The primary goal of this project was to identify G-protein coupled receptors that could serve as targets for imaging and therapeutic agents in medulloblastoma, and this has been successful. GPCR expression patterns also have the potential to elucidate initiating and proliferative mechanisms. The results of this study indicate that GPCR expression patterns delineate five groups of medulloblastoma tumors, two of which correlate with high fidelity to the WNT and the SHH subgroups of medulloblastoma [5, 7, 10, 14, 17, 18, 32]. Distinct GPCRs are uniquely over-expressed in the WNT and SHH subgroups, as well as in three other groups of tumors, strongly suggesting that GPCR targets specific to each medulloblastoma subgroup can be identified. Additionally, these data indicate that the unique GPCR expression patterns found may help clarify important mechanistic differences between the groups.
Development of new, or utilization of known ligands to uniquely over-expressed GPCRs offer the potential to provide patient specific information. Subgroup targeted imaging, using radiolabeled GPCR ligands, would offer a non-invasive method to simultaneously diagnose medulloblastoma and characterize molecular subgroups. Importantly, targeted imaging would also afford a sensitive technique for follow-up imaging to determine response to therapy and the presence of metastatic lesions. A benefit in developing imaging agents targeting over-expressed receptors is that the downstream action of the targeted receptor need not correspond to specific tumorigenesis mechanisms. Rather, the viability of an imaging agent is dependent on the following criteria: high affinity and specificity for the target receptor, reliably and highly differential expression of the target receptor between tumor and normal tissue, limited off-target or off-tissue effects, and size constraints. In regard to medulloblastoma, the ability to cross the blood–brain barrier is also crucial. The utility of such a GPCR-targeted imaging modality has already been proven: The Octreoscan, which employs SPECT imaging to detect radiolabeled somatostatin receptor analogues has the ability to differentiate medulloblastoma from low-grade cerebellar tumors and provides an imaging modality to differentiate recurrent medulloblastoma from scar tissue, as well as to localize metastatic lesions [24, 25, 28]. Positron emission tomography (PET) imaging provides a higher degree of sensitivity than SPECT imaging  and somatostatin receptor targeted agents are being adapted for use with PET imaging . Recent evidence has shown that high expression of somatostatin receptors, particularly in non-SHH subgroup tumors, is correlated to an increased survival and may have potential as a prognostic marker . However, previous studies report that somatostatin receptor expression is uniformly high in all medulloblastoma tumors [37, 38]. Though these studies were performed prior to the advent of medulloblastoma subgrouping, they indicate that somatostatin receptor targeted agents will fail to distinguish between the subgroups of medulloblastoma. Somatostatin receptor, type 2, expression trends towards over-expression in all subgroups, however does not reach significance in our current data set (WNT subgroup: 7.6-fold, p = 0.24; SHH subgroup: 5.1-fold, p = 0.34; Non-WNT/SHH: 8.1-fold, p = 0.23).
GPCR-targeted therapeutics, either radioablative or chemically based, have the potential to reduce the need for external beam radiation, and the highly toxic effects associated with cranial spinal radiation treatment . Therapeutically, GPCR antagonists represent the most instinctive approach to counteracting the proliferative signals transduced by some GPCRs within the context of cancer . As such, GPCR antagonists have previously been investigated as potential chemotherapeutic targets in a variety of malignancies . Our data indicate that drugs targeting specific GPCRs may not display the same efficacy in all medulloblastoma tumors, and that subgroup specific GPCR targets would likely result in more beneficial outcomes. GPCR targeted radiotherapy, in which an isotope, such as lutetium-177 or yttrium-90, is attached to a receptor ligand, antibody or other molecule in order to target the radioactivity to the desired GPCR, has been successful in the treatment of neuroendocrine tumors and other malignancies [40, 41].
While each of the differentially expressed GPCRs in our data set deserves active investigation into its potential as a target, several candidate receptors are outlined below. Twenty-six GPCRs exhibited significantly altered expression levels in the WNT subgroup tumors; thirteen of these were unique to the WNT subgroup, and eight of these thirteen displayed overexpression, as compared to normal cerebella, and thus are candidate receptors for targeting. Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) is significantly over-expressed in the WNT subgroup of tumors (120-fold, p =0.01); additionally, our data suggest that it is mildly under-expressed in other groups of medulloblastoma, providing the benefit of being highly differentially expressed not only between WNT tumors and normal cerebella, but also between WNT tumors and other medulloblastoma groups (Additional file 3: Table S2; Figure 3). Furthermore, LGR5 holds the intuitive advantage of being involved in the WNT signaling pathway; LGR5, a known marker of certain adult stem cells, forms a complex with Frizzled/LRP and acts to potentiate the WNT signal . The role of LGR5 in cancer biology has been well-described, especially in the realm of gastrointestinal cancers, where antibodies to LGR5 can be used to identify colorectal cancer stem cells [43, 44]. Recently, R-spondins, a class of four large, secreted proteins known to enrich WNT signaling, have been identified as high affinity ligands for LGR5 (and LGR4) . R-spondin proteins hold limited potential as imaging or therapeutic agents in medulloblastoma due to their large size (35 kDa)  and likely inability to cross the blood–brain barrier [47, 48]. However, the emergence of high-throughput screening facilities provides the resources to potentially identify key binding elements of the R-spondin proteins. Our data suggest that the development of small-molecule agents targeting the LGR5 receptor is worthy of attention.
