In a carcinoma, the purinome of the tumor cell differs from that of the infiltrating immune cells and vascular endothelial cells, such that the overall tumoral response to eATP represents the sum total of each cells’ purinomic response. This response is further dependent on the concentration of the agonist, the affinity of each receptor for the agonist and functional interactions between homomeric and heteromeric P2 receptors [10, 61]. In addition, P2 receptor signaling is controlled/influenced by two families of ectoenzymes that degrade nucleotide phosphates leading to the generation of new ligands that are capable of activating other nucleotide receptor families, such as P1 (adenosine) and P0 (adenine) receptors [11, 62]. Thus, purinergic signaling should not only be considered as an “outside-in” signaling cascade, but also one capable of lateral signaling. In future experiments, it will be necessary for researchers to broadly consider the whole purinome as a single entity, whose responses can be modulated through pharmacological or genetic manipulation. Indeed, an active area of research includes the real-time measurement of extracellular nucleotide levels in vivo [59, 63], so that researchers can identify pathological conditions where purinergic signaling is dysregulated and conceive of therapeutic interventions that can be targeted to the most relevant component(s) of the purinome.

Figure 1. Extracellular purinome signal integration.

Cell-specific responses to extracellular nucelotides result from the combined activation of P2X ATP-gated ion channels, P2Y G protein-coupled nucleotide receptors and P1 G protein-coupled adenosine receptors. Through the action of ectonucleotide triphosphate diphosphohydrolases (ENTPDs) and the ecto-5’-nucleotidase CD73, extracellular ATP is broken down to generate nucleotide di- and monophosphates and adenosine to shift the purinergic response from ATP-activated P2X and P2Y receptors to UDP and ADP-activated P2Y receptors and eventually to P1 adenosine receptors. Adenosine is then recycled through cell-surface nucleoside transporters that terminate the extracellular signal. Cell- and tissue-specific expression of P2R, P1R, ENTPD, CD73 and nucleoside transporters make up the local extracellular purinome and intracellular integration of ion flux and G protein-mediated signaling cascades determines the overall cellular response. Figure created with BioRender.com.

Although beyond the scope of this review, it should be noted that while P2Rs generally mediate pro-inflammatory responses, whereas P1Rs generally mediate anti-inflammatory responses, the ratio of P2R:P1R signaling likely influences the observed responses in multicellular complex systems [64, 65]. This balance between pro- and anti-inflammatory purinergic responses may be important in the tumor microenvironment, where the host anti-tumor inflammatory response can be harnessed for cancer treatment. Chemotherapeutic treatments such as the PD-1 antagonists pembrolizumab and nivolumab boost the host T cell anti-tumor response by blocking immune tolerance mechanisms induced by PD-L1-expressing tumor cells [66]. Therefore, modulating eATP-mediated immune responses may have beneficial or deleterious effects on cancer treatments. Indeed, previous studies have shown that P2X7Rs expressed in host immune cells modulate the anti-tumor response [25, 67] and endothelial P2Y₂Rs are key modulators of lymphocytic infiltration into inflamed tissues [68–71]. Thus, any P2 receptor-targeting chemotherapeutic strategy must balance purinergic responses of the tumor with those of the host cell purinomes. Further exploration of this concept will push the boundaries of targeted therapy, perhaps through increased exploration of pharmacological delivery strategies (i.e., intratumoral injection vs. systemic application) or the use of cell-specific gene-targeting strategies.

4. P2Y Receptor Subtypes in Cancer

Recent work on the role of P2Y receptors in cancer pathogenesis over the last 2 decades has taken advantage of the continued development of P2Y subtype-selective agonists and antagonists, as well as specific gene targeting techniques such as siRNA and CRISPR-Cas9, to significantly advance our understanding of the individual contributions of P2Y receptor subtypes to cancer progression. Furthermore, the use of more sophisticated in vitro and in vivo animal models continues to expand our translational understanding of how P2Y receptors can be targeted therapeutically. Below, we review the current understanding of the contribution of these receptors to key oncogenic processes and discuss the prospects for targeting them to treat cancer.

4.1. P2Y₁R

The 373 amino acid P2Y₁R, whose activation by ADP (EC₅₀ = 10 nM) is coupled to the Gα_q signaling cascade, was among the first P2Y receptors to be cloned [72]. Activation of P2Y₁R in cancer cells is generally associated with anti-proliferative responses and P2Y₁Rs expressed in platelets may contribute to cancer cell metastasis, as is described below in greater detail with P2Y₁₂Rs. Immunohistochemical analysis of human melanoma tissue has shown abundant, uniform expression P2Y₁ and P2Y₆ receptors, whereas P2Y₂ receptors were expressed primarily at the proliferative margins of the tumor. Furthermore, treatment of the human melanoma cell line A-375 with the P2Y₁R-selective agonist 2-MeS-ADP inhibited proliferation, whereas the P2Y₂R-selective agonist UTP enhanced proliferation in a dose-dependent manner, suggesting opposing roles for these two P2YR subtypes in melanoma growth [73].

Although the human P2Y₁R is basally expressed in many different tissues, its expression was found to be the strongest in the human prostate [74]. Additionally, PC-3 human prostate cancer cells expressed the P2Y₁R at greater levels than other ADP-activated P2Y receptor subtypes, where its activation with the P2Y₁R-selective ADP analog MRS2365 significantly inhibited cell proliferation and markedly increased caspase-3-dependent apoptosis that was attenuated by siRNA-mediated P2Y₁R knockdown [75]. Due to its established anti-proliferative role, a recent study utilized a P2Y₁R structural model to virtually screen a library of 1-indolinoalkyl 2-phenolic compounds and identify potential P2Y₁R agonists as novel therapeutics for prostate cancer [51]. In PC-3 and DU-145 human prostate cancer cells, two P2Y₁R agonists with high binding affinities and docking scores were found to dose-dependently increase [Ca²⁺]_i, inhibit cell proliferation, enhance apoptosis and generate reactive oxygen species (ROS) to a greater extent than the well-characterized P2Y₁R agonist MRS2365 [51], suggesting that these compounds should be further explored for their potential therapeutic use in the treatment of prostate cancer.

