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. 2019 Nov;110(11):3486-3496.
doi: 10.1111/cas.14187. Epub 2019 Sep 20.

Tyrosine Kinase Eph Receptor A6 Sensitizes Glioma-Initiating Cells Towards Bone Morphogenetic Protein-Induced Apoptosis

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Free PMC article

Tyrosine Kinase Eph Receptor A6 Sensitizes Glioma-Initiating Cells Towards Bone Morphogenetic Protein-Induced Apoptosis

Erna Raja et al. Cancer Sci. .
Free PMC article

Abstract

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation- and apoptosis-inducing effects on glioma-initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin-producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP-2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP-2-induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK-2, which was associated with BMP-induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain-of-function of EPHA6 together with BMP-2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP-2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.

Keywords: EPHA6; apoptosis; bone morphogenetic protein-2; cancer stem cell; glioblastoma.

Figures

Figure 1
Figure 1
Erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) is a glioblastoma tumor suppressor, which functions with bone morphogenetic protein 2 (BMP‐2) to induce apoptosis. A, EPHA and EPHB phosphorylation in patient‐derived glioma‐initiating cells (GIC) in response to BMP‐2 treatment evaluated by receptor tyrosine kinase (RTK) phospho‐array analysis. TGS‐01, TGS‐04, and TGS‐05 were untreated (control, black bars) or treated with BMP‐2 (gray bars) for 24 h prior to analysis. Phosphorylation of the control conditions was normalized to 1 in each RTK. B, EPHA6 expression in normal brain or glioblastoma tissues in The Cancer Genome Atlas (TCGA) datasets. ***P < .001. Glioblastoma was classified into four subtypes according to Verhaak et al.34 C, Kaplan‐Meier analysis of disease‐free survival of patients with glioblastoma from TCGA dataset (n = 112). Survival analysis was carried out using a log‐rank test. D, E, WST cell survival assay in TGS‐03 and TGS‐04 cells, respectively, after adenoviral transduction of LacZ control (ad‐LacZ) or wild‐type EPHA6 (ad‐EPHA6‐WT) in the presence or absence of BMP‐2 for 6 d. Data represent mean ± standard deviation (SD) of n = 3 independent experiments. *P < .05, **P < .01 and n.s. (not significant). F, G, Apoptosis assays by FACS analysis in TGS‐03 and TGS‐04, respectively. The same experimental setting as (D, E) was used, and cells were labeled with Annexin‐V and propidium iodide. Data represent mean ± SD of n = 4 (TGS‐03) and n = 3 (TGS‐04) independent experiments
Figure 2
Figure 2
Bone morphogenetic protein 2 (BMP‐2) induces erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) tyrosine phosphorylation. A, Immunoblot analysis of TGS‐03 cells (left) and TGS‐04 cells (right) infected with ad‐LacZ or wild‐type EPHA6 (ad‐EPHA6‐WT) with or without BMP‐2 stimulation for 24 h. Cell lysates were subjected to immunoprecipitation (IP) with anti‐FLAG antibody for EPHA6, followed by phospho‐tyrosine or FLAG immunoblotting. pTyr, phospho‐tyrosine; pSMAD1/5, phospho‐SMAD1/5; TCL, total cell lysate. B, Amino acid sequence in the conserved activation loop region of EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, and EPHA8. The tyrosine residue recognized by phospho‐EPHA (pEPHA) antibody when phosphorylated is highlighted in yellow (Y779 for EPHA3 and Y925 for EPHA6). C, Immunoblots of TGS‐03 cells infected with ