Involvement of the Catecholamine Pathway in Glioblastoma Development

Abstract

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor-ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2-ADRBK1/GRK2; dopamine receptor D2-DRD2; and synaptic vesicle monoamine transporter-SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary-recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

Keywords: DNA CpG methylation; catecholamine pathway; gene expression; sequential glioblastoma.

Figure 1

Representative staining for each marker. This figure shows representative images of the IHC preparations with the four selected markers, namely, ADRA1D, ADRBK1, DRD2 and SLC18A2, from the controls and a GBM sample (sample numbers 315 vs. 16534; 7442 vs. 16534; 7244 vs. 16534; 7166 vs. 16534, respectively. The GBM sample was a recurrent GBM).

Figure 2

IHC complex score (CS) values of the markers in HC, GBM1 and GBM2. This figure shows the median and interquartile range values of the IHC complex scores (CSs) of selected markers in the healthy control (HC), GBM1 and GBM2 groups. The Wilcoxon singed rank test (GBM1 vs. GBM2) and Mann–Whitney-U test (HC vs. GBM1 or GBM2) p-values are indicated (* p < 0.05, ** p < 0.01).

Figure 3

Promoter and gene methylation levels of the four markers in MC, GBM1 and GBM2. This figure presents the median and interquartile range values of the DNA CpG methylation levels in the promoters and genes of the selected markers in the methylation control (MC) (from Klughammer et al. 2018 [50]) and the GBM1 and GBM2 groups. The Wilcoxon singed rank test (GBM1 vs. GBM2) and Mann–Whitney-U test (HC vs. GBM1 or GBM2) p-values are indicated (* p < 0.05, ** p < 0.01).

Figure 4

Promoter and gene methylation levels of the four markers in the database sequential GBM cohort. This figure depicts the median and interquartile range values of the DNA CpG methylation levels in the promoters and genes of the selected markers in a database of 112 sequential GBM pairs (Klughammer et al. 2018 [50]).

Figure 5

Involvements of the studied neurotransmitters in tumor development. This schematic figure illustrates as to how the signal transduction pathways of catecholamines and their associated receptors contribute to tumorigenesis. (A) Signaling pathway of dopamine via DRD2; (B) signaling pathway of norepinephrine or epinephrine via ADRA1D; and (C) interaction of GRKs with GPCRs and non-GPRCs to regulate cell biology and gliomagenesis. VMAT2 = vesicular monoamine transporter 2; GPCR = G protein-coupled receptor; GRK = G protein-coupled receptor kinase; DRD2 = dopamine receptor D2; ADRA1D = alpha-1D adrenoreceptor.