Whittier et al. Acta Neuropathologica Communications 2013, 1:66 http://www.actaneurocomms.org/content/1/1/66 RESEARCH Open Access G-protein coupled receptor expression patterns delineate medulloblastoma subgroups Kelsey L Whittier1,2, Erin A Boese1, Katherine N Gibson-Corley4, Patricia A Kirby4, Benjamin W Darbro1, Qining Qian1, Wendy J Ingram5, Thomas Robertson6, Marc Remke7, Michael D Taylor7 and M Sue O’Dorisio1,2,3* Abstract Background: Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results: Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions: Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors. Keywords: Medulloblastoma subgroups, G-protein coupled receptors, Therapeutic targets, Imaging targets Background that are currently offered can result in physical, neuro- Medulloblastoma is an embryonal tumor of the cerebel- logical and intellectual disabilities [1-3]. Historically, me- lum that accounts for 20% of all pediatric brain tumors dulloblastoma tumors have been treated according to a and is the most common cause of death from CNS ma- morphology-based classification system that divides tu- lignancy in children [1]. Furthermore, survivors face a mors into three principle histopathologic classes: classic, multitude of long-term sequelae secondary to treatment; desmoplastic/nodular and large cell/anaplastic (LCA) exposing a developing brain to the cytotoxic therapies [4]. The histopathological class informs prognosis, for example tumors displaying LCA morphology generally * Correspondence: [email protected] have the worst prognosis [4]. However, recent advances 1Department of Pediatrics, Carver College of Medicine, University of Iowa, have utilized genetic profiling to classify medulloblas- Iowa City, IA 2524 JCP, USA toma tumors and these techniques have identified me- 2Neuroscience Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, IA, USA dulloblastoma subgroups that differ in both molecular Full list of author information is available at the end of the article and clinical profiles [5-7]. Various groups have identified © 2013 Whittier et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Whittier et al. Acta Neuropathologica Communications 2013, 1:66 Page 2 of 15 http://www.actaneurocomms.org/content/1/1/66 between four and five potential subgroups [6,8-13]; how- While the molecular expression patterns of many ever, a recent consensus conference determined that genes and proteins in medulloblastoma subgroups have evidence supported four distinct subgroups and acknowl- been discerned, subgroup-specific GPCR expression pat- edged the potential for multiple subtypes within each terns have not previously been investigated. A subset of subgroup [7]. The two most well-defined subgroups are GPCRs appear on commonly used gene chips, such as the characterized by overactive signaling in the WNT and Affymetrix U133 chip; however these chips do not allow Sonic hedgehog (SHH) mitogenic pathways. Less is known for the detection of under-expressed genes [29,30]. Our about the underlying tumorigenesis mechanisms of the approach, using quantitative GPCR arrays (Taqman), al- remaining two tumor subgroups, Group 3 and Group 4; lows for the assessment of both over- and under-expressed however, specific genetic aberrations and gene expression GPCRs. characteristics have been found, and epigenetic origins to The aim of this study was to discover G-protein coupled these tumors have been proposed [14-16]. These four receptors that could serve as targets for imaging and principle medulloblastoma subgroups differ in terms of therapeutic agents in medulloblastoma, and we have suc- demographics, predominant histology, likely cell of origin, cessfully identified potential receptor targets. Elucidating DNA copy number aberrations and molecular markers tumorigenic and potentiating mechanisms in medulloblas- [5,7,17]. Importantly, the genetic profile has prognostic sig- toma subtypes has been a secondary benefit to our study. nificance leading investigators to urge translation of genetic classification into clinical therapeutic trials [7,14,18,19]. Methods Tumors of the WNT subgroup have the most favorable Human tumor cohort outcomes and SHH tumors have an intermediate response Tumors analyzed for GPCR expression consisted of snap- to current therapies. The recent development of small mol- frozen tumor tissues from 41 medulloblastomas, repre- ecule inhibitors of the SHH pathway holds promise for the senting primary surgical resection tissue. Normal pediatric treatment of these tumor subgroups [20,21]. Group 3 tu- cerebellum was used as control tissue. Both specimen mors appear to have the worst prognosis using current types were acquired from the Cooperative Human Tissue therapeutic approaches [5]; however, Groups 3 and 4 are Network (Columbus, OH), The Queensland Children’s less well-characterized, both clinically and genetically, Tumour Bank (Queensland, AUS), The Children’s Cancer resulting in a lack of potential targets that has hin- Research Unit at the Children’s Hospital at Westmead dered the development of novel therapeutic strategies. (Westmead, AUS), the Knight Cancer Institute Biolibrary Identification of tumor subgroup using molecular classifi- at Oregon Health and Sciences University (Portland, OR) cation is expected to become an important component of and from patients of the University of Iowa Hospitals and medulloblastoma diagnosis and staging in the near future. Clinics (UIHC) Children’s Hospital. Basic clinical data in- Molecular classification will also likely be used to guide cluding age and sex were also obtained. The histopatho- therapeutic options, to measure response to therapy and to logical reports were acquired with the majority of tumor provide early detection of relapse. samples and more extensive pathology reports including G-protein coupled receptors (GPCRs) are key regula- cytogenetics were available for some patients. UIHC speci- tors and points of control in both the SHH and WNT mens were acquired under an Institutional Review Board signal transduction pathways, as well as many other cell (IRB) approval. Specimens acquired from other sources signaling mechanisms [22]. GPCRs possess characteris- were de-identified and use of these tissues was declared tics that make them ideal targets for molecular imaging “Not Human Research” by the University of Iowa IRB. and therapeutics; including that they are membrane- bound, their ligands bind with high affinity and specificity, RNA isolation and GPCR expression arrays and that the receptor-ligand complex is subsequently RNA was isolated from snap-frozen tumor tissue using endocytosed carrying the ligand into the tumor cell [23]. the PerfectPure RNA Tissue Kit (5Prime); the quantity The utility of targeting GPCRs in medulloblastoma has and quality of RNA was evaluated using a Nanodrop been demonstrated with the advent of somatostatin recep- 1000 Spectrophotometer (Thermo Fischer Scientific) tor targeted imaging and therapy [24,25] and Octreoscans and an Agilent 2100 Bioanalyzer. RNA of sufficient qual- are now able to differentiate medulloblastoma from low- ity was defined as having an RNA Integrity Number grade cerebellar