Paired related homeobox 1 transactivates dopamine D2 receptor to maintain propagation and tumorigenicity of glioma-initiating cells

Abstract

Glioblastoma multiforme (GBM) is a highly invasive brain tumor with limited therapeutic means and poor prognosis. Recent studies indicate that glioma-initiating cells/glioma stem cells (GICs/GSCs) may be responsible for tumor initiation, infiltration, and recurrence. GICs could aberrantly employ molecular machinery balancing self-renewal and differentiation of embryonic neural precursors. Here, we find that paired related homeobox 1 (PRRX1), a homeodomain transcription factor that was previously reported to control skeletal development, is expressed in cortical neural progenitors and is required for their self-renewal and proper differentiation. Further, PRRX1 is overrepresented in glioma samples and labels GICs. Glioma cells and GICs depleted with PRRX1 could not propagate in vitro or form tumors in the xenograft mouse model. The GIC self-renewal function regulated by PRRX1 is mediated by dopamine D2 receptor (DRD2). PRRX1 directly binds to the DRD2 promoter and transactivates its expression in GICs. Blockage of the DRD2 signaling hampers GIC self-renewal, whereas its overexpression restores the propagating and tumorigenic potential of PRRX1-depleted GICs. Finally, PRRX1 potentiates GICs via DRD2-mediated extracellular signal-related kinase (ERK) and AKT activation. Thus, our study suggests that therapeutic targeting the PRRX1-DRD2-ERK/AKT axis in GICs is a promising strategy for treating GBMs.

Keywords: dopamine D2 receptor; glioblastoma; glioma-initiating cells; paired related homeobox 1.

Figure 1

Prrx1 maintains stemness of cortical NPCs of embryonic cerebrum. (A) Prrx1 in situ hybridization of coronal sections of mouse embryonic dorsal forebrain (cortex) using digoxigenin-labeled RNA probe. Bottom panels showed boxed area in corresponding upper panels. Arrowheads point to epidermis. Dashed lines demarcate boundaries between VZ/SVZ and IZ. Scale bar, 500 μm (upper) and 50 μm (bottom). (B) E11.5 cortical NPCs were transduced with lentiviruses expressing scramble or Prrx1 shRNAs before plating into 96-well plates for limiting dilution assays. Cultures were maintained until day 10, when the number of wells containing spheres for each cell plating density (number of positive cultures) was recorded, calculated, and plotted using online ELDA analysis program (Hu and Smyth, 2009). Bottom, representative sphere images in each group. Right, incidences of sphere-forming NSCs and P-values between groups. Scale bar, 25 μm. (C and D) Plasmids expressing the indicated shRNAs were in utero electroporated into E13.5 mouse cortex, and embryos were sacrificed at E16.5. BrdU was injected 30 min before sacrificing. Cryo-sectioned brains were subjected to immunofluorescent stainings of Pax6 (C) and BrdU (D), with transduced cells labeled with ZsGreen. PRRX1B^r contains wobble mutations resistant to shPrrx1#1. Scale bar, 50 μm. (E) Percentile of Pax6+, BrdU+, and NeuroD2+ cells in transduced cells in the VZ/SVZ. Histograms represent mean ± SD of cortex from three mouse embryos. LV, lateral ventricle; GE, ganglionic eminence.

Figure 2

PRRX1 is overrepresented in glioma and labels GICs/GSCs. (A) PRRX1 expression in normal and GBM samples using data retrieved from TCGA Affymetrix dataset. Left, comparison of PRRX1 expression levels between 10 normal and 557 GBM samples (***P < 0.001, Student's t-test); right, comparison of PRRX1 expression levels among GBM subtypes (*P < 0.05, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Tukey's multiple-comparisons test). (B) Scattered dot plot showing PRRX1 relative expression in three normal cortex samples and 31 glioma samples. Expression data were presented as mean ± SEM. *P = 0.038, unpaired Student's t-test. (C) Kaplan–Meier survival plot for GBM cases in TCGA Affymetrix dataset. PRRX1^high and PRRX1^low are defined as GBM samples within top and bottom quartiles of PRRX1 expression levels in the TCGA dataset (median survival: 12.6 months in PRRX1^high vs. 14.9 months in PRRX1^low, *P = 0.04). (D) Representative immunohistochemistry images showing PRRX1 expression in normal cortex and glioma samples. Scale bar, 50 μm. (E) Representative immunofluorescent images of two GBM samples showing co-expression of PRRX1, CD133, and SOX2. Merged images are superimposed PRRX1/CD133/SOX2 images. Scale bar, 20 μm.

Figure 3

PRRX1 maintains tumorigenic potential of glioma cells. (A) Colony formation and soft-agar sphere assays showing effects of PRRX1 depletion on U251 glioma cell proliferation. Scale bar, 500 μm. (B) Quantification of colony and cell numbers in A. Histograms represent mean ± SD of three independent experiments. (C) MTT assay showing effects of PRRX1 knockdown on U251 and U87MG glioma cell proliferation. (D) Upper, images of nude mice at 3 weeks after subcutaneous inoculation of 2×10⁶ U251 glioma cells. Bottom, growth curves for xenografted U251 cells transduced with scramble and shPRRX1#1 lentiviruses. (E) Upper, image of tumor mass at 3 weeks after subcutaneous inoculation of 2×10⁶ U251 glioma cells. Bottom, scattered dot blot showing tumor weight distributions. (F) Gross (upper) and fluorescent (bottom) brain images at 4 weeks after intracranial xenografting with 2×10⁵ U251 cells, which were transduced with the indicated lentiviruses before inoculation. Brains in (a) and (c) were from 4% PFA perfused animals, whereas brains in (b) and (d) were fresh dissected.

