Purinergic Signaling in Colorectal Cancer

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Gabriela Gonçalves Roliano	--	5153	2022-11-17 20:31:34	\|
2	format corrected.	Beatrix Zheng	+ 1 word(s)	5154	2022-11-21 03:20:53	\|

This entry is adapted from the peer-reviewed paper 10.3390/cancers14194887

Colorectal cancer is a leading cause of cancer-related death. Activated immune cells have the potential to eliminate tumor cells, but cancers gain the ability to suppress immune cell functions and escape immune attack. The researchers explored one mechanism that cancers use to evade immune cells in colorectal cancer. This mechanism alters levels of molecules known as purines. Purines are key players in cellular energetics and many cellular processes and can also lead to immune suppression in cancer.

colorectal cancer purinergic signaling immune system tumor microenvironment

1. Ectonucleotidases—General Aspects

Purinergic signaling involves the biological effects of extracellular purines and pyrimidines on purinoceptors. This mechanism is tightly controlled by ectonucleotidases, enzymes that aid in the conversion of adenosine triphosphate (ATP) to ADO within the extracellular space ^[1]. The role of ectonucleotidases in the production of immunosuppressive ADO maybe critical to understanding the progression of CRC. ADO can be produced via two adenosinergic pathways: the canonical and non-canonical pathways. The canonical adenosinergic pathway includes enzymes that convert ATP to AMP (Figure 1). These enzymes include ecto-nucleoside triphosphate-diphosphohydrolases (E-NTPDases), such as the NTPDase1, also known as CD39, which converts ATP to ADP and ADP to AMP, ecto-pyrophosphate-phosphodiesterases (E-NPP) which convert ATP to AMP, alkaline phosphatases (ALPs) which convert ATP to ADP, ADP to AMP, and AMP to ADO, ecto-5′-nucleotidase/CD73 (CD73) which converts AMP to ADO, and adenosine deaminase (ADA) which converts ADO to inosine ^[2]^[3]^[4]. ADO can also be generated by the non-canonical adenosinergic pathway, which involves the enzymes nicotinamide adenine dinucleotide (NAD+)-glycohydrolase/CD38 which converts NAD+ to ADP-ribose (ADPR), and NPP1/CD203a (PC-1) which converts ADPR to AMP. This final step generates extracellular AMP which is metabolized to ADO by CD73. Therefore, CD73 represents the common link between the canonical and non-canonical adenosinergic pathways ^[5].

Figure 1. Purinergic signaling cascade: Intracellular ATP is externalized by the PNX1 channel and activates P2 receptors. P2Rs are divided into two major categories: P2X1-7 and P2Y_{1,2,4,6,11–14}. ATP is hydrolyzed by E-NTPDase1/CD39 to ADP, which also binds to P2YR and is further hydrolyzed by CD39 to AMP. AMP is also formed through an alternative pathway that involves the enzymes NAD+-glycohydrolase/CD38, which converts NAD+ to ADP-ribose, and NPP1/CD203a, which metabolizes ADP-ribose to AMP. Once AMP is formed by the canonical and/or non-canonical pathway, it is hydrolyzed by ecto-5′-nucleotidase/CD73 to ADO, thus connecting both adenosinergic pathways. ADO binds to the P1 receptors, which are divided into A1, A2A, A2B and A3, each with different ligand affinities. ADO is internalized into the cell through ENTs or CNTs and/or it is deaminated to INO by ADA. Abbreviations: ATP (adenosine triphosphate); ADP (adenosine diphosphate); AMP (adenosine monophosphate); ADO (adenosine); INO (inosine); NAD+ (nicotinamide adenine dinucleotide); PNX1 (Pannexin-1); P2X (ionotropic purinergic receptors); P2Y (metabotropic purinergic receptors); P1 (adenosine metabotropic receptors); E-NTPDase1/CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1); ENTs (nucleoside equilibrative transporters); CNTs (concentrative nucleoside transporters); ADA (adenosine deaminase).