Analysis of lung squamous cell carcinoma datasets from The Cancer Genome Atlas (TCGA) found that the P2Y₁R and P2Y₁₂R had the highest frequency of gene copy number gain in lung cancer patients [76] and P2Y₁R expression is significantly increased in bronchoalveolar fluid cells isolated from metastatic non-small cell lung cancer (NSCLC) patients, as compared to non-metastatic patients [77]. However, the pathophysiological significance of these observations was not further explored. Additionally, in ZL-55 human malignant mesothelioma cells, ADP also inhibited cell proliferation, enhanced cisplatin-induced cytotoxicity and stimulated extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent matrix metalloprotease (MMP)-2/9 activity [78], although definitive links to P2Y₁R were not further explored.

Although a handful of studies have highlighted the role of P2Y₁R in inhibiting cancer cell proliferation, its role in cancer metastasis and chemoresistance remains largely unknown. A recent study suggested that P2Y₁R could play a major role in blocking tumor progression [79]. Specifically, P2Y₁R was identified as a target of a microRNA (miR-23b), which is highly upregulated in multiple metastatic tumor cell lines, including human pancreatic neuroendocrine tumors [79]. The role of P2Y₁R was verified by knockdown of P2Y₁R in the pancreatic β-tumor cell line, βTC3, resulting in significant inhibition of metastasis to the liver [79]. Regulation of P2Y₁R expression by microRNAs has also been linked to multidrug chemoresistance in 5637 and EJ bladder cancer cells, where miR-34b-3p, a microRNA belonging to the well-established miR34 family, attenuated multidrug chemoresistance [80]. Higher expression of miR-34b-3p correlated with decreased P2Y₁R gene expression in EJ bladder cancer cells and heterologous overexpression of the P2Y₁R conferred increased chemoresistance to 5 chemotherapeutic drugs [80]. More importantly, intratumoral injection of miR-34b-3p repressed paclitaxel chemoresistance and in vivo tumor growth of 5637 bladder cancer cells [80]. Taken together, these studies suggest that P2Y₁R expression can be targeted by miRNAs to inhibit oncogenic processes.

P2Y₁Rs expressed in endothelial cells have also been suggested to enhance vascularization of breast cancer tumors through a unique mechanism involving the release of nucleoside diphosphate kinase (NDPK) from breast cancer cells to modulate eATP concentrations [81, 82]. NDPK is constitutively secreted from numerous human breast cancer cell lines and NDPK activation enhances ADP-induced vascular endothelial growth factor receptor 2 (VEGFR-2) and ERK1/2 phosphorylation in human endothelial cell cultures [81]. Furthermore, NDPK- or 2-MeS-ATP-induced endothelial cell migration can be blocked by the P2Y₁R antagonist MRS2179. MDA-MB-231 human breast cancer xenografts also secrete NDPK in the in vivo tumor microenvironment, where blockade of NDPK or P2Y₁R with ellagic acid or MRS2179, respectively, significantly inhibited tumorigenesis and metastasis [82], suggesting the presence of a unique signaling mechanism to promote breast tumor growth and angiogenesis. However, because NDPK activity acts opposite to ectonucleotidases to generate ATP from ADP and the preferred ligand for P2Y₁R is ADP, it is likely that extracellular NDPK promotes the co-activation of other ATP-preferring receptors, such as the P2Y₂R, rather than enhancing P2Y₁R activation.

Lastly, P2Y₁Rs expressed in nervous tissue of the spinal cord and dorsal root ganglia have been shown to mediate ERK1/2 phosphorylation and nociceptive transmission in a rat model of breast cancer-induced bone pain, where intrathecal injection of the P2Y₁R antagonist MRS2179 into the spinal canal reduced tactile allodynia and spontaneous pain [83]. These findings are similar to those with the ADP-preferring P2Y₁₂R that has also been shown to modulate nociception in cancer-induced pain models [84].

4.2. P2Y₂R

The most studied P2Y receptor in the cancer field is the 377 amino acid, G_q-coupled P2Y₂ receptor that is activated equipotently by extracellular ATP and UTP (EC₅₀ ~ 0.5–3 μM) [85]. One of the first P2Y receptors to be cloned and characterized, the P2Y₂R is unique in its ability to stimulate numerous signaling pathways through distinct structural motifs. In addition to activation of the canonical Gα_q/PLC/IP₃/Ca²⁺ signaling cascade, the P2Y₂R can activate Gα_o/Rho and Gα₁₂/Rac through an Arg-Gly-Asp (RGD) motif in its 1^st extracellular loop that interacts directly with α_v/β_3/5 and α₅/β₁ RGD-binding integrins [15, 16, 86] to enable extracellular nucleotides to activate integrin complex-associated G_o and G₁₂ binding proteins. P2Y₂Rs also stimulate MAPK signaling (i.e., ERK1/2 phosphorylation) [48, 87, 88] and intracellular Src homology 3 (SH3) binding domains in the C-terminal tail bind Src to promote P2Y₂R-mediated Src kinase autophosphorylation, which enables nucleotide-induced transactivation of growth factor receptors [50, 89, 90]. P2Y₂R-mediated activation of the matrix metalloproteases ADAM10 and ADAM17 contributes to nucleotide-induced release of membrane-bound growth factors and subsequent EGFR signaling [48, 49, 91]. Given its association with such diverse signaling pathways, it is not surprising to find that P2Y₂Rs contribute to tumorigenesis, migration, metastasis and drug resistance in numerous types of cancers.

