Androgen Receptor Activation in Glioblastoma Can Be Achieved by Ligand-Independent Signaling through EGFR-A Potential Therapeutic Target.

Nomi Zalcman,Tal Shahar,Michael Weller,Mijal Gutreiman,Iris Lavon

doi:10.3390/ijms222010954

Abstract

Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a potential therapeutic target. In prostate and breast cancers, AR activation can be achieved also by a ligand-independent signaling through receptor tyrosine kinases such as epidermal growth factor receptor (EGFR). Considering its major role in glioblastoma, we explored whether EGFR is involved in AR signaling in this tumor. Analysis of mRNA expression in 28 glioblastoma samples with quantitative real-time reverse-transcription polymerase chain reaction revealed a positive and significant correlation between AR and EGFR mRNA expression levels (R = 0.47, p = 0.0092), which was validated by The Cancer Genome Atlas dataset (n = 671) analysis (R = 0.3, p = 0.00006). Using Western blotting and immunofluorescence staining, we showed that the transduced overexpression of EGFR or its variant EGFRvIII in the U87MG cells induced AR protein overexpression and nuclear translocation and Protein kinase B (AKT) S473 and AR S210/213 phosphorylation. The EGFR kinase inhibitor afatinib and the AKT inhibitor MK2206 reduced AR nuclear translocation. Afatinib diminished AKT phosphorylation at 30 min and 6 h in the EGFR- and EGFRvIII-overexpressing cells, respectively, and decreased AR phosphorylation in EGFR-overexpressing cells at 4 h. Afatinib or MK2206 combination therapy with the AR antagonist enzalutamide in the EGFR and EGFRvIII-overexpressing cells had synergistic efficacy. Our findings suggest that EGFR signaling is involved in AR activation in glioblastoma and buttresses the concept of combining an EGFR signaling inhibitor with AR antagonists as a potential glioblastoma treatment.

Highlights

Considering the conflicting results on the correlation between epidermal growth factor receptor (EGFR) and Androgen receptor (AR) expression in various cancer types, we explored their expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in glioblastoma samples from 28 patients
The EGFR and AR expression data obtained by RNA sequencing (RNA-seq) of The Cancer Genome Atlas (TCGA) dataset (n = 671) (Figure 1B) yielded similar results (R = 0.3; p = 0.00006)
EGFR or EGFRvIII Overexpression Induces AR Overexpression and Nuclear Translocation, U87MG WT cells using an anti-AR antibody by Western blot analysis and enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

Glioblastoma is the most common and most aggressive primary brain tumor, with a very poor prognosis. One of the most common genetic aberrations associated with glioblastoma is epidermal growth factor receptor (EGFR) amplification [1]. EGFR is overexpressed in approximately 60% of tumors. EGFR belongs to the four-member of Human Epidermal Growth Factor Receptor (HER); receptor tyrosine kinase (RTK) family: EGFR (HER1), HER2, HER3, and HER4. EGFR signaling results in downstream signaling pathway activation, e.g., the phosphoinositide-3 kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) pathway. In glioblastoma, this pathway downstream of EGFR is often hyperactivated by overexpression or activating EGFR mutations and by Phosphatase and tensin homolog (PTEN) deletion or downregulation, which antagonizes

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