Extracellular ADO levels are also regulated by nucleoside equilibrative transporters (ENTs) and concentrative nucleoside transporters (CNTs). ENTs and CNTs are expressed in the cell membrane and help transport ADO into the cells ^[6]^[7]. Within the cell, ADO is phosphorylated by ADO kinase (AdoK) and adenylate kinases for subsequent conversion into ADP ^[8]. ADO can also be deaminated to inosine (INO) by adenosine deaminase (ADA) ^[3]^[4]. ADA is expressed in most human tissues, with the highest levels found in the lymphoid system, including lymph nodes, spleen, and thymus ^[9]. ADA binds to the cell surface through an ADA binding protein termed CD26 ^[10]. The complexity of extracellular ATP metabolism by ectoenzymes has been increasingly appreciated and reviewed in last years ^[2].

1.1. Ectonucleotidases—The Role of CD39 in CRC Progression

Several studies have investigated the role of Tregs in the TME of CRC with the aim of characterizing Treg subpopulations and their immunosuppressive potency ^[11]^[12]^[13]. Multiple lines of evidence highlight CD39+FOXP3⁺ T cells as the as the major regulatory T cell infiltrate in tumor tissue. Furthermore, Tregs are also elevated in peripheral blood from patients with CRC compared to healthy donors ^[11]^[12]^[13]^[14]^[15]^[16]^[17]. Levels of CD39+Tregs in peripheral blood from CRC patients correlate with TNM staging and clinicopathological features, and the expression of CD39 gradually increases in Tregs from initial to advanced stages of CRC development ^[12]^[15]^[18]. Similar to FOXP3, Helios is a marker that characterizes immunosuppressive Tregs ^[19] and its co-expression with CD39 was found in multiple studies ^[13]^[17]. Additionally, CTLA-4 and PD-1 checkpoint inhibitor molecules were co-expressed with CD39 in Tregs from both CRC tumor bulk and the peripheral circulation, suggesting that this lymphocyte subpopulation participates in CRC-associated immunosuppression ^[11]^[12]^[13]. In line with this, Khaja et al. proposed simultaneous blockade of CD39 and PD-1 as a new modality of treatment for patients with CRC (2017).

In addition to demonstrating the presence of Tregs in CRC samples, the immunosuppressive activity of CD39+ Tregs was also investigated. CD39+ γδTregs (a subtype of T cells located mainly in the GI tract) isolated from CRC biopsies promoted a higher levels of inhibition of in vitro CD3⁺ effector T cell proliferation and function when compared to other T cells subsets extracted from the same tissue ^[12]. ADO receptor blockade dramatically reversed this immunosuppressive effect, while inhibition of IL-10, TGF-β, CTLA-4, and PD-1 signaling did not substantially change levels of Treg-induced immunosuppression ^[12]. Adenosinergic signaling has also been reported to mediate differentiation of tumor-infiltrating CD39+ γδTregs induced by tumor-derived TGF-β1 ^[12].

The primary location of CRC has also received considerable attention, due to differences in cellular/molecular characteristics, response to therapy, and patient outcomes based on location in the right (ascending) or left (descending) colon ^[20]. Several lines of evidence point towards worse outcomes in right-sided compared to left-sided CRC, causing location to be classified as a patient risk factor ^[21]. In line with the role of CD39 in tumor promotion, one group observed an increased frequency of CD39+ γδTregs in right-sided compared to left-sided CRC ^[22]. Additionally, CD39 expression in γδ-TILs increased in vivo tumor growth, metastasis, and invasion ^[22]. In human clinical biopsies, low CD39 expression in right-sided CRC was correlated with improved patient outcomes, while in left-sided CRC low CD39 expression was associated with worse patient outcomes ^[22]. High frequency of CD39 positivity in Tregs positively correlates with Treg immunosuppressive activity, which can be reversed by siRNA knockdown of CD39 ^[15].

CD39^high Tregs can also suppress T cell migration in CRC. One study found that the CD39^high Treg subpopulation derived from CRC patients reduced CD4+ and CD8+ T cell transendothelial migration (TEM), whereas CD39^high Tregs derived from healthy volunteers did not exert these effects on TEM. CD39 enzyme activity contributes to ADO-mediated reduction of T cell migration, as addition of ADO reduced TEM, whereas blockade of CD39 or ADO receptors enhanced TEM. Furthermore, the effects of ADO on TEM were shown to be mediated by monocytes, as the addition of ADO decreased the ability of monocytes to activate the endothelium. Cellular expression of CD73 was low and did not differ between patients and controls ^[11]. Although more studies of the effects of CD39 expression on Treg CD39^high cells in CRC are needed, these data indicate that this is an important regulator of immunosuppression in the TME, with the ability to reduce T effector cell migration into the tumor.