Analysis of breast cancer datasets from TCGA indicated that P2Y₂R expression is increased in breast tumor tissue compared to normal mammary tissue, where its increased expression was positively correlated with patient tumor, node, metastasis (TNM) stage [92], indicating that P2Y₂Rs may contribute to breast cancer cell metastasis. Independent immunohistochemical analyses also demonstrated increased P2Y₂R expression in breast tumors compared to adjacent healthy tissue, where colocalization with cancer stem cell markers [93] and general localization at the invasive edge of the tumor and in tumor cells infiltrating surrounding adipose mammary tissue were observed [94]. P2Y₂R expression was the most abundant among all P2Y receptor subtypes in MCF7 and Hs578T human breast cancer cell lines and P2Y₂R-targeting siRNA blocked eATP-induced cell migration, extracellular matrix invasion and upregulation of epithelial-mesenchymal transition (EMT)-associated genes [94, 95], cellular mechanisms that contribute to cancer cell metastasis [96]. Similarly, P2Y₂R-targeting shRNA attenuated eATP-induced migration and invasion of MDA-MB-231 human breast cancer cells, likely through disruption of P2Y₂R-mediated β-catenin signaling [92]. Furthermore, P2Y₂R knockdown reduced tumorigenesis of MDA-MB-231 xenografts and abolished spontaneous metastasis to the liver and lungs [92, 97]. P2Y₂Rs may also contribute to cancer cell metastasis by enhancing the enzymatic activity of lysyl oxidase (LOX), which catalyzes the crosslinking of the extracellular matrix proteins collagen and elastin during premetastatic niche formation [98]. Hypoxic conditions found in the tumor microenvironment enhance the release of ATP from MDA-MB-231 cells, where subsequent P2Y₂R activation induced LOX secretion and collagen cross-linking that was attenuated by shRNA-mediated P2Y₂R knockdown [99].

In addition to enhancing tumor cell migration and invasion, P2Y₂Rs in host endothelial cells also contribute to metastasis. P2Y₂R activation in endothelial cells has been shown to increase VEGFR-2-dependent expression of cell adhesion molecules and decrease tight junction stability through the upregulation of VCAM-1 and the phosphorylation of VE-cadherin, respectively, which promotes the attachment and transendothelial migration of cells from the blood into target tissues [68, 70, 90]. ATP released from MDA-MB-231 breast cancer cells enhanced their adhesion to human umbilical vein endothelial cells (HUVECs) through increased expression of ICAM-1 and VCAM-1 and induced phosphorylation of VE-cadherin, responses that were blocked by siRNA-mediated knockdown of endothelial P2Y₂Rs [97]. Additionally, tumor cells activate platelets to induce nucleotide release from platelet dense granules, thereby enhancing endothelial P2Y₂R-mediated transendothelial migration of tumor cells, and reduced lung metastases were observed in P2Y₂R^−/− mice following tail-vein injection of B16 mouse melanoma or Lewis Lung Carcinoma (LLC) mouse lung cancer cells [23].

While most often associated with activation of the P2X7R, recent reports indicate a role for P2Y₂Rs in promoting inflammasome activation in breast cancer cells, where ATP-induced caspase-1 activity and IL-1β release from wild type and radiotherapy-resistant MDA-MB-231 cells was reduced by P2Y₂R knockdown [100]. Further studies demonstrated that both TNFα and eATP increased IL-1β release and expression of the inflammsome components NLRC4, ASC and caspase-1 in a P2Y₂R-dependent manner [101], but notably unaffected was expression of NLRP3, whose activity is regulated by P2X7Rs [52]. Although the mechanism of P2Y₂R-mediated inflammasome activation requires further study, K⁺ efflux is necessary for P2X7R-mediated NLRP3 inflammasome activation [53] and P2Y₂R-mediated changes in [Ca²⁺]_i modulate the calcium-dependent K⁺ channel K_Ca3.1 in SK-OV-3 ovarian cancer cells [102], suggesting a possible ionic mechanism. P2Y₂Rs have also been suggested to inhibit estrogen-induced proliferation of MCF-7 human breast cancer cells [103], although opposing results were demonstrated in 5637 and T24 human bladder cancer cell lines, where an estrogen-responsive P2Y₂R enhancer RNA increased P2Y₂R expression and promoted estrogen-induced cell proliferation and migration that was attenuated by CRISPR-mediated P2Y₂R knockout [104].

