Activation of Dopamine Receptor 2 Prompts Transcriptomic and Metabolic Plasticity in Glioblastoma

Abstract

Glioblastoma (GBM) is one of the most aggressive and lethal tumor types. Evidence continues to accrue indicating that the complex relationship between GBM and the brain microenvironment contributes to this malignant phenotype. However, the interaction between GBM and neurotransmitters, signaling molecules involved in neuronal communication, remains incompletely understood. Here we examined, using human patient-derived xenograft lines, how the monoamine dopamine influences GBM cells. We demonstrate that GBM cells express dopamine receptor 2 (DRD2), with elevated expression in the glioma-initiating cell (GIC) population. Stimulation of DRD2 caused a neuron-like hyperpolarization exclusively in GICs. In addition, long-term activation of DRD2 heightened the sphere-forming capacity of GBM cells, as well as tumor engraftment efficiency in both male and female mice. Mechanistic investigation revealed that DRD2 signaling activates the hypoxia response and functionally alters metabolism. Finally, we found that GBM cells synthesize and secrete dopamine themselves, suggesting a potential autocrine mechanism. These results identify dopamine signaling as a potential therapeutic target in GBM and further highlight neurotransmitters as a key feature of the pro-tumor microenvironment.SIGNIFICANCE STATEMENT This work offers critical insight into the role of the neurotransmitter dopamine in the progression of GBM. We show that dopamine induces specific changes in the state of tumor cells, augmenting their growth and shifting them to a more stem-cell like state. Further, our data illustrate that dopamine can alter the metabolic behavior of GBM cells, increasing glycolysis. Finally, this work demonstrates that GBM cells, including tumor samples from patients, can synthesize and secrete dopamine, suggesting an autocrine signaling process underlying these results. These results describe a novel connection between neurotransmitters and brain cancer, further highlighting the critical influence of the brain milieu on GBM.

Keywords: cancer stem cell; cellular plasticity; dopamine; glioblastoma.

Copyright © 2019 the authors 0270-6474/19/391982-12$15.00/0.

Figures

Figure 1.

Chemotherapeutic stress induces DRD2 expression in the patient-derived xenograft glioma lines. A, B, H3K27ac and H3K27me3 marks distribution around the transcription start sites of dopamine receptors following therapy. ChIP-Seq was performed for the open chromatin state H3K27 acetylation in PDX line GBM43 after 4 d of single exposure to TMZ (50 μm), or treated with one fractionated dose of radiotherapy (2 Gy). DMSO was used as a vehicle control. The red box in DRD2 highlights a key residue near the promoter that uniquely exhibited increased H3K27ac following TMZ treatment. (MACS2 peak score: 45.0, fold-enrichment: 4.023, p < 0.0001). Table summarizes statistical information for each of the five DRDs. Figure 1-1 also shows peak calling of all monoamine receptors. C, Western blot analysis of dopamine receptors expression in PDX cells treated with TMZ over 8 d. D, FACS analysis of DRD2 on different subtypes of PDX cells treated with vehicle control DMSO or TMZ (50 μm). Figure 1-2 includes analysis of all five DRDs demonstrating elevated DRD2 expression in human samples. Bars represent means from three independent experiments and error bars show the SD. Student t tests were performed for each day separately. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2.

Glioma initiating cells preferentially express DRD2 and specifically respond to receptor activation with hyperpolarization. A, Two different subtypes of PDX lines freshly isolated from animals were cultured in tumor sphere maintenance media (neurobasal media containing EGF and FGF) or differentiation media (DMEM containing 1% FBS) and subjected to immunoblot analysis for the expression of DRD2 and various GIC markers. B, PDX cells were subjected to FACS analysis for GIC marker CD133 and DRD2. Using controls, we established gates for CD133+ and CD133- populations. We then quantified the mean fluorescence intensity (MFI) for DRD2 expression in each of these populations. C, Human glioma U251 cells expressing RFP under the control of the CD133 promoter were patch-clamped and electrophysiological recordings were taken during exposure to highly selective DRD2 agonist. Puffing DRD2 agonist (30 nm; yellow) onto fluorescently labeled GIC (brightly labeled cell, left; red membrane potential trace, middle) induces a more robust hyperpolarization of the membrane potential (mV) than non-GICs exposed to the same agonist (nsGBM; non-labeled cell, left; blue trace, middle), or versus puffing vehicle control (n = 4 each). Graph: DRD2 agonist hyperpolarizes GIC more strongly than non-stem GBM cells (right). D, Electrophysiological recordings of PDX cells confirms that only certain cells respond to the agonist with hyperpolarization. Bars represent means from three independent experiments and error bars show the SD. Student t tests were performed for each day separately. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.