CD39 expression was also studied in non-regulatory T cell subsets derived from CRC patients. Two different subsets of CD8+ T cells were identified in CRC tumors, one referred to as the bystander population, which did not exhibit tumor-specific antigen specificity, and the other referred to as tumor-specific TILs, which exert a tumor antigen-specific response. A marked lack of CD39 expression was demonstrated in the CD8+ bystander population (CD8+CD39⁻), whereas tumor-specific CD8+ TILs expressed high levels of CD39. The CD8+CD39+ TILs exhibited high expression of genes related to proliferation and exhaustion, which is characteristic of chronically stimulated T cells and indicates that these are an exhausted T cell population. Furthermore, CD8+CD39+ TILs expressed genes related to antigen presentation and processing. The authors proposed the use of CD39 as a biomarker for CD8+ T cells, where the lack of CD39 expression could be used to identify T cells with bystander roles ^[23]. Increased CD8+CD39^high TILS were also found in tumor compared to surrounding non-tumor tissue at initial stages of CRC (I-II). These cells were characterized by high PD-1 expression and low INFγ production, which was correlated with exhausted T cell phenotypes and suppressed CD4+ T cell proliferation ^[24]. In tumor samples, TIL CD4/CD8 presented a CD39 increase and CD73 decrease, suggesting that CD39 expression can be an inhibitor of T cells. CD39 was similar in mucosa, tumor, and tumor margin, suggesting that the other cells can express this enzyme. In contrast, CD39 was decreased and CD73 increased in advanced TNM, suggesting that advanced tumors had CD39 missed and CD73 overexpressed. The CD39 inhibition in autologous spheroids of CRC increased T cell capacity to attack these cells, by increasing movement and the destruction of spheroids ^[25].

Recently, a specific mucosal-associated invariant T cell (MAIT cell) was investigated in CRC ^[26]. MAIT cells are capable of recognizing microbial metabolites and can exert effects on anti-tumor immunity. Exhausted MAIT cells demonstrated reduced polyfunctionality with regard to the production of important anti-tumor effector molecules, including IFN-γ and GrB. PD-1 blockade partially improved in vitro activation of tumor-infiltrating MAIT cells ^[26]. However, whether CD39 participates as a source of immunosuppressive ADO was not investigated in this context.

T cells have been the predominant immune cell type investigated in the context of cancer-associated purinergic signaling, primarily due to CD39/CD73 expression, which has been proposed as a marker for immunosuppressive Tregs. However, as described above, other immune cell populations are present in the TME and yield important contributions to the creation and maintenance of an immunosuppressive microenvironment ^[27]. One study sought to define the role of the CD39/CD73 axis in MDSC populations in CRC. MDSCs were investigated during the course of treatment of patients with advanced stage metastatic CRC (mCRC) receiving FOLFOX plus bevacizumab therapy. A higher level of circulating MDSCs, in particular granulocytic MDSCs (gMDSCs), was observed in untreated mCRC and were associated with decreased overall survival (OS) and progression-free survival (PFS). FOLFOX plus bevacizumab treatment reduced gMDSC levels and increased OS and PFS. This research also investigated the expression of PD-L1, CD39, and CD73 in MDSCs, and found that gMDSCs from mCRC patients that expressed these markers exhibited potent immunosuppressive effects relative to other myeloid cell populations present in the blood. These immunosuppressive effects could be reversed by a blockade of the CD39/CD73 and PD1/PD-L1 axes, suggesting the potent immunosuppressive activity of these cells in CRC in an ADO-pathway and PD1/PD-L1 dependent process ^[28]. In accordance with other studies, the authors suggest the use of ATP ectonucleotidase inhibitors and/or PD-1/PDL1 inhibitors as potential therapeutic combinations with FOLFOX-bevacizumab ^[28]. An additional study sought to characterize MDCSs in CRC and demonstrated that the number of Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs in peripheral blood were markedly increased in CRC patients compared to healthy donors. MDSCs exhibited elevated CD39 expression and were positively correlated with tumor metastasis. MDSCs cells also demonstrated potent immunosuppressive activity, including the ability to inhibit CD3⁺ T cell proliferation from two stage IV CRC patients ^[29].