In other cancer types, P2Y₂Rs have been more definitevly linked to cell proliferation. P2Y₂R expression has been shown to be significantly increased in pancreatic cancer tissue [105], where analysis of TCGA datasets and independent immunohistochemical analysis of pancreatic ductal adenocarcinoma (PDAC) patients indicates that increased P2Y₂R expression is associated with decreased overall survival [106]. In the PANC-1 and CFPAC-1 human PDAC cell lines, P2Y₂R activation enhanced Src kinase- and PI3K/Akt-dependent cell proliferation [107] that was blocked by the selective P2Y₂R antagonist AR-C118925 [108]. Similar results were reported in the AsPC-1 and BxPC-3 human PDAC cell lines, where P2Y₂R activation enhanced PI3K/Akt-dependent cell proliferation and glycolysis through platelet-derived growth factor receptor-β (PDGFR-β) transactivation mediated by the Src family kinase Yes1 [106]. Furthermore, intraperitoneal AR-C118925 administration significantly impaired tumor xenograft growth and extended overall survival in a syngeneic, orthotopic mouse model of PDAC [106]. When combined with the classical chemotherapeutic drug gemcitabine, AR-C118925 treatment significantly improved the antitumor effects of this first-line therapy [106]. Likewise, eUTP induced EGFR transactivation and proliferation in CAL27 and FaDu human head and neck squamous cell carcinoma (HNSCC) cell lines that was attenuated by CRISPR-mediated P2Y₂R knockout [48]. Furthermore, growth of P2Y₂R^−/− CAL27 and FaDu tumor xenografts in mice was significantly reduced, compared to wild type xenografts, and AR-C118925 treatment reduced tumorigenesis in the MOC2 syngeneic mouse model of HNSCC [48]. P2Y₂R-mediated EGFR transactivation also enhances cell migration and extracellular matrix invasion of PC-3 and DU-145 human prostate cancer cell lines [87] and siRNA-mediated P2Y₂R knockdown reduces prostate tumor xenograft growth and liver metastases via a mechanism involving interleukin-8 secretion and expression of the EMT-associated genes E-cadherin and snail [109]. Lastly, a study of lung cancer resistance to the anaplastic lymphoma kinase (ALK) inhibitor crizotinib identified P2Y₂R, EGFR (ErbB1) and NRG1 (neuregulin growth factor, an ErbB3/4 ligand) as top resistance gene candidates [110], suggesting that P2Y₂R-mediated transactivation of growth factor receptors may contribute to chemotherapeutic drug resistance. This mechanism may also be relevant in HNSCC resistance to the anti-EGFR monoclonal antibody cetuximab [48, 49]. Taken together, these studies emphasize the crosstalk that exists between P2Y₂ and growth factor receptors and highlight the role of P2Y₂Rs in extracellular nucleotide-induced cancer cell proliferation and drug resistance.

In hepatocellular cancer (HCC), increased P2Y₂R expression was demonstrated in primary cells isolated from HCC tumors, as compared to healthy control hepatocytes [111], and in HCC tumors compared to adjacent tumor-free liver tissue [112]. In HepG2 human HCC cells, eATP-induced cell proliferation and migration was blocked by shRNA-mediated P2Y₂R knockdown or inhibition of store-operated Ca²⁺ channels. Furthermore, intraperitoneal administration of ATP (10 mg/kg/day) enhanced the growth of wild type HepG2 xenografts, but not P2Y₂R- or stromal interaction molecule 1 (STIM1)-knockdown xenografts, suggesting that P2Y₂R-mediated changes in [Ca²⁺]_i contribute to HCC progression [111]. While one of the cell lines (BEL-7404) used in this study was later shown to be a HeLa derivative [113], similar responses were demonstrated between BEL-7404 and HepG2 cells. In a chemically-induced mouse model of liver cancer, P2Y₂R^−/− mice showed significantly reduced tumorigenesis following intraperitoneal diethylnitrosamine (DEN) administration, as compared to wild type mice, likely due to diminished DEN-induced hepatocyte proliferation and ATP-induced DNA damage in the P2Y₂R^−/− mice [114].

Using whole genome DNA microarrays, P2Y₂R expression was shown to be increased in gastric tumors, as compared to adjacent healthy gastric mucosa [115], and in MKN-74 human gastric adenocarcinoma cells, as compared to non-cancerous GES-1 gastric mucosal cells [116]. Additionally, eATP and eUTP stimulated proliferation of MKN-74 and AGS gastric cancer cells that was attenuated by P2Y₂R antagonism with AR-C118925 [116]. Similar findings were noted in non-melanoma skin cancers, where highly expressed P2Y₂Rs colocalized with proliferating cell nuclear antigen (PCNA) in human skin squamous cell carcinomas and eATP and eUTP equipotently induced proliferation of A431 human epidermoid carcinoma cells [39]. P2Y₂R expression and function were also demonstrated in primary human colorectal cancer (CRC) cells and in the human CRC cell line, HT-29, where eATP and eUTP equipotently increased [Ca²⁺]_i [117]. Conflicting studies report increased P2Y₂R expression in CRC tumors, as compared to adjacent tumor-free tissue [118], and decreased P2Y₂R expression in CRC tumors, as compared to benign colon tissue isolated from patients with diverticulitis [119]. Perhaps these divergent findings reflect the different control tissues used for comparisons. Lastly, while P2Y receptors contribute to many oncogenic processes, they are generally not considered to be bona fide oncogenes. However, one study generated a retroviral cDNA expression library from the human CRC cell line, RKO, that when used for subsequent transformation of mouse 3T3 fibroblasts followed by focus formation and soft-agar growth assays identified P2Y₂R as a transforming oncogene [120], suggesting that P2Y₂ receptors have transforming potential under certain conditions.

4.3. P2Y₄R

The human P2Y₄R is a 365 amino acid G_q protein-coupled receptor whose activation by its preferred ligand UTP (EC₅₀ = 73 nM) releases Gα_q leading and subsequent PLCβ activation [121]. In contrast to the extensive studies on the role of UTP-activated P2Y₂Rs in cancer, the P2Y₄R has garnered far less attention. Due to the convergent signaling elicited by UTP through both P2Y₄R and P2Y₂R and overlap between effector G proteins, the studies on the role of P2Y₄Rs in cancer have yet to adequately delineate P2Y₄R- vs. P2Y₂R-mediated responses. Similar to the P2Y₂R, P2Y₄R expression was increased in CRC tumors, as compared to adjacent tumor-free tissue [118]. P2Y₄R expression also has been demonstrated in human HT-29 and Caco-2 CRC cell lines, where along with the P2Y₂R, P2Y₄R likely contributes to UTP-induced Src kinase and MAPK activation [122, 123]. Studies have also demonstrated that P2Y₂R and P2Y₄R mediate activation of Src kinase, MAPKs and downstream transcription factors in MCF-7 breast cancer cells [124]. In the SH-SY5Y human neuroblastoma cell line, P2Y₄Rs were found to mediate UTP-induced neurite outgrowth [125], although P2Y₂R activation has also been shown to stimulate neurite outgrowth in mouse primary cortical neurons [126]. ATP promotes cell survival of the NSCLC cell lines A549 and H23, where P2Y₄R expression was increased compared to the control cell line BEAS-2B, although P2Y₄R-specific responses were not established [44]. In the ovarian cancer cell line SK-OV-3, UTP-induced release of lysophosphatidic acid was attributed to P2Y₄R-mediated phospholipase D activation [127]. In human hepatocellular carcinoma, bioinformatics analysis of TCGA datasets found that P2Y₄R expression was increased in HCC tumor samples, although further cancer prognostic value was not evaluated [128], and P2Y₄R was found to be a hub-gene for the cytotoxic effects of emodin in the HCC cell line, HepG2 [129].