Activation of DRD2 signaling increases the self-renewal capacity of GBM cells. A, PDX GBM cells treated with either DMSO or TMZ were sorted based on DRD2 expression and neurosphere assays were performed. Using the extreme limiting dilution assay algorithm, frequency of GICs was calculated (DMSO, DRD2 + 1/458, and DRD2- 1/2091, p < 0.001; TMZ, DRD+ 1/196, and DRD2 − 1/515, p < 0.001). B, In vivo engraftment of 500 cells sorted on DRD2 ± expression demonstrates DRD2+ cells engraft more efficiently. Figure 3-1 includes more images of engrafted tumors from other mice. PDX lines treated with dopamine agonist demonstrate elevation in key stem cell genes via Western blot. Figure 3-1 includes Western blots from other PDX lines demonstrating similar trends. C, PDX GBM cells were treated with either DRD2 agonist (30 nm) or equimolar vehicle control DMSO and plated for neurosphere assays. Using the extreme limiting dilution assay algorithm, frequency of GICs was calculated [GBM6: DMSO stem cells 1/62 and agonist stem cells 1/29.2 (p < 0.01); GBM39: DMSO stem cells 1/181.9 and agonist stem cells 1/87.8 (p < 0.05); GBM5: DMSO stem cells 1/118.9 and agonist stem cells 1/67.4 (p < 0.05)]. Figure 3-1 includes a nonresponding GBM12 neurosphere assay. D, Two PDX lines were treated with either CPM (1 μm) or equimolar DMSO and plated for neurosphere assays as above. Fraction Nonresponding graph is shown for GBM 39. Difference between groups was determined by score test of heterogeneity. Error bars represent upper and lower limit of 95% confidence interval computed for stem-cell frequency. E, Viral knock-out of DRD2 using two different SH-RNA constructs in GBM43 demonstrates reduced stem-cell frequency. Student t tests were performed for each comparison. **p < .001, ****p < 0.0001.

Figure 4.

GBM cells synthesize and secrete dopamine. A, Western blot analysis of TH expression, the rate-limiting enzyme in dopamine synthesis. β-Actin was used as a loading control. B, PDX cells were implanted intracranially into athymic nude mice. Upon sickness from tumor burden, mice were killed and whole brains extracted. FACS analysis of HLA+ tumor cells demonstrates that GBM tumors continue to express TH in vivo. Each dot represents individual mice; FACS analyses were run in technical triplicates. Inset, Scatter plots are representative for FACS results in each cell line tested. C, Conditioned media from PDX GBM cells growing in purified monocultures were analyzed by HPLC-MS for dopamine. Unconditioned DMEM media with and without 1% FBS was used as a control. D, HPLC-MS was performed on GBM samples from patients undergoing resection. Cortical tissue from an epilepsy surgery was used as the control. E, PDX GBM6 cells were growing in either 1% FBS containing media or neurobasal media (NBM). Conditioned media were collected at various days and dopamine levels were calculated using HPLC. F, PDX GBM43 cells were cultured in the presence of either 20% or 1% oxygen (hypoxia) for 3 h. Conditioned media was collected, and dopamine levels determined. Dots represent values from each time point and/or sample tested. Figure 4-1 includes other genes involved in dopamine synthesis (VMAT2, DAT, DDC, TH) measured in patient tumor samples as well as HPLC performed on various PDX subtypes undergoing a TMZ treatment time course.

Figure 5.

DRD2 signaling activates hypoxia inducible factors in normoxic cultures. A, Correlation between DRD2 and 12042 genes from TCGA was determined by Pearson correlation coefficients. Forty-nine genes with coefficients >0.5 or <−0.5 and FDR <0.05 were selected. Genes fitting these parameters were then analyzed using Enrichr. Top hits are shown in the graph, with combined score and adjusted p value. B, PDX GBM cells were treated with DRD2 agonist (30 nm) for 1, 2, or 4 d and then probed for expression of HIF1α by immunoblot analysis. DMSO-treated cells as vehicle-treated control and cells exposed to hypoxic conditions (1.5% O2) were used as a positive control for HIF expression, and β-actin was used as a loading control. C, Western blots were used to analyze the level of proteins in the VHL–PHD regulatory pathway after treatment with either DMSO or DRD2 agonist (20 or 30 nm). β-Actin was used as a loading control. D, PDX GBM6 were exposed to DRD2 agonist (30 nm) and treated with HIF inhibitor for 24, 48, and 72 h; SOX2, MMP2, and CMYC protein levels were assayed.

Figure 6.

DRD2 triggers alterations in gene expression and metabolic phenotype. A, Microarray analysis of gene expression was performed in PDX cells treated with DRD2 agonist after 4 d. GeneVenn analysis was performed on gene transcripts found to be significantly elevated in the two cell lines, revealing subtype-specific alterations in expression profiles. B, GSEA was performed on each cell line for canonical hypoxia response signaling. C, D, Microarray results were then limited to genes with confirmed hypoxia response elements. E, F, Seahorse analysis was performed to quantify the glycolytic rate in two PDX lines; (E) GBM39, which responds to DRD2 activation with increased sphere-formation, and (F) GBM12, which are unresponsive to DRD2 activation in neurosphere formation. Cells were treated with either DMSO or 30 nm DRD2 agonist and subjected to Seahorse analysis after 96 h. Figure 6-1 includes a responding PDX line, GBM5, as an assay control demonstrating similar effects. Figure 6-1 also includes FACS analysis of glucose uptake and fatty acid uptake in GBM12 and 39 as further validation of seahorse assays. G, Summary schematic of dopamine's role within the tumor microenvironment and at the single-cell level. Microarray was performed on biological triplicates. Bars represent means from three independent experiments and error bars represent SD. Student t tests were performed for each separate cell line. ***p < 0.001.