The role of CD39 in CRC progression and metastasis was investigated in CD39⁺^/− mice, CD39⁺^/+ mice, or over-expressing CD39 transgenic mice (htCD39) all in a BALB/c background. No differences in primary tumor growth were observed between these groups. However, in a metastatic dissemination model, tumors derived from the MC-26 murine colon adenocarcinoma cell line grew significantly faster in htCD39 mice compared to CD39⁺^/− mice, suggesting that CD39 overexpression in the TME favors tumor spread. In both primary and metastatic CRC models, CD39 was expressed at high levels around the tumor borders, including expression in stromal cells, endothelial cells, and immune cells ^[30]. In a separate study, increased CD39 expression was also reported in tumor tissue from CRC patients when compared to normal border, and in vitro inhibition of CD39 impaired tumor cell proliferation ^[31]. In addition, the presence of variant allele CD39 in patients with mCRC was an indicator of favorable response to chemotherapy plus bevacizumab, suggesting that this may be used as a predictive marker ^[32].

In a conflicting study, clinical CRC biopsies showed lower CD39 levels when compared to non-neoplastic tissue, particularly in early stages of tumor development. However, throughout tumor progression, CD39 levels became more comparable to levels on normal border (T3N ± M1). Additionally, P2Y₂ and P2X7 expression was markedly decreased in tumor tissues when compared to normal borders ^[30].

In sum, multiple lines of evidence point to a strong association between CD39 expression and a suppressive immune cell profile, which was mainly characterized in Tregs but also extended to MDSCs. Nevertheless, the mechanisms underlying CD39-mediated immunosuppression, and its correlation with tumor progression and prognosis in CRC, remain to be more deeply understood. Further cell-type specific studies are needed to more fully characterize how CD39 expression in tumor, stromal, and epithelial cells within the tumor and the tumor borders impacts CRC progression and metastasis ^[30].

1.2. Ectonucleotidases—Participation of CD73 in CRC Progression

CD73 is an enzyme that can be anchored to the plasma membrane in the C-terminal portion by a residue of glycosyl-phosphatidylinositol (GPI), or can be found in soluble form in the cytosol or the extracellular medium ^[7]^[33]. Physiologically, CD73 is widely distributed in different tissues, including the colon, kidney, brain, liver, heart, lung, vascular endothelium, spleen, lymph nodes, and bone marrow ^[34]. In the immune system, CD73 is found on the surface of Tregs, neutrophils, MDSCs, DCs, NK cells, and macrophages ^[8]. An increasing number of studies point to a role for CD73 in cancer pathogenesis.

CD73 expression was reduced in a highly liver-metastatic human CRC cell subline (SW48LM2 cells) when compared with less malignant tumor cells, both in normoxic and hypoxic conditions. In addition, metastatic CRC cells (mCRC) exhibited a decrease in extracellular nucleoside levels (ADO, guanosine and INO) and a parallel increase in nucleotides (AMP, GMP and IMP), which indicate that loss of CD73 expression and subsequent changes in purine metabolism are beneficial for tumor progression ^[35]. In contrast, low expression of CD73 was associated with improved progression free survival (PFS) in patients that received single or combined therapy with cetuximab or FOLFIRI/FOLFOX ^[36]^[37]. KRAS status also impacts the predictive role of CD73 in CRC therapy efficacy. For example, CD73 levels were predictive of PFS and OS benefit for cetuximab therapy of KRAS wild type tumors, while no predictive effects for CD73 were observed in KRAS mutant tumors ^[37].

In contrast, higher expression of CD73 in CRC has been reported in several studies ^[38]^[39]^[40]^[41]^[42]. Pathologically, CD73 is overexpressed in several types of tumors, including CRC ^[43]^[44]^[45]^[46]^[47]^[48]. CD73 overexpression was associated with increased cell proliferation ^[40], nerve invasion, lymph node and distant metastasis, and advance tumor staging ^[41]. In liver metastasis, CD73 expression was also associated with worse pathological features and poorer response to preoperative therapy ^[42]. CD73 expression was significantly lower in chemotherapy-responsive CRC patients, and RAS-MAPK-inhibition induces CD73 upregulation and an immunosuppressive TME ^[49]. CRC patients with CD73^high tumor expression have shorter OS when compared to those with CD73^low expression ^[38]^[39]^[41]^[42]^[49]. For these reasons, some authors suggest CD73 as a biomarker for poor prognosis ^[39]^[40].