4.4. P2Y₆R

The human P2Y₆R is a 328 amino acid G protein-coupled receptor that is perhaps best known for its role in immunomodulation and neuroinflammatory processes, including microglial cell activation and phagocytosis [130]. P2Y₆R activation by its preferred agonist UDP (EC₅₀ = 15 nM) promotes Gα_q-mediated activation of the PLC/IP₃/Ca²⁺ second messenger system [131], although studies have shown that the P2Y₆R also couples to Gα_12/13 proteins to activate the small GTPase Rho and induce migration of A549 lung and Caco-2 colorectal cancer (CRC) cells [17]. In CRC cells, p53 has been suggested to modulate P2Y₆R expression at the transcriptional level, where wild type p53 and the inflammatory bowel syndrome-associated p53^R273H mutant differentially interact with the P2Y₆R promotor [132]. P2Y₆R activation has been shown to protect human HT-29 CRC cells from inflammatory cytokine-induced apoptosis, through the stabilization of X-linked inhibitor of apoptosis (XIAP), and the selective P2Y₆R agonist MRS2693 reduced the cytotoxic effects of the chemotherapeutic drug 5-fluorouracil [133], suggesting that P2Y₆R activation promotes CRC growth and chemoresistance. Indeed, P2Y₆R^−/− mice develop fewer, smaller and less-vascularized chemically-induced CRC tumors following azoxymethane and dextran sulfate sodium treatment, where nuclear β-catenin localization was observed in dysplastic colonic regions of WT, but not P2Y₆R^−/−, mice [133]. P2Y₆R activation has also been suggested to modulate β-catenin signaling in human gastric cancers, where decreased P2Y₆R expression has been demonstrated in tumor tissue, as compared to adjacent normal tissue, and low P2Y₆R expression was associated with poor prognosis in gastric cancer patients [134]. In contrast to findings in CRC cells, eUTP or eUDP suppressed in vitro proliferation of MKN-45 human gastric adenocarcinoma cells and intratumoral injection of UTP or UDP inhibited tumorigenesis of SGC-7901 xenografts that was rescued by the selective P2Y₆R antagonist MRS2578 or by P2Y₆R-targeting shRNA [134]. However, the SGC-7901 gastric cancer cell line was later shown to be a HeLa derivative [113], although similar in vitro responses were demonstrated between MKN-45 and SGC-7901 cells. P2Y₆R antagonism with MRS2578 has also been shown to suppress proliferation and LPS-induced cytokine secretion in human glioma cell lines [135].

In contrast to gastric tumors, P2Y₆R expression is increased in breast cancer tissue, as compared to adjacent normal mammary tissue, where its expression was positively correlated with the TNM stage and decreased overall survival in breast cancer patients [136, 137]. eUDP enhanced the expression and activity of MMP-9 and induced migration and extracellular matrix invasion of MDA-MB-231 human breast cancer cells in vitro [136]. Further in vivo experiments demonstrated that intraperitoneal injection of UDP promoted metastasis of MDA-MB-231 cells from tumor xenografts, responses that were blocked by co-administration of MRS2578 or by P2Y₆R knockdown in tumor cells using shRNA [136]. Moreover, P2Y₆R expression is increased during hypoxia-induced EMT and MRS2578 attenuated vimentin upregulation in MDA-MB-468 breast cancer cells [137]. Because the cellular changes associated with EMT are recognized as significant contributors to dissemination of cancer cells from the primary tumor to distant sites throughout the body [96], these studies highlight the potential of targeting P2Y₆Rs to inhibit breast cancer cell metastasis.

Increased P2Y₆R expression has also been shown in pancreatic cancer tissues, as compared to normal pancreatic tissue isolated from organ donors [105], and P2Y₆R was identified as part of a 7-mRNA signature of differentially regulated genes in pancreatic adenocarcinoma that were associated with reduced overall survival [138], suggesting that increased P2Y₆R expression could serve as a useful prognostic indicator in pancreatic cancer patients.

Interestingly, nucleotides have been shown to be released from melanoma and lung cancer cells following exposure to γ-radiation [139, 140], where subsequent activation of P2Y₆ receptors promoted DNA repair mechanisms that were blocked by the P2Y₆R antagonist MRS2578, leading to reduced survival of irradiated A549 lung cancer cells [140, 141]. Other recent studies have also demonstrated a role for P2 receptors in the cellular response to γ-radiation [142] and purinergic signaling has been suggested to mediate the radiation-induced bystander effect in adjacent, non-irradiated tissues [143].