GPR64 displays a favorable differential expression profile for a WNT subgroup target (2200-fold, p = 0.04; Additional file 3: Table S2, Figure 3). GPR64 is an orphan receptor that belongs to a family of adhesion proteins and is normally highly expressed only in the epididymis; however it has recently been found to be expressed in Ewing’s sarcoma (ES), as well as other carcinomas, and represents a marker of invasiveness and metastatic potential in ES . The exact signaling mechanism that follows GPR64 activation is yet unknown and a direct connection between GPR64 and WNT signaling is not readily apparent; however, the development of imaging and radiotherapeutic targets is not dependent on the role of downstream mechanisms in proliferation or apoptosis. Due to its differential expression in medulloblastomas, as well as the fact that it is normally only expressed in the epididymis, GPR64 represents a promising candidate for the development of imaging or radiotherapeutic agents that could be potentially efficacious not only in WNT subgroup medulloblastomas, but also Ewing’s sarcoma.
PTGER4 is a GPCR that was uniquely over-expressed (16-fold, p = 0.02) in the SHH group of tumors (Additional file 3: Table S2). It was also over-expressed in “Cluster C” GPCR-grouped medulloblastomas (5.66-fold, p = 0.01; Table 1), however these tumors fell into the Non-WNT/SHH subgroup and the same pattern of PTGER4 expression was not seen in that subgroup as a whole. PTGER4, or EP4, is a receptor for prostaglandin E2 (PGE2). PGE2 has been shown to act as a growth promoting molecule that stimulates proliferation, angiogenesis and invasion , and is present at high levels in a variety of malignancies [51, 52]. Furthermore, the role of PGE2, and its receptors, has been investigated within the context of medulloblastoma . PGE2 induces medulloblastoma cell proliferation in vitro, while inhibition of PGE2 activity was suppressive both in vitro and in vivo
. While Baryawno and colleagues  found that PGE2 receptors EP1-3 were most important in stimulating medulloblastoma cell growth, our data suggest that tumor subgrouping may affect PGE2’s role. Small molecule antagonists to EP4 are currently in development for the treatment of inflammatory pain ; EP4 represents a particularly viable therapeutic target, as blockage at this site does not interfere with the production of other important prostanoids, and thus avoids the cardiovascular side effects that can be seen with blockage of this pathway . EP4 represents a viable potential target in medulloblastoma, a possibility that is furthered by the fact that inhibition of the prostaglandin cascade has been shown to enhance the cytotoxic effects of radiotherapy  presenting the possibility of synergistic combination therapy. Interestingly, PGE2 has been shown to potentiate the WNT signaling cascade, both in colorectal cancer cells , as well as in normal adult hematopoietic stem cells , and it was recently found that PGE2 upregulates LGR5 . This finding highlights the important crosstalk between the WNT and SHH signaling cascades .
While over-expressed GPCRs provide potential targets, their under-expressed counterparts are equally pertinent when probing unanswered mechanistic questions. GPCRs are responsible for initiating intracellular signaling for multiple pathways; these receptors act at the cell surface to integrate and coordinate diverse communicative stimuli between cells, and converge on shared downstream modulators and effectors. Identifying GPCRs down-regulated in medulloblastoma subgroups may pinpoint receptors critical for growth suppression or inhibition, whose under-expression can lead to, or potentiate, the development of cancer. Less is known about the initiating mechanisms at play in Groups 3 and 4 medulloblastomas ; identifying differentially under-expressed GPCRs may help identify additional pathways that contribute to tumorigenesis in these subgroups. MTNR1A, a GPCR for melatonin, is significantly under-expressed only in the Non-WNT/SHH group of medulloblastoma tumors (0.0065-fold, p = 0.02). Melatonin has been postulated to be a tumor suppressor gene due to its oncostatic effect in various cancers [33, 57, 58], as such, expression of MTNR1A was found to be frequently silenced through methylation of CpG islands surrounding the MTNR1A promoter in cases of oral squamous cell carcinoma (OSCC) and other primary cancers . Furthermore, forced expression of MTNR1A in cells led to growth suppression, suggesting that loss of MTNR1A activity plays a role in the pathogenesis of OSCC ; similar results have also been found in breast cancer cell lines  and in prostate epithelial cells . The anti-proliferative effect observed in prostate epithelial cells was demonstrated to be due to MTNR1A-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) with a subsequent increase in p27 (kip1) gene transcription. The p27 gene encodes for cyclin-dependent kinase inhibitor 1B, a protein that prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, thus regulating cell cycle progression. A similar mechanism may be at play in Non-WNT/SHH medulloblastomas.
Another GPCR, the adenosine A1 receptor (ADORA1) has a known role in growth suppression [62, 63]. In colon cancer cells, adenosine, via ADORA1, induces apoptosis by activating caspases . Additionally, it has been reported that deletion of ADORA1 leads to an increase in glioblastoma tumor growth, however this observed effect was believed to be mediated through tumor-adjacent microglia . ADORA1 was under-expressed in the Non-WNT/SHH group (0.088-fold, p = 0.03), again suggesting that loss of ADORA1 activity may play a role in the pathogenesis of a Non-WNT/SHH medulloblastoma tumors, especially those seen in Cluster “E” (Table 1).
Our data identify GPCRs whose expression is significantly altered in subgroups of medulloblastoma; while many of these alterations reach significant levels, a limitation of our study was the restricted sample size available. To partially alleviate this concern, we worked with the Medulloblastoma Advanced Genomics International Consortium (MAGIC), an international consortium that aims to stratify and characterize medulloblastoma through genomics. Our key findings, specifically the over-expression of LGR5 and GPR64 in the WNT subgroup tumors and F2R and FZD2 in all medulloblastoma, were mirrored in three independent international cohorts of subgrouped medulloblastoma (Figures 3 and 4). Though our data cannot be quantitatively combined with these larger data sets, a qualitative comparison adds substantial confidence and weight to our results.