CD73 has also been associated with CRC development and progression. CD73 inhibition in a mouse model of colitis-associated tumorigenesis reduced histologic evidence of colon damage. Furthermore, using the same model, the CD73 inhibitor AB680 unlocked the anticancer immune response, leading to increased CD8+T cell activation and improved Treg and exhausted T cell function (Kim et al., 2021). In a study with a microRNA termed miR30a that inhibits in vitro and in vivo CRC cell proliferation and promotes apoptosis, the authors suggest that the antitumor effects of miR30a are mediated by CD73 downregulation ^[50]. Furthermore, cancer associated fibroblasts (CAFs) express high levels of CD73 in CRC and are strongly correlated with poor prognosis ^[51]. In this paper, the authors demonstrated that elevated ADO upregulates CD73 via an A2B-mediated pathway, inducing an immunosuppressive TME ^[51]. Targeting this circuit significantly improved antitumor immunity in CAF-rich tissues ^[51]. The authors described CD73 as an immune checkpoint protein in a feedforward circuit, which is enhanced by tumor-derived ADO ^[51].

The levels of CD73 in CRC have also been correlated with macrophage phenotypes. A study showed that alternate-day fasting for two weeks led to a subsequent decrease in in vivo CRC growth and decreased M2-TAM polarization. In vitro, fasting conditions of low-glucose/low serum-containing media induced autophagy, CD73 suppression, and decreased generation of extracellular ADO in the CT26 mouse CRC cell line. Decreased ADO levels led to impairment of M2-TAM polarization and was associated with inactivation of JAK1/STAT3 pathway under fasting conditions, suggesting that antitumor immunity induced by fasting is mediated by blockade of ADO signaling ^[52].

Overall, these studies indicate that CD73 expression promotes CRC growth and metastasis. However, the tumor-promoting effects of CD73 are dependent on tumor stage and on its cell-type specific expression levels within the TME. Finally, considering that the majority of the above investigations were focused on lymphocytes, additional studies to the CD73 expression and function in innate immune cells present in the TME of CRC are also needed.

2. Purinoceptors—General Aspects

Purinergic signaling is initiated by the release of nucleotides and nucleosides into the extracellular space. Under normal conditions, ATP is in the millimolar range in the cytoplasm (3–10 mM) and in the nanomolar range in the extracellular space ^[53]. However, ATP can be released through the plasma membrane by lytic (cell death) and nonlytic mechanisms (vesicles and membrane channels formed by the pannexin-1/connexins) ^[54]^[55]. Following release into the extracellular space, purinergic nucleotides and nucleosides exert their effects through interactions with specific membrane receptors called purinergic receptors or purinoceptors ^[56]. These receptors are responsible for purinergic message transmission. Purinoceptors are divided into two groups: P1 receptors (P1Rs) and P2 receptors (P2Rs). P1Rs utilize the main endogenous agonist ADO, whereas P2Rs are sensitive to multiple di- and triphosphate nucleosides, such as ATP, ADP, UTP, and UDP ^[57].

2.1. ATP as an Agonist of Purinoceptor-Mediated Protumor and Antitumor Actions

The role of extracellular ATP in CRC is still not well-understood, and the literature on this subject is conflicted. This may be due to different functions depending on the receptor and cell type involved. Multiple studies have shown that high concentrations of ATP and P2R agonists reduce cell viability and proliferation at the S phase of cell cycle through inhibition of protein kinase C (PKC) in CRC cell lines ^[58]^[59]. In contrast, a separate study indicates that exposure of Caco-2, a human CRC cell line, to ATP increases multidrug resistance-associated protein 2 (MRP2) expression, which was shown to confer resistance to etoposide, cisplatin and doxorubicin, leading to enhanced cell survival. It is possible that this mechanism is mediated by P2Y receptors, but further investigation is needed to elucidate which receptors may be involved ^[60].