4.5. P2Y₁₁R

The P2Y₁₁ receptor is unique among P2Y receptors in that its activation couples to both Gα_q and Gα_s signaling, which activates both PLC and adenylyl cyclase [14]. Although ATP is the preferred P2Y₁₁R agonist (EC₅₀ = 65 μM and 17 μM for IP₃ and cAMP production, respectively), the dinucleotide NAD⁺ has also been shown to be an agonist, but its physiological role is less clear [144]. P2Y₁₁R activation in immune cells has been shown to modulate apoptotic and migratory responses in the innate [145] and adaptive [146] immune system, supporting a physiological role for P2Y₁₁Rs in immunoregulation. Functional P2Y₁₁Rs are also expressed in HCC, PDAC and cholangiocarcinoma cell lines, as well as in tumor-derived endothelial cells, which similarly modulate cell migration and invasion [147–149].

Although barely detectable in normal human liver tissue, the P2Y₁₁R is highly expressed in human hepatocellular carcinoma tissue [148]. In the human HCC cell lines Huh-7 and HepG2, both ATP and the selective P2Y₁₁R agonist NF 546 induced increases in [Ca²⁺]_i that were blocked by pretreatment with the selective P2Y₁₁R antagonist NF 340 or P2Y₁₁R knockdown using siRNA. Furthermore, ATP-induced transwell migration of Huh-7 cells was also blocked by NF 340 [148], suggesting that P2Y₁₁Rs promote HCC cell migration. Conversely, experiments using BxPC-3 human PDAC cells suggest that eATP alone does not enhance cell migration in scratch or transwell assays, but did potentiate protease-activated receptor 2-induced migration that was attenuated by the selective P2Y₁₁R antagonist NF 157 [150], suggesting that P2Y₁₁Rs may have direct and/or indirect roles in cancer cell migration.

Cholangiocarcinoma (bile duct carcinoma) is associated with deciliation of epithelial cholangiocytes that line the bilary tree of the liver [151]. In addition to their chemosensory functions, cholangiocyte cilia may have a role in tumor suppression [151], as demonstrated when deciliation of normal human cholangiocytes induced a malignant-like phenotype with enhanced proliferation, anchorage-independent growth and invasive migration [152]. While extracellular ATP inhibited cilia-dependent migration and invasion of normal human ciliated cholangiocytes (NHCs), eATP enhanced invasive migration of deciliated NHCs and the human cholangiocarcinoma cell line HuCCT-1, responses that were attenuated following P2Y₁₁R knockdown by shRNA [149]. These P2Y₁₁R-mediated responses were regulated by protein kinase A activation and subsequent phosphorylation of the tumor suppressor proteins, liver kinase B1 (LKB1) and phosphatase and tensin homolog (PTEN), resulting in AKT inhibition that reduced F-actin-dependent filopodia formation [149]. Although the P2Y₁₁R on cilia could be a potential target for treatment of cholangiocarcinoma, LKB1 phosphorylation was also activated by hesperidin menthyl chalcone, thus bypassing the need for primary cilia [149].

Modulation of cell migration by P2Y₁₁R activation in endothelial cells may also inhibit vascularization of breast cancer tumors. In endothelial cells isolated from human breast carcinoma (BTECs), high [eATP] (> 20 μM) strongly inhibits their migration and 3-D tubulogenesis in Matrigel, but had no effect on the non-malignant human microvascular endothelial cell line HMEC-1 [147]. Furthermore, pharmacological inhibition of P2Y₁₁R or P2X7R with NF 157 or brilliant blue G, respectively, or genetic knockdown by siRNA, partially restored the migratory capacity of BTECs [147]. Increased intracellular [cAMP], which has previously been shown to inhibit endothelial cell migration [153], was suggested to mediate the observed responses to P2Y₁₁R activation. The additional observation that low [eATP] (~ 1 μM), which did not inhibit migration, increased paracellular permeability of BTEC monolayers to dextran, whereas high [eATP] (> 20 μM) decreased BTEC paracellular permeability, is surprising [147] given that high [eATP] is thought to promote vascular permeability [154]. Taken together, these studies further highlight the cell-specific contributions of P2Y₁₁R and other P2R subtypes to cancer progression.

4.6. P2Y₁₂R

Activation of the 342 amino acid P2Y₁₂R by its preferred agonist ADP (EC₅₀ = 60 nM) is coupled to Gα_i/o signaling that inhibits adenylyl cyclase activity to reduce [cAMP]_i [155]. The P2Y₁₂R is best known for its crucial role in mediating platelet aggregation, where ATP/ADP release induced by primary platelet activators (i.e., thrombin) secondarily activates P2Y₁ and P2Y₁₂ receptors to promote and stabilize the aggregation response [156]. Platelet P2Y₁₂Rs are the target of numerous United States Food and Drug Administration (FDA)-approved antithrombotics, including the irreversible P2Y₁₂R antagonists clopidogrel, ticlopidine and prasugrel that require bioactivation in the liver and the reversible P2Y₁₂R antagonists ticagrelor and cangrelor that do not require bioactivation [156]. Notably, clopidogrel (Plavix) has been one of the most prescribed drugs over the past decade and was recently added to the World Health Organization’s List of Essential Medicines [157]. With regards to cancer, platelet activation has been shown to promote tumor cell metastasis and transendothelial migration [23, 156, 158]. Therefore, platelet P2Y₁₂Rs indirectly contribute to cancer progression [24], although other studies have described a role for macrophage P2Y₁₂Rs in the host anti-tumor response [159] and activation of P2Y₁₂Rs in cancer cells can modulate chemotherapeutic drug cytotoxicity [160].

