Purinoreceptors are important to tumor pathogenesis given that ATP is so ubiquitous in the tumor microenvironment (TME) and critical to driving cell proliferation. The concentration of ATP in the TME is approximately 100–500 μM, while 10–100 μM in normal tissues and blood (Hu et al., 2019; De Marchi et al., 2020; Mimoto et al., 2020). The mechanism underlying ATP release in the TME is multifarious as it may be actively secreted, passively released upon cell death, or generated de novo extracellularly (Di Virgilio et al., 2018a). Regardless, the dysregulation of extracellular ATP acts in a paracrine or autocrine fashion via purinoreceptors to alter the functionality of numerous nearby cell subtypes.

In combination with the changes in extracellular ATP concentration, a variety of P2XRs are upregulated in disparate cancer types, including glioblastoma (GBM), hepatocarcinoma, hepatobiliary carcinoma, breast cancer, multiple myeloma, prostate cancer, and renal cancer (Farrell et al., 2010; Di Virgilio et al., 2018a; Khalid et al., 2018; Asif et al., 2019; Huo and Chen, 2019). The specific tumor type appears to define whether upregulation of P2X may be protective or prohibitive against a tumorigenic phenotype. The mechanism of action of P2XRs in cancer proliferation and growth is multifold. Interestingly, P2XR activation lies on a spectrum as physiological ATP concentrations can promote proliferation and metastasis, whereas excessive ligand binding can induce apoptosis (Virgilio, 2012).

Another effect of P2XRs on cancer pathophysiology is tumor migration and invasiveness. One of the hallmarks of late-stage and metastatic tumors is their ability to spread. This confers a significant disease burden to the tumors as infiltration into non-native tissue can be a common cause of mortality. Therefore, if P2XRs are involved in this process, it is a promising therapeutic avenue to “antagonize” the receptor to diminish tumor motility. In particular, P2XRs have been implicated in tumor epithelial-to-mesenchymal transition (EMT). The EMT confers a morphology to tumor cells that allow for extracellular proliferation and distant metastasis (Thiery, 2002).

Purinergic receptor activation can also secondarily modify tumor proliferation through its activity in the immune system. Firstly, P2XR subtypes are widely expressed in a variety of immune cell subtypes, ranging from macrophages to lymphocytes (He et al., 2017). In human T-cells, ATP release was shown to increase IL-2 expression and p38 MAPK activation (Loomis et al., 2003). IL-2 is a key pro-proliferative cytokine that induces the proliferation of B- and T-lymphocytes. Therefore, increased expression of IL-2 would support an immunogenic response. This conclusion is supported by evidence that “antagonism” of P2X signaling led to T-cell anergy, or functional inactivation (Schenk et al., 2008). The following subsections details the various research on P2XR subclasses and their relationship to cancer pathogenesis.

As with our discussion of other ligand-gated ionotropic receptors (below), we turn to recent developments in P2XR “antagonism” for tumor therapy. While purinergic signaling is a more recent target for chemotherapeutics, the targeting of purine/purinergic incorporation into rapidly dividing cancer cells has been a highly utilized cancer therapeutic mechanism. For example, the 5-Fluorouracil (5-FU) prodrug, capecitabine, both incorporate incorrect fluoronucleotides into RNA and DNA while simultaneously inhibiting the action of thymidylate synthase (Longley et al., 2003). By this mechanism of action, rapidly dividing cancer cells are preferentially killed. Returning to purinergic signaling, much of the literature surrounding P2XR “antagonism” concerns abrogation of the P2X₇R subtype. In a rat model of C6 glioma, pharmacological inhibition of P2X₇R by brilliant blue G (BBG) inhibited tumor growth (Ryu et al., 2011). Mohammed et al. showed that the use of the P2X₇R antagonists, A438079 and AZ10606120, reduced caspase-3, caspase-1, p21, and Cdc25c in a mouse model of pancreatic cancer which resulted in decreased cancer proliferation (Mohammed et al., 2017; Zhang et al., 2021). Similarly, B16 melanoma cells and B16 melanoma-bearing mice when treated with oxidized ATP (purinergic receptor inhibitor) displayed decreased tumor cell proliferation (Hattori et al., 2012). However, one must also weigh the anti-immune effects of P2X antagonism when considering these anti-tumorigenic effects.

While having anti-tumorigenic potential, purinergic therapeutics also may be used in symptom management. Different types of pain are associated with P2XR activation. For example, the P2X₃R subtype expressed on sensory neurons has been associated with the transmission of neuropathic pain (Bele and Fabbretti, 2015). Similarly, inhibition of the P2X₄R subtype in spinal cord microglia through intrathecal injection of bone marrow stromal cells attenuated neuropathic pain in a murine model (Ulmann et al., 2008; Teng et al., 2019). The mechanism of this nociception is thought to be activation of P2X₄R on microglia by afferent neurons leading to MAPK signaling, which in turn leads to the synthesis and excretion of brain-derived neurotrophic factor (BDNF) (Ulmann et al., 2008; Trang et al., 2009).