The ATP-based chemotherapy response assay (ATP-CRA) is a method used to measure tumor response to therapy., since tumor cells quickly deplete ATP. In order to evaluate the utility of this assay in CRC, ATP levels were tested in peripheral blood samples from advanced CRC patients undergoing adjuvant chemotherapy with FOLFOX or Mayo clinic regimen (5-FU and leucovorin). Response for both treatments as indicated by ATP-CRA (>40% ATP reduction) positively correlated to OS and PFS, suggesting that the ATP-CRA is a useful test to guide individualized chemotherapy ^[61]. In contrast, in vivo tests with an immunogenic cell death (ICD)-inducing therapy resulted in enhanced ATP secretion, in addition to other danger signaling molecules, which correlated to tumor reduction and increased CD8+ T-mediated antitumor response ^[62]. Therefore, extracellular ATP levels may not be a consistent indicator of treatment responses.

The variety of pathways involved in ATP release to the extracellular environment, including cell membrane channels, cell membrane damage, or cell death, may explain the conflicting results in the above studies. To summarize, in vitro analyses point to the dual role of ATP, which can reduce CRC cell viability and proliferation ^[58]^[59] while simultaneously promoting chemoresistance ^[62]. Decreased ATP levels were a clinical indicator of chemotherapy responsiveness in CRC ^[61], whereas increased ATP secretion was reported in immunogenic cell death in vivo studies and was associated with an improved immunologic response ^[62]. Taken together, these studies leave open questions surrounding whether extracellular ATP benefits or harms CRC progression.

2.2. P1 Receptors and Their Relationship with CRC Progression

P1Rs differ in their affinity for ADO. A1R, A2AR and A3R are high affinity receptors, whereas A2BR is a low affinity ADO receptor ^[63]. Under physiological concentrations, ADO is present at low levels in both the intracellular and extracellular space (nanomolar range) and mediates effects via A1R, A2AR, and A3R. When ADO concentrations reach elevated levels (micromolar range), such as under inflammatory conditions or in the TME, A2BR becomes the principal receptor that mediates ADO signaling ^[64]. Studies have shown that P1Rs are differentially expressed in CRC cell lines and tissue ^[65]^[66].

A1R and A3R are coupled to Gi or Go proteins and lead to decreased intracellular cyclic AMP (cAMP) levels, whereas A2AR and A2BR are coupled to Gs protein and result in increased levels of intracellular cAMP. Stimulation of A1R and A3R can trigger the release of Ca²⁺ ions from intracellular stores, whereas A2BR receptor stimulation can activate phospholipase C (PLC) ^[57]^[64]. All P1Rs are coupled to MAPK/ERK signaling pathways ^[67].

The A1R was investigated in one study involving the anti-diabetic drug metformin. Increased A1R expression was observed in HCT116 and SW480 CRC cell lines following treatment with metformin and corresponded with induction of apoptosis. This effect was dependent on AMPK-mTOR pathway, a key player in cancer cell survival/proliferation, which resulted in cell cycle arrest and apoptosis ^[65].

The A2BR exhibits a dual role in CRC progression. A2BR expression was considerably higher in CRC tissues compared to normal colonic mucosa and was present in five CRC cell lines (DLD1, SW480, HCT-15, LOVO, and COLO205). A similar cancer-specific increase was not detected for the other P1Rs (A1R, A2AR and A3R). Furthermore, A2BR was increased under hypoxic compared to normoxic conditions, whereas no increase in A1R, A2AR or A3R expression was demonstrated under hypoxic conditions ^[68]. It is possible that the overexpression of A2BR during hypoxia contributes to cancer cell growth and angiogenesis ^[68]. In contrast, the expression of A2BR was also associated with cell death in Saos-2 cancer cell line ^[69]. This research showed that activation of p53 in TetOn-p53-WT Saos-2 cells directly stimulates A2BR gene expression, which in turn contributes to p53-induced apoptosis. Moreover, A2BR was shown to be involved in hypoxia and chemotherapy-induced cell death in Saos-2 cells by regulating Bcl-2 family members, a group that mediates intrinsic cell death ^[69].

High A2AR expression has been demonstrated in CRC and associated with worse prognosis and the presence of tumor infiltrating lymphocytes (TILs) ^[66]^[70]. One study analyzing 204 tumor specimens from CRC patients demonstrated that both PD-L1 and A2AR expression was higher in tumor than in adjacent non-tumor tissue. Additionally, both PD-L1 and A2AR expression levels were correlated with higher TNM stage and lower OS, showing the independent prognostic predictor value of both immunosuppressive markers in CRC ^[66]. RNA sequencing and whole exome sequencing data from 453 CRCs demonstrated a positive association between TIL levels and expression of immune checkpoint molecules and A2AR in colon adenocarcinomas. These data are in accordance with the protumor role of the A2AR receptor ^[70].