Figure 4

PRRX1 is essential for self-renewal of GICs. (A) PRRX1 is expressed in GIC cells (TPC2-80) and co-localizes with SOX2. Upon 2% FBS-induced differentiation for 5 days, PRRX1 and SOX2 expression levels decreased significantly. Scale bar, 50 μm. (B) Immunofluorescent stainings of PRRX1 in TPC1115 GICs transduced with the indicated lentriviruses (ZsGreen+). Scale bar, 50 μm. (C) TPC1115 GICs were transduced with the indicated lentiviruses before plating into 96-well plates for limiting dilution assays. Cultures were maintained until day 10, when the number of wells containing spheres for each cell plating density (number of positive cultures) was recorded, calculated, and plotted using online ELDA analysis program. Bottom, representative sphere images in each group. Right, incidences of sphere-forming GICs and P-values between groups. Scale bar, 25 μm. (D) Adherent cultured GIC cells (TPC1115, TPC2-80, and TPC0209) were transduced with the indicated shRNAs, and cell numbers were counted every other day. Line graphs represent mean ± SEM (n = 3), and statistical significance was determined by one-way ANOVA followed by Tukey's multiple-comparisons test. Comparisons between cells transduced with scramble and PRRX1 shRNAs, were showed, and no statistical difference was found among shRNAs against PRRX1 (sh#1, sh#2, sh#3, sh#5) (***P < 0.001, ****P < 0.0001). (E) Brain sections stained with hematoxylin and eosin (H&E) showing intracranial xenografts derived from TPC2-80 GICs transduced with the indicated lentiviruses. Scale bar, 1 mm.

Figure 5

PRRX1 transactivates the expression of DRD2 in GICs. (A) Gene ontology (GO) analyses of downregulated genes upon PRRX1 knockdown in TPC1115 GICs. (B) Changes of DRD2 expression upon PRRX1 knockdown and overexpression in RNA-seq assay. RPKM, reads per kilobase per million. (C and D) qRT-PCR verifying changes of DRD2 expression upon PRRX1 knockdown and overexpression in TPC2-80 (C) and TPC0209 (D) GICs. (E) qRT-PCR indicating changes of DRD2 expression upon PRRX1 knockdown and wild-type or homeodomain-mutated (HDm) PRRX1A or PRRX1B overexpression in TPC1115 GICs. (F) Schematic diagram of human DRD2 TSS vicinity region, DNA fragments used for luciferase assay, and amplified regions in ChIP-qPCR. (G) Dual-luciferase assays were performed using DNA fragments (D2-PrM and D2-PrS) showed in F. Two panels on the left, vectors expressing PRRX1A, PRRX1B, or DRRF were co-transfected with pGL3-D2-PrM or pGL3-D2-PrS into H4 glioma cells. Two panels on the right, vectors expressing wild-type or homeodomain-mutated (HDm) PRRX1A or PRRX1B were co-transfected with pGL3-D2-PrS into 293T and HeLa cells. (H) Vectors expressing PRRX1A or PRRX1B were co-transfected with truncated DRD2 promoter regions into 293T cells for luciferase assays. (I) ChIP-qPCR assays showing enrichment of corresponding DNA fragments (illustrated in F) precipitated in TPC1115 GICs transduced with FLAG-PRRX1A, FLAG-PRRX1B, or control (untagged PRRX1A and PRRX1B co-expression) lentiviruses. ChIP-qPCR for SOX9 promoter region was also performed.

Figure 6

DRD2 is overrepresented in GICs and is required for GIC propagation. (A) Magnetic sorted CD133+ glioma cells were subjected to RNA isolation and qRT-PCR for the indicated genes. CD133- glioma cells from the same sample were used as controls. (B) Representative immunofluorescent images of three glioma specimens showing co-expression of PRRX1, DRD2, and SOX2. Merged images are superimposed PRRX1/DRD2/SOX2 images. Scale bar, 20 μm. (C) GICs (TPC0411, TPC1115, and TPC0209) were transduced with lentiviruses expressing DRD2 shRNAs, and cell numbers were counted every 3 days (TPC0411) or every other day (TPC1115 and TPC0209). (D) GICs were treated with DRD2 antagonists, risperidone and haloperidol, at the indicated concentrations. Cell numbers were counted every 3 days (TPC0411) and every other day (TPC0209).

Figure 7

PRRX1 depends on DRD2 to sustain propagation and tumorigenicity of GICs. (A) TPC2-80 GICs were transduced with the indicated lentiviruses before plating into 96-well plates for limiting dilution assays. Cultures were maintained until day 10, when the number of wells containing spheres for each cell plating density (number of positive cultures) was recorded, calculated, and plotted using online ELDA analysis program. Bottom, representative sphere images in each group. Right, incidences of sphere-forming GICs and P-values between groups. Scale bar, 50 μm. (B) Representative images showing BrdU incorporation in treated TPC1115 GICs. BrdU incorporation rates in each group were presented as mean ± SEM (n = 3). Scale bar, 50 μm. (C) Brain sections stained with H&E showing intracranial xenografts derived from TPC2-80 GICs transduced with the indicated lentiviruses. Boxed regions in the right striata were enlarged at the bottom-left corners. Asterisks in red indicate necrotic regions. Arrowheads point to invasive fronts. Scale bar, 1 mm and 100 μm (enlarged images). (D) TPC2-80 GICs were treated with lentiviruses and quinpirole (Q) as listed. Cell extracts were subjected to immunoblot assays using antibodies as indicated. (E) A diagram summarizing the PRRX1−DRD2−p-ERK/p-AKT axis in promoting GIC self-renewal and tumorigenesis.