Non-THC cannabinoids, such as CBD, show a reduced affinity for the classical CBRs, acting as an antagonist or inverse agonist of CBRs, with an inhibitory constant (Ki) of 1458.5 ± 158.5 nM for CB1R and 372.4 ± 57.5 nM for CB2R [
71]. Therefore, it is unlikely that CB1R and CB2R play a significant role in the anti-cancer effects of CBD. Transient receptor potential vanilloid (TRPV) calcium selective ion channels are targets for cannabinoids [
72,
73] (discussed in
Section 3.2.1). The majority of reports highlight a role for non-THC cannabinoids as ligands for de-orphaned GPCRs, such as G-protein-coupled receptor 55 (GPR55) (discussed in
Section 3.2.2) and GPR119. However, GPR119 is only activated by eCBs analogues, including oleoylethanolamide and palmitoylethanolamide, rather than pCBs or sCBs. Therefore, GPR119 is not discussed in detail here, other than to outline that its agonism by specific ligands (such as MBX-2982 or GSK1292263), re-sensitises breast cancer cells that have developed resistance to gefitinib [
74]. Uniquely, CBD is a ligand for the orphan GPCRs, GPR3, GPR6 and GPR12 (discussed in
Section 3.2.3).
3.2.1. Transient Receptor Potential Channels of the Vanilloid Subtype–TRPV1/2
The transient receptor potential (TRP) superfamily of transmembrane ion channels TRPV1–TRPV4, and TRPA1 and TRPM8, are termed the ionotropic CBRs. The activation of TRPV1 by CBD, and of TRPV2 by CBD and ∆
9-THC, inhibits human glioma cell proliferation and viability in vitro [
58]. TRPV1 receptors regulate calcium influx to trigger apoptosis via the mitochondrial intrinsic and p38 MAPK-dependent pathways (
Figure 1) [
75]. TRPV1 also degrades EGFR (epidermal growth factor receptor), which is known to be overexpressed and activated in a variety of cancers; findings that highlight the tumour-suppressor capabilities of TRPV1, as well as identifying a role for CBD in cancer cell types where EGFR is overexpressed or mutated [
76]. Commensurate with the tumour-suppressor capabilities of TRPV1, the expression of
TRPV1 has been shown to be significantly decreased in the high-grade glioma, glioblastoma multiforme (GBM), thereby potentially reducing the therapeutic benefits of CBD in advanced patients [
75]. In addition to TRPV1, TRPV2 plays a role in regulating glioma cell survival and proliferation, with high levels of TRPV2 expression detected in benign astrocytes, with progressively less expression of TRPV2 in high-grade gliomas corresponding with histological grade [
77]. Indeed, the repression of
TRPV2 abundance in human glioma cell lines was in turn demonstrated to result in the increased expression of cyclin E1, cyclin-dependent kinase 2 (
CDK2), transcription factor E2F1 (
E2F1), RAF proto-oncogene serine/threonine-protein kinase (
RAF1) genes, and the anti-apoptotic gene, Bcl-xL (
BCL2L1) [
58]. Increased
cyclin E1,
CDK2,
E2F1,
RAF1 and
BCL2L1 gene expression reduced the abundance of the cell death receptors and of proteins encoded by apoptosis-related genes,
Fas and
procaspase-8, an expression change that enhanced cell survival and proliferation (
Figure 1). Interestingly, CBD has also been shown to enhance the cytotoxicity of certain chemotherapeutics via triggering TRPV2-dependent calcium (Ca
2+) influx, and this in turn increased the degree of chemotherapeutic uptake, while simultaneously reducing the incidence of chemoresistance in GBM cells. No such effect has been reported for normal human astrocytes (
Table 1) [
58]. A limitation of this study is, however, that parallel assessment of the TRPV2-dependent Ca
2+ influx and sensitisation to temozolomide (TMZ), carmustine (BCNU) or doxorubicin (DOXO) was not performed following ∆
9-THC treatment.