A specific subtype of cancer-related pain is cancer-induced bone pain (CIBP) which represents a significant burden on the quality of life. CIBP is the product of metastasis of non-bone tumors to the bone resulting in debilitating pain. Interestingly, antagonism of the P2X_2/3R by minodronic acid, a third-generation bisphosphonate, resulted in an analgesic effect in a murine model of CIBP as measured by nociceptive behaviors (Kakimoto et al., 2008).

Particularly unique to the discussion of the glutamatergic superfamily is the fact that the ligand, glutamate, is not only implicated as an extracellular messenger but also as a key intracellular modulator of tumor proliferation. Otto Warburg noticed that cancer cells take up glucose at almost a tenfold rate compared to normal cells and shunt glucose through the anaerobic pathway to produce lactate (e.g., the Warburg effect) (Koppenol et al., 2011; Liberti and Locasale, 2016). As glucose is frequently shunted to glutamate, it is natural that increased glutamate levels in various cancers correlate to poorer prognoses. For example, in prostate cancer, glutamate concentrations directly correlated with advanced Gleason scores, a clinical prostate staging system (Koochekpour et al., 2012). Researchers are also analyzing whether glutamate levels can be used as a diagnostic approach in breast cancers due to a similar logic as in pancreatic cancers (Budczies et al., 2015). The tumor microenvironment generates high concentrations of ROS. Basal levels of ROS lead to cancer proliferation, whereas high levels of ROS induce a cytotoxic response (LeBoeuf et al., 2020). Cancer cells co-opt the elevated levels of glutamate to produce glutathione which can act as a “sponge” of ROS to maintain the optimal range of ROS concentration, allowing for cancer growth (Hayes et al., 2020).

Seizures are one of the most common presenting features of CNS tumors. It is thought that the release of glutamate from the tumor microenvironment (TME) via a cystine-glutamate exchanger (SLC7A11, xCT) contributes to this clinical presentation (Sørensen et al., 2018). It has been shown that high levels of glutamate induce seizures through its excitatory effect (Buckingham et al., 2011). Perampanel, a non-competitive AMPAR antagonist, has already been established to treat patients with seizures (Lange et al., 2021). Therefore, it can be easily translated to cancer-induced seizures with the potential added benefit of inhibiting tumor progression through the aforementioned mechanisms. Aside from directly antagonizing the iGluRs, SLC7A11/xCT can be antagonized through sulfasalazine (cystine-glutamate exchanger antagonist) which reduces extracellular glutamate release, thereby decreasing seizure incidence (Figure 3; Verbruggen et al., 2021). An additional benefit of this therapeutic modality is that there is potential for a synergistic anti-tumor effect, treating the cause and symptoms simultaneously. Highlighting this point, SLC7A11 antagonism caused synthetic lethality in KRAS-mutant lung adenocarcinoma (Hu et al., 2020).

Glutamatergic signaling has also been shown to play a critical role in the peripheral sensitization and mechanical hypersensitivity associated with CIBP. Zhu et al. (2020) showed that glutamate injection-induced neuronal excitation in dorsal root ganglia (DRG) is similar to that shown to be induced by CIBP. In a breast cancer model of CIBP, sulfasalazine reduced nociceptive perception and delayed onset of behavioral pain signs (Ungard et al., 2014; Ellingson and Vanderah, 2020; Zhu et al., 2020). Furthermore, in a rat model of CIBP, direct inhibition of NMDAR in the spinal cord led to nociceptive relief (Dai et al., 2017). The proposed mechanism of action was through reduced phosphorylation of PKCγ and ERK1/2 in the spinal cord (Dai et al., 2017).

GABA_AR subunits expression is enhanced in both CNS and systemic cancers, including pediatric and adult brain tumors (Cho et al., 2011) and gastric, pancreatic, ovarian, and breast cancers (Hujber et al., 2018; Jiang et al., 2020). In addition, GABA_ARs are not only significantly upregulated in medulloblastoma, a pediatric brain cancer, and melanoma but also are a functional GABA-responsive receptor (Sengupta et al., 2014; Pomeranz Krummel et al., 2021). The putative pathway of GABA in the proliferation of these cancers has yet to be fully elucidated, but one prevailing theory is that the lack of a GABA switch with subsequent retention of a depolarizing phenotype in many cancers allows for pro-tumorigenic pathways to be upregulated. In support of this theory is research done on pancreatic cancer cells which showed that enhancing GABA_AR activity with an agonist-induced a Ca²⁺ influx, resulting in subsequent MAPK/ERK pathway activation (Takehara et al., 2007). This MAPK/ERK activation was reduced by the administration of a calcium chelator, further implicating calcium in this pathway. Aside from inducing proliferation, GABAergic signaling and metabolism may aid tumor metastases to survive in the brain microenvironment. Neman et al. (2014) showed that breast-to-brain metastatic cells displayed increased GABA metabolism and a strong association between HER2 and reelin, a protein expressed by GABAergic neurons, implicating this phenotype with control over cell motility and cytoskeletal shape. These studies imply that a hyperactivation of GABA_AR may be partially responsible for a tumorigenic phenotype.

Conversely, other research has shown that GABA agonism may inhibit cancer proliferation and growth which complicates the therapeutic landscape given the discrepancy with the aforementioned literature. GABA signaling has been implicated in the maintenance of embryonic (ESC) and neural stem cells (NSC) in the stem cell niche, preventing proliferation (Wang et al., 2008). Paradoxically, this mechanism is found to be a hyperpolarizing effect of GABA_AR despite occurring in stem cells, which presumably have not undergone a GABA-switch (Wang et al., 2008). GABA_AR is thought to activate S-phase checkpoint regulatory kinases such as PIKK which in turn phosphorylate the histone H2AX, correlated with decreased proliferation in ESC and NSC cells (Andäng et al., 2008). This mechanism may provide an explanation as to why in human hepatocellular carcinoma, prostate cancer, and some brain tumors the downregulation of GABA_AR allows for increased tumor proliferation (Young and Bordey, 2009).

Given GABA’s documented role in proliferation and stem cell maintenance, is the GABAergic system a possible target for anti-tumor activity? GABA_ARs are already a highly targeted neurotransmitter receptor subtype for the treatment of anxiety, insomnia, and epilepsy. Although benzodiazepines have received “bad press” as of late, due to their addictive potential (Oransky, 2005). An epidemiological study conducted by Kleinerman et al. revealed that patients on diazepam fared better, e.g., exhibited smaller invasive tumors and less lymph node involvement, than patients not taking the anxiolytic (Kleinerman et al., 1984). This retrospective study inspired our lab to explore the mechanism of benzodiazepine action in cancer cells. Benzodiazepine treatment in medulloblastoma and melanoma cells and respective murine models for these cancers impairs viability via mitochondrial depolarization and the intrinsic (mitochondrial) apoptotic pathway (Figure 4; Sengupta et al., 2014; Jonas et al., 2016; Kallay et al., 2019; Pomeranz Krummel et al., 2021). This anti-proliferative effect of a positive allosteric modulator, a benzodiazepine, is consistent with the earlier discussion of GABA_AR and the maintenance of the stem cell niche. Given that benzodiazepines are often used as an anxiolytic during radiation treatment for a multitude of tumors, having this as a potential anti-cancer therapeutic in the “clinicians’ toolbox” would be valuable.

Importantly, benzodiazepines not only alone can impair the viability of cancer cells but also appear capable of sensitizing the cancer cells to radiation, chemotherapeutic, and an immune checkpoint inhibitor. If benzodiazepines can reduce the dose necessary to achieve the efficacy of these treatments, this may represent an approach to improve quality of life or reduce patient co-morbidities, because of these treatments. Underlying the basis for such sensitization by modulating the GABA_AR may be due to an interaction of the receptor with an intracellular protein called GABARAP or GABA Type-A Receptor-Associated Protein (Pérez-Hernández et al., 2019). GABARAP has been reported to be an important contributor to the autophagosomes mediating autophagy, the process by which lysosomes degrade and recycle damaged organelles as a means for cells to control proliferation and the formation of radical inorganic species (Liu et al., 2016). This provides a putative mechanism linking the activation of GABA_AR to the autophagy pathway.

Another potential pathway by which GABAergic signaling can influence tumor cell death is by modulating cells of the immune system. GABA_ARs appear to have roles in many immune cell subtypes, including T-cells, dendritic cells, macrophages, and neutrophils (Jin et al., 2013; Bhattacharya et al., 2021). Moreover, several immune cells have the capability of synthesizing and secreting GABA, including T-cells, macrophages, and dendritic cells (Bhat et al., 2010; Dionisio et al., 2011).

Treatment of cancer comes with a host of constitutional symptoms that significantly impact the quality of life of patients, including anxiety, insomnia, bone pain, and chemotherapy-induced nausea or vomiting (Triozzi et al., 1988). Anxiety may arise from the overall prospect of the disease itself or context-specific situations, such as radiotherapy. Benzodiazepines are commonly used as an anxiolytic during events such as radiation therapy. Bone pain can present as a consequence of a cancer metastasis. Nausea and vomiting are common side effects of chemotherapeutics. Regardless of the origin of symptoms, benzodiazepines and other GABA_AR modulators are useful therapeutics, as they simultaneously treat all of the above symptoms.

Work in a rat model of CIBP found that GABA was downregulated in CIBP rats in combination with the upregulation of GABA transporter type-1 (GAT-1) in spinal cord astrocytes (Ge et al., 2019). Interestingly, administration of exogenous GAD and NO-711 (a GAT-1 specific-inhibitor) significantly reversed CIBP-induced mechanical allodynia (Ge et al., 2019). This suggests that the upregulation of GABAergic signaling has the potential to be an analgesic in CIBP. Moreover, given the effects that GABA agonism has been shown to have on tumor regression by Sengupta et al. (2014) this suggests a dual benefit to GABA treatment for cancer—addressing the symptoms and root cause simultaneously. This treatment must also be contextualized in light of the research by Neman et al. (2014) and Takehara et al. (2007) which implicated GABA signaling in tumor proliferation. Likely tissue-specific factors will determine the efficacy of GABA agonism or antagonism therapy.