The Endocannabinoid System as a Target in Cancer Diseases: Are We There Yet?

The endocannabinoid system (ECS) has been placed in the anti-cancer spotlight in the last decade. The immense data load published on its dual role in both tumorigenesis and inhibition of tumor growth and metastatic spread has transformed the cannabinoid receptors CB1 (CB1R) and CB2 (CB2R), and other members of the endocannabinoid-like system, into attractive new targets for the treatment of various cancer subtypes. Although the clinical use of cannabinoids has been extensively documented in the palliative setting, clinical trials on their application as anti-cancer drugs are still ongoing. As drug repurposing is significantly faster and more economical than de novo introduction of a new drug into the clinic, there is hope that the existing pharmacokinetic and safety data on the ECS ligands will contribute to their successful translation into oncological healthcare. CB1R and CB2R are members of a large family of membrane proteins called G protein-coupled receptors (GPCR). GPCRs can form homodimers, heterodimers and higher order oligomers with other GPCRs or non-GPCRs. Currently, several CB1R and CB2R-containing heteromers have been reported and, in cancer cells, CB2R form heteromers with the G protein-coupled chemokine receptor CXCR4, the G protein-coupled receptor 55 (GPR55) and the tyrosine kinase receptor (TKR) human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2). These protein complexes possess unique pharmacological and signaling properties, and their modulation might affect the antitumoral activity of the ECS. This review will explore the potential of the endocannabinoid network in the anti-cancer setting as well as the clinical and ethical pitfalls behind it, and will develop on the value of cannabinoid receptor heteromers as potential new targets for anti-cancer therapies and as prognostic biomarkers

NCS-1 associates with adenosine A2A receptors and modulates receptor function

Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM—the A2A adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A2A—NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signaling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signaling

Heteromerization between α2A adrenoceptors and different polymorphic variants of the dopamine D4 receptor determines pharmacological and functional differences. Implications for impulsive-control disorders.

Polymorphic alleles of the human dopamine D4 receptor gene (DRD4) have been consistently associated with individual differences in personality traits and neuropsychiatric disorders, particularly between the gene encoding dopamine D4.7 receptor variant and attention deficit hyperactivity disorder (ADHD). The α2A adrenoceptor gene has also been associated with ADHD. In fact, drugs targeting the α2A adrenoceptor (α2AR), such as guanfacine, are commonly used in ADHD treatment. In view of the involvement of dopamine D4 receptor (D4R) and α2AR in ADHD and impulsivity, their concurrent localization in cortical pyramidal neurons and the demonstrated ability of D4R to form functional heteromers with other G protein-coupled receptors, in this study we evaluate whether the α2AR forms functional heteromers with D4R and weather these heteromers show different properties depending on the D4R variant involved. Using cortical brain slices from hD4.7R knock-in and wild-type mice, here, we demonstrate that α2AR and D4R heteromerize and constitute a significant functional population of cortical α2AR and D4R. Moreover, in cortical slices from wild-type mice and in cells transfected with α2AR and D4.4R, we detect a negative crosstalk within the heteromer. This negative crosstalk is lost in cortex from hD4.7R knock-in mice and in cells expressing the D4.7R polymorphic variant. We also show a lack of efficacy of D4R ligands to promote G protein activation and signaling only within the α2AR-D4.7R heteromer. Taken together, our results suggest that α2AR-D4R heteromers play a pivotal role in catecholaminergic signaling in the brain cortex and are likely targets for ADHD pharmacotherapy

Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation

Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes

Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders

The endocannabinoid system as a target in cancer diseases: are we there yet?

The endocannabinoid system (ECS) has been placed in the anti-cancer spotlight in the last decade. The immense data load published on its dual role in both tumorigenesis and inhibition of tumor growth and metastatic spread has transformed the cannabinoid receptors CB1 (CB1R) and CB2 (CB2R), and other members of the endocannabinoid-like system, into attractive new targets for the treatment of various cancer subtypes. Although the clinical use of cannabinoids has been extensively documented in the palliative setting, clinical trials on their application as anti-cancer drugs are still ongoing. As drug repurposing is significantly faster and more economical than de novo introduction of a new drug into the clinic, there is hope that the existing pharmacokinetic and safety data on the ECS ligands will contribute to their successful translation into oncological healthcare. CB1R and CB2R are members of a large family of membrane proteins called G protein-coupled receptors (GPCR). GPCRs can form homodimers, heterodimers and higher order oligomers with other GPCRs or non-GPCRs. Currently, several CB1R and CB2R-containing heteromers have been reported and, in cancer cells, CB2R form heteromers with the G protein-coupled chemokine receptor CXCR4, the G protein-coupled receptor 55 (GPR55) and the tyrosine kinase receptor (TKR) human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2). These protein complexes possess unique pharmacological and signaling properties, and their modulation might affect the antitumoral activity of the ECS. This review will explore the potential of the endocannabinoid network in the anti-cancer setting as well as the clinical and ethical pitfalls behind it, and will develop on the value of cannabinoid receptor heteromers as potential new targets for anti-cancer therapies and as prognostic biomarkers

Detection of Heteromers Formed by Cannabinoid CB 1

Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells

Orexin-Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking

Reversible photocontrol of dopaminergic transmission in wild-type animals

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a time scale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illuminationdependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wildtype animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes

Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist-antagonist binding modulation

Many G-protein-coupled receptors (GPCRs) are expressed on the plasma membrane as dimers. Since drug binding data are currently fitted using equations developed for monomeric receptors, the interpretation of the pharmacological data are equivocal in many cases. As reported here, GPCR dimer models account for changes in competition curve shape as a function of the radioligand concentration used, something that cannot be explained by monomeric receptor models. Macroscopic equilibrium dissociation constants for the agonist and homotropic cooperativity index reflecting the intramolecular communication within the dopamine D1 or adenosine A2A receptor homodimer as well as hybrid equilibrium dissociation constant, which reflects the antagonist/agonist modulation may be calculated by fitting binding data from antagonist/agonist competition experiments to equations developed from dimer receptor models. Comparing fitting the data by assuming a classical monomeric receptor model or a dimer model, it is shown that dimer receptor models provide more clues useful in drug discovery than monomer-based models