Early in vivo mouse experiments demonstrated the ability of the recently described platelet aggregation inhibitor ticlopidine to attenuate lung metastases following tail vein-injection of B16 melanoma cells and reduce the spontaneous metastasis of LLC cells from primary tumors [161]. While the molecular identity of the drug’s target was not yet known, further in vitro experiments demonstrated that exposing platelet-rich plasma to B16 melanoma cells or the rat HCC cell line AH130 caused significant platelet aggregation that was inhibited by ticlopidine [161]. Similar effects on platelet aggregation were later demonstrated with the human ovarian cancer cell line 59M, where 59M cells dose-dependently increased platelet aggregation, which was blocked by the P2Y₁₂R antagonist cangrelor, the P2Y₁R antagonist MRS2179 or apyrase, an ectodiphosphohydrolase [162]. Furthermore, interaction with platelets also induced gene changes in the ovarian cancer cells, where 59M cells exhibited upregulated expression of anti-apoptotic and proliferative genes [162] and SK-OV-3 cells demonstrated EMT-associated gene expression changes and enhanced extracellular matrix invasion activity [163].

In HT-29 human colorectal cancer cells, in vitro exposure to platelets also induced EMT-associated gene changes and enhanced their metastasis to lungs following tail-vein injection into mice, responses that were blocked by pre-incubation of platelets with aspirin or ticagrelor [164]. Similar results have been reported with LLC and B16 melanoma cells, where in vitro exposure to platelets isolated from WT, but not P2Y₁₂R^−/−, mice induced EMT-associated gene changes and increased the cells’ invasiveness through a mechanism involving platelet TGF-β secretion [24]. Additionally, P2Y₁₂R^−/− mice developed fewer spontaneous lung metastases from primary LLC tumors and fewer experimental lung metastases following tail-vein injection of B16 melanoma cells, further confirming earlier studies that used the P2Y₁₂R antagonist ticlopidine [161]. Ticagrelor also reduced experimental lung and liver metastasis of B16 melanoma cells, reduced spontaneous lung metastasis of orthotopically-injected mouse 4T1 breast cancer cells and improved overall survival of tumor-bearing mice, suggesting that the ability of aggregating platelets to enhance cancer metastasis through P2Y₁₂R signaling is not specific to cancer origin, species or organ type [158, 165]. Notably, these studies collectively demonstrate that systemic P2Y₁₂R antagonism did not enhance the growth of primary xenogeneic or syngeneic tumors.

One study demonstrated that P2Y₁₂Rs in platelets may enhance the growth of primary tumors, since ticagrelor administration attenuated platelet-induced proliferation of A2780 human ovarian cancer cells and reduced the growth of orthotopic tumor xenografts [166]. Interestingly, in a syngeneic ovarian cancer model using mouse ID8 cells, tumor growth was reduced ~ 90% in P2Y₁₂R^−/− mice, but was restored following adoptive transfer of WT hematopoetic cells into P2Y₁₂R^−/− mice. This effect was further demonstrated to be independent of P2Y₁₂R expression in the cancer cells, since CRISPR-mediated P2Y₁₂R knockout in ID8 cells had no effect on tumor growth [166], further highlighting the indirect role of platelet P2Y₁₂Rs in cancer progression.

Several studies have described more direct roles for P2Y₁₂R signaling in cancer cells, where P2Y₁₂R antagonists can enhance cell death. In the C6 human glioma cell line, the P2Y₁₂R antagonist cangrelor blocked nucleotide-induced MAPK activation and cell proliferation [167]. Additionally, P2Y₁₂R antagonism with ticlopidine, prasugrel or clopidogrel synergistically enhanced autophagic death of LN-71 human glioblastoma cells induced by imipramine, a tricyclic antidepressant, by increasing [cAMP]_i levels leading to enhanced autophagy [168]. Furthermore, the combined administration of imipramine + ticlopidine significantly enhanced survival and reduced tumor malignancy grade in multiple in vivo models of glioma [168]. In the human pancreatic cancer cell lines AsPC-1 and BxPC-3, P2Y₁₂R expression was increased, compared to non-malignant hTERT-HPNE pancreatic cells, where P2Y₁₂R inhibition with ticagrelor or knockdown with P2Y₁₂R-targeting siRNA reduced EGFR and Akt phosphorylation, suggesting that P2Y₁₂Rs participate in growth factor receptor crosstalk similar to P2Y₂Rs. Furthermore, ticagrelor synergized with the chemotherapeutic drugs cisplatin, gemcitabine and paclitaxel in cytotoxicity assays in vitro and combined gemcitabine + ticagrelor treatment significantly reduced tumor growth in BxPC-3 xenografts and in a syngenic pancreatic cancer model, compared to monotherapy with either drug [169]. Synergism of P2Y₁₂R antagonists with cisplatin has also been demonstrated in 4T1 mouse breast cancer cells [160]. Taken together, these studies suggest that P2Y₁₂R signaling may play a role in cancer progression beyond its well-described effects in platelets. Indeed, P2Y₁₂R expression has been detected in human melanoma tumor-associated macrophages, where its activation may promote migration towards damaged tumor cells [159], responses that have previously been ascribed to P2Y₂Rs in other tissues [170]. Interestingly, polymorphisms in the P2Y₁₂R gene have been associated with increased pre- and post-operative cancer pain [171], a finding that is bolstered by studies showing that P2Y₁₂Rs in microglial cells contribute to neuropathic pain induced by bone and oral cancers through p38 MAPK activation [84, 172].

