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.

Long Non-Coding RNA LncKdm2b Regulates Cortical Neuronal Differentiation by Cis-Activating Kdm2b

“…pCALNL was a gift from Dr. Xiaoqun Wang (Institute of Biophysics, Chinese Academy of Sciences). Luciferase reporter vector was constructed according to the previous study (Li et al, 2017b). Briefly, the T5 Forward, T5 Reverse, T5-mini Forward, or T5-mini Reverse were PCR amplified from the genomic DNA of C57BL/6 mice and cloned into pGL3-Basic Vector using Mlu I and Xho I. Kdm2b promoter and Kdm2b promoter with the CArG box deletion were cloned into pGL3-Basic Vector using Sac I and Xho I.…”

“…Digoxigenin labeled riboprobes were transcribed using the DIG-RNA Labeling Mix (Roche). In situ Hybridization was performed as described (Li et al, 2017b). In brief, all solutions were prepared properly to avoid RNase contamination.…”

“…IF and immunoblotting were performed as described (Li et al, 2017b). For immunofluorescent staining, 4% paraformaldehyde (PFA) fixed 14 μm sections or cells were permeabilized and blocked with blocking buffer (3% heat-inactivated normal goat serum, 0.1% bovine serum albumin and 0.1% Triton-X 100 in 10 mM Tris-HCl, pH 7.4; 100 mM NaCl) for one hour at R/T.…”

“…In utero microinjection and electroporation were performed essentially at E13.5 as described (Li et al, 2017b). In brief, pregnant CD-1 mice were anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg).…”

“…Primary culture of embryonic cortical NPCs was performed as described (Li et al, 2017b). In brief, E11.5 or E12.5 mouse cortices (dorsal forebrain) tissues were washed with and minced in filter-sterilized hibernation buffer (30 mM KCl; 5 mM NaOH; 5 mM NaH2PO4; 5.5 mM glucose; 0.5 mM MgCl 2 ; 20 mM Na-pyruvate; 200 mM Sorbitol, pH 7.4) followed by dissociating into single cells using pre-warmed Papain (Worthington Biochemical) enzyme solution (1 × DMEM; 1 mM Na-pyruvate; 1 mM L-Glutamine; 1 mM N-Acetyl-L-Cysteine; 20 U/mL Papain; 12 μg/mL DNase I).…”