Glioblastoma Multiforme Cancer Stem Cells Express Components of the Renin–Angiotensin System

Amy Ruth Bradshaw,Alice M Chibnall,Agadha Crisantha Wickremesekera,Swee T Tan,Paul F Davis,Tinte Itinteang,Helen D Brasch

doi:10.3389/fsurg.2016.00051

Abstract

To investigate the expression of the renin-angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM). 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively. DAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2. This study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.

Highlights

Glioblastoma multiforme, a grade IV astrocytoma, contributes to about 50% of all malignant gliomas [1, 2]
The cancer stem cells (CSCs) concept proposes that a cancer originates from a small population of CSCs, which are generated by upregulation of certain genes in putative resident stem or progenitor cells [11, 12]
Cancer stem cells have been identified in many cancer types [43,44,45,46,47,48] and were first characterized in GBM by Singh et al [8, 49]

Summary

Introduction

Glioblastoma multiforme, a grade IV astrocytoma, contributes to about 50% of all malignant gliomas [1, 2] It almost invariably recurs following surgical resection, radiotherapy, and chemotherapy [3,4,5,6]. The CSC concept proposes that a cancer originates from a small population of CSCs, which are generated by upregulation of certain genes in putative resident stem or progenitor cells [11, 12] These genetic alterations confer, upon these cells, the capacity to proliferate and differentiate in an uncontrolled manner resulting in tumorigenesis [11,12,13,14]. CSCs can be identified using markers associated with embryonic stem cells (ESCs) [15, 16], including ESC markers such as transcription factors NANOG, SALL4, and OCT4, transcription co-factor SOX2 and signaling molecule pSTAT3 [17,18,19,20,21]

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