Dual anti-platelet therapy using P2Y₁₂R antagonists

Dual anti-platelet therapy (DAPT; combined aspirin + P2Y₁₂R antagonist) is commonly prescribed for patients who have received intervention for coronary dysfunction or patients with stable coronary artery disease to reduce risk of cardiovascular complications [173]. In a large-scale animal study of inflammation-associated hepatocellular cancer induced by hepatitis B virus, DAPT with aspirin and clopidogrel was shown to reduce HCC tumor development and improve overall survival [174], suggesting that DAPT reduces the risk of cancer development. Interestingly, this effect appeared to be specific to HCC driven by chronic hepatitis, as DAPT had no effect on HCC induced by the chemical carcinogen CCl₄ [174]. However, analysis of data from several large clinical trials investigating the long-term cardiovascular benefits of prolonged DAPT unexpectedly observed an increased incidence of solid cancer development and a significantly increased risk of cancer-related death [173, 175–177]. A retrospective study of acute coronary syndrome patients found that cancers of the genital, digestive, urinary and respiratory systems were the most common in patients receiving DAPT, although no comparison to non-DAPT control subjects was made [178]. Interestingly, different P2Y₁₂R antagonists used in DAPT correlated with different cumulative levels of cancer incidence, clopidogrel having the highest and ticagrelor the lowest (2.2 vs. 0.3 per 100 patients/year, respectively) [178]. However, follow-up clinical trials and meta-analyses found no significant association between P2Y₁₂R antagonists and cancer incidence or cancer-related death [179–181]. A separate study of colorectal, prostate and breast cancer patients in the United Kingdom further showed no significant association between clopidogrel use and cancer-related death [182]. Additionally, an analysis of an Israeli population-based historical cohort of over 180,000 subjects comparing the rate of cancer development in those receiving DAPT, aspirin alone or no treatment actually demonstrated a significantly decreased risk of cancer development with aspirin or DAPT [183]. Similarly, a case-control study using primary care data from Spain found that low-dose aspirin and clopidogrel, taken as monotherapy or dual-therapy, were both associated with decreased risk of developing colorectal cancer [184]. These findings are in line with FDA analyses on the safety of clopidogrel [173, 183] and recommendations from the United States Preventive Services Task Force on the use of low-dose aspirin for the prevention of cardiovascular disease and colorectal cancer [185].

4.7. P2Y₁₃R and P2Y₁₄R

The P2Y₁₃ receptor (P2Y₁₃R) is activated by ADP (EC₅₀ = 60 nM) and the P2Y₁₄ receptor (P2Y₁₄R) by UDP-glucose (EC₅₀ = 80 nM) and are considered to be P2Y₁₂R-like due to their high sequence homology and coupling to the Gα_i/o signaling cascade [186, 187]. Despite these receptors having been cloned over 20 years ago and their expression identified in many human tissues and cell types [188, 189], little is known about their role in cancer. Nonetheless, decreased P2Y₁₃R expression was observed following EGF-induced EMT-like changes in MDA-MB-468 human breast cancer cells, but no further mechanistic studies were performed [190]. A recent comprehensive analysis of datasets from the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO) and The Cancer Genome Atlas revealed that P2Y₁₃R expression was downregulated in lung cancer tissues, compared to control tissue [191]. Interestingly, this analysis also found decreased expression of P2Y₁₂R and P2Y₁₄R, along with P2X1R and P2X7R, all associated with a favorable prognosis in lung cancer patients [191]. The opposite was observed for acute myloid leukemia (AML) specimens, where transcriptome analysis found increased P2Y₁₃R expression in AML cells, as compared to umbilical cord-blood derived CD34⁺ progenitor cells [192]. Similarly, P2Y₁₄R expression was increased in acute leukemia cell lines that were resistant to PKI-587, an inhibitor of the PI3K/mTOR pathway that is often dysregulated in leukemia, and high P2Y₁₄R expression was correlated with reduced overall survival in FLT3-ITD-positive AML and acute lymphoblastic leukemia patients [193]. Finally, ~ 5% of colon adenocarcinoma tumors harbor mutations in the P2Y₁₃R gene [194], however functional ramifications of these mutations remain unexplored. Thus, further studies on the roles of P2Y₁₃R and P2Y₁₄R in cancer development and progression are warranted

5. Concluding Remarks

In summary, the effects of extracellular nucleotides on cancer cell growth are highly dependent on nucleotide concentration and the cell-specific expression of P1/P2 receptors and ectonucleotidases, termed the extracellular purinome. P2Y receptors expressed in tumor cells influence oncogenic processes directly, whereas P2Y receptors in host immune cells, endothelial cells and platelets modulate these processes indirectly. P2Y₁R activation in cancer cells generally inhibits proliferation, whereas P2Y₂Rs, the most widely studied P2Y receptor subtype in the cancer field, promote proliferation of numerous cancer cell types and may enhance metastasis directly by promoting cancer cell migration and tissue invasiveness or indirectly by mediating transendothelial migration and extravasation of circulating tumor cells. P2Y₆R activation in cancer cells also modulates proliferation and migration and activates DNA repair processes in response to γ-radiation, whereas the contributions of P2Y₄Rs to cancer progression remain unclear, due to their large signaling overlap with P2Y₂Rs that has not been satisfactorily parsed. P2Y₁₁Rs modulate migration of cancer cells and tumor-derived endothelial cells. Due to P2Y₁₂R’s central role in mediating platelet aggregation and the well-described effects of platelets on circulating cancer cells, P2Y₁₂Rs can enhance metastasis of cancer cells indirectly. Therefore, anti-thrombotic P2Y₁₂R antagonists have been explored to reduce the risk of developing certain cancer types. The roles of the P2Y₁₃R and P2Y₁₄R in oncogenic processes remain largely unexplored, although their expression is dysregulated in lung and blood cancers.

The continued development and utilization of P2Y receptor subtype-specific agonists and antagonists will drive the translation of this basic research into clinical settings, where the therapeutic implications of purinergic modulation will be fully realized. Further refinement of techniques to measure changes in extracellular nucleotide levels in vivo will help researchers identify relevant pathological systems where blockade of P2Y receptor subtypes can provide effective therapies. In considering the extracellular purinome as a single entity moving forward, it appears that the field of purinergic signaling in cancer progression will come full circle by fully characterizing the cell-specific contributions of individual P2Y receptors to oncogenesis.