3.2.2. De-Orphaned G-Protein-Coupled Receptor 55
GPR55 is widely expressed in the mammalian brain with its abundance concentrated in large dorsal root ganglions (DRGs) [
78], and is also expressed in peripheral tissues such as the endocrine pancreas [
79]. GPR55 is a direct target of the pCBs, CBD and ∆
9-THC, the eCBs, AEA and 2-AG, and the sCB, JWH-015 [
63,
80]. The downstream effects of GPR55 agonism depend on the cannabinoid present. For example, ∆
9-THC, AEA, JWH-015, bind GPR55 to drive intracellular release of Ca
2+ in HEK293 cells, transiently transfected to express human GPR55 (GPR55-HEK293), with this process regulated by G protein, G
q/11-phospholipase C (PLC) [
77]. This effect can be abolished via the transient transfection of HEK293 cells with a dominant-negative mutant of
Gq/11, or via use of the CB1R/GPR55 antagonist, SR141716A [
78]. On the other hand, CBD, WIN-55,212–2 or 2-AG, all failed to increase the intracellular release of Ca
2+, with the CB2R antagonist, SR144528, also failing to reduce the degree of induction of JWH-015-mediated Ca
2+ flux in large DRG neurons. Additional GPR55-HEK293 transgenic studies showed that the inverse agonist of CB1R, AM251, and l-α-lysophosphatidylinositol (LPI; a ligand of GPR55), evoked GPR55-mediated Ca
2+ oscillation [
81]. The endogenous lysophospholipid, LPI, is a well-established mitogenic mediator of oncogenic signalling [
82,
83]. LPI binding to GPR55 drives the proliferation of cancer cells, often in an autocrine fashion (reviewed in [
84]), via the activation of the G protein-RhoA/Rho kinase (Rho/ROCK)-PLC signalling axis, resulting in intracellular Ca
2+ release from the endoplasmic reticulum (ER). This leads to the activation and subsequent nuclear translocation of the transcription factor NFAT (nuclear factor of activated T-cells), which in turn regulates the transcriptional activity of a number of genes [
81]. LPI-mediated Ca
2+ release was abolished using the Rho/ROCK inhibitor, Y-27632, or by transiently transfecting GPR55-HEK293 cells with a dominant-negative mutant of
RhoA [
81].
These ground-breaking studies have laid the foundations for future research characterising the role GPR55 plays in cancer cell proliferation and anchorage-independent growth [
85], angiogenesis [
86], migration [
87] and metastasis [
88,
89], with evidence that LPI-induced GPR55 activation plays a role in prostate, ovarian, GBM, breast, skin and pancreatic cancers (reviewed in [
84]). Indeed, LPI (10 μM) induced activation of GPR55 in prostate and ovarian cancer cell lines drives the rapid intracellular release of Ca
2+ to increase phosphorylation of the extracellular signal-regulated kinases, ERK and Akt [
85]. Topically, LPI-induced ERK signalling was reduced by pre-treating human ovarian and prostate cancer cell lines with CBD (3.0 μM/0.94 mg/L), or SR141716A (2.0 μM), the Rho/ROCK inhibitor Y-27632 (20 μM), or via the molecular inhibition of
GPR55 [
84]. Interestingly, unlike CBD and SR141716A treatments, the inhibition of Rho/ROCK did not reduce LPI induced Ca
2+ oscillation [
84]. This finding recognises an important role for CBD in cancer cell types characterised by high GPR55 activity.
Elevated GPR55 gene expression in breast cancer is associated with reduced disease-free survival, overall survival, and metastasis-free survival, with the highest levels of expression observed in aggressive basal/triple-negative breast tumours [
89]. Pro-metastatic properties in this context were exacerbated through an LPI-induced GPR55-G
q/11 coupling, and activation of RhoA, leading to actin cytoskeleton remodelling, changes in cell dynamics, and increased metastasis. Further, ERK activation in turn stimulates the expression of the transcription factor, ETV4/PEA3 [
89] (
Figure 1); a gene expression pattern associated with a higher risk of distant metastasis in basal/triple-negative breast cancers [
90]. Importantly, previous studies have shown that CBD (5 mg/kg) inhibits the growth of MDA-MB-231 breast cancer cells in vivo using xenograft mouse models [
13], once again highlighting a potential role for CBD in the treatment of GPR55 overexpression and aggressive cancer types.
Recently, using the KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), Ferro et al. demonstrated that GPR55 signalling could enhance pancreatic cancer cell growth and proliferation in vivo, via the activation of the mitogen-activated protein kinase (MAPK) signalling pathway (
Figure 1) [
91]. The knockdown of
GPR55, or its pharmacological inhibition using CBD, reduced ERK and ribosomal protein S6 phosphorylation, to decrease anchorage-dependent and -independent growth. In addition, inhibition of GPR55 using CBD decreased cyclin D1 and D2 expression and activated the tumour suppressor retinoblastoma (RB) to block cell cycle progression (
Figure 1) [
91]. The standard of care chemotherapy for pancreatic cancer centres on the use of the nucleoside analogue gemcitabine (GEM), which has previously been shown to increase ERK activation, a suggested mechanism of acquired resistance [
92]. GEM can act via inhibition of the enzyme ribonucleotide reductase 1 (RRM1), and resistance to GEM can also be associated with increased RRM1 and RRM2 expression (
Figure 1). The expression of both reductases however, was shown to reduce following CBD treatment in vitro [
91]. Importantly, treatment using CBD (100 mg/kg), to inhibit GPR55, in combination with GEM (100 mg/kg), improved the survival rate of a PDAC mouse model by a factor of three (mean 52.7 vs. 18.6 days, median 56 vs. 20 days), compared to the vehicle control, and also longer than mice given GEM alone (mean 52.7 vs. 27.8 days, median 56 vs. 23.5). Furthermore, this combined treatment approach, when applied in vitro and in vivo, was also demonstrated to counteract the development of resistance to GEM [
91], offering an exciting clinical prospect for the application of CBD in this setting.