High expression of A3R was demonstrated in HT-29 CRC cell line and in human CRC tissue compared to normal adjacent mucosa using [18F]FE@SUPPY as a PET-tracer for A3R ^[71]. Conversely, the A3R agonist (N6-(2,2-diphenylethyl)-2-hexynyladenosine) inhibited Caco-2 CRC cell proliferation in a concentration and time dependent manner. However, A3R knockdown did not reverse or prevent the antitumor effects induced by the A3 agonist, indicating that these effects were likely off-target ^[72]. Therefore, the role of A3R in CRC remains relatively unexplored.

2.3. P2 Receptors—P2X Participation in CRC Development

P2Rs consist of two major categories of receptor, P2X1-7 ionotropic receptors that are sensitive to extracellular ATP and P2Y_{1,2,4,6,11–14} metabotropic receptors that are stimulated by ATP, ADP, UTP, UDP, and UDP-glucose ^[57]. P2XR receptors have intracellular N- and C-subunits that bind to protein kinases and also have two transmembrane regions, which are involved in channel activation. P2XR subtypes differ in their rates of desensitization, ion conductivity, and sensitivity to agonists, antagonists, and allosteric modulators ^[56]^[73].

The P2X1 receptor is widely expressed in smooth muscle cells; it also mediates the actions of ATP and Ca²⁺ influx on platelet aggregation ^[74]^[75]. P2X2 receptors are widely expressed in central and peripheral neurons and have been implicated in neurotransmission ^[76]^[77]^[78]. P2X3 receptors are expressed in sensory neurons ^[79]^[80]^[81]. The receptors P2X4 and P2X6 are expressed in the central nervous system, endothelial cells, and thymus ^[54]^[82]^[83]^[84]. The P2X7 receptor is expressed in immune cells, pancreas, skin, and microglia and leads to cell death in the presence of high levels of ATP ^[73]^[85]^[86]^[87]^[88]^[89]^[90]^[91]. P2XR cell signaling is mediated by gating of primarily Na^+, K⁺, and Ca²⁺ and, occasionally Cl⁻ channels ^[92]^[93].

P2X7 has been suggested as a target for CRC treatment ^[94]^[95]. Analysis of P2X7 levels in human tumor and adjacent tissue revealed that its expression was higher in tumors, and was an independent variable associated with worse prognosis, shorter survival, and higher TNM stage ^[95]. Increased P2X7 expression was found in mCRC tissues when compared to primary tumors, suggesting its association with metastasis. In line with this, higher P2X7 levels were also reported in CRC cell lines compared with normal colon cell lines, and in metastatic-derived CRC cell lines compared with primary tumor-derived cells ^[95]. In addition, induction of P2X7 signaling with use of a selective agonist resulted in PI3K/Akt and NF-κB pathway activation and further induction of CRC cell proliferation ^[94]^[95]. P2X7 overexpression also mediated in vivo tumor growth, by stimulating angiogenesis, cancer stem cell (CSC) properties, and macrophage/TAM infiltration ^[96]. Also consistent with promotion of CRC malignancy by P2X7, antagonism or knockdown of P2X7 impaired in vitro and in vivo cell proliferation, invasion, and migration and promoted apoptosis ^[97]. Interestingly, a recent study performed in a model of colitis-associated colorectal cancer revealed that P2X7 sensitization in combination with signals from a dysbiotic microbiota promotes CRC development by inducing the inflammasome activation and further inflammatory cascade amplification ^[98]. In contrast, P2X7 blockade in an in vivo colitis-associated cancer model promoted Treg and neutrophil infiltration and epithelial cell growth and decreased apoptosis, suggesting a protective factor of P2X7 against cancer formation ^[99].

Analysis of colon cancer tissue and normal colon tissue from two independent datasets (GEO and TCGA) indicated that P2X5 was among the top four high risk genes. Most of the differentially expressed genes that were identified are involved in protein transport, apoptosis and neurotrophin signaling pathways ^[100].

Out of all the P2X receptors listed, only papers on P2X7 and P2X5 were available for this research, demonstrating the lack of information about P2XR role in CRC biology. Overall, the available literature points to a role for these receptors in promoting CRC.

2.4. P2 receptors—P2Y Participation in CRC Development

P2Y₁, P2Y₁₂, and P2Y₁₃ are activated by ATP and ADP, P2Y₂ and P2Y₄ are activated by UTP, P2Y₆ is activated by UDP, and P2Y₁₄ receptor is activated by UDP-glucose ^[77]. P2YR-mediated activation of protein Gq leads to the stimulation of PLC, with subsequent production of inositol-(1,4,5)-trisphosphate and diacylglycerol (DAG). Inositol-(1,4,5)-trisphosphate leads to increased intracellular Ca²⁺ levels, and DAG stimulates PKC and may inhibit adenylate cyclase ^[101]. P2YRs are expressed in a variety of tissues and organs, including lung, kidney, pancreas, adrenal gland, heart, vascular endothelium, skin, muscles, and brain.

The purinergic receptors are very complicated to investigate, due to factors including methodologic limitations, complexity of receptor subtypes and activation, and cell-type specific mechanisms of action. Few studies of the P2YR were identified for this research and only included P2Y₂, P2Y₆, and P2Y₁₂. The P2Y₂ receptor was investigated in one in vitro study, which analyzed resistance to apoptosis mediated by treatment with ursolic acid in CRC HT-29 cells. Results demonstrated that ursolic acid treatment induced ATP production, which is probably released and binds to P2Y₂ receptors. The P2Y₂ receptor then activates tyrosine kinase Src, leading to p38 phosphorylation. The p38 pathway induces the expression of COX-2 and results in chemoresistance in HT-29 CRC cell line ^[102].

The P2Y₆ receptor was analyzed in two articles, one in vitro and one both in vitro and in vivo. The role of P2Y₆ receptors in cell migration was investigated in lung and colon cancer cell lines. Stimulation of a Caco-2 cell monolayer with a P2Y₆-selective agonist (MRS2693) induced filling in the plaque of the cells and formation of focal adhesions and filopodia. Treatment with P2Y₆ antagonist (MRS2578) had the opposite effect, demonstrating a potential role for this receptor in the tumor growth ^[103]. Meanwhile, treatment of HT-29 cells with the P2Y₆ agonist prevented the pro-apoptotic effects of TNF-α, leading to increased expression of X-linked inhibitor of apoptosis protein (XIAP), which was correlated with Akt/PI3K phosphorylation ^[104].

In vivo experiments with P2Y₆^+/+ and P2Y₆^−/− mice demonstrated that P2Y₆^−/− animals had significantly reduced number and size of colorectal tumors. Besides this, P2Y₆^−/− animals had a significantly lower dysplastic score and reduced vascularization when compared to P2Y₆^+/+ mice, indicating a potential role for this receptor in promoting tumorigenesis. Expression of β-catenin was investigated to further understand the influence of P2Y₆ loss. β-catenin was observed in the plasma membrane of cells derived from P2Y₆^−/− animals, versus demonstrating plasma membrane, cytosolic, perinuclear, and nuclear staining in cells derived from P2Y₆^+/+ mice. Furthermore, the presence of β-catenin in the nucleus correlated with abnormal expression of proto-oncogene c-MYC. These data indicate that the expression of P2Y₆ is associated with the localization of β-catenin, which is related to increased proliferative and invasive phenotypes and poor outcomes in CRC patients ^[104]. However, the authors suggest a cautious interpretation of their results, as the sample size was small, and further investigations are needed.

The P2Y₁₂ receptor in platelets is well known for its role in arterial thrombosis, and antagonists are used for the prevention and treatment of cardiovascular diseases ^[105]. The efficacy of one of P2Y₁₂ antagonist (ticagrelor) therapy for breast and CRC was tested, as platelets participate in cancer-associated thrombosis and metastasis ^[106]. Ticagrelor significantly inhibited formation of tumor cell-induced platelet aggregation in vitro and reduced platelet aggregation and activation in patients. These findings suggest the role of P2Y₁₂ in cancer-associated platelet aggregation and put forth P2Y₁₂ inhibition as a potential therapy for patients with a high risk of cancer-associated thrombosis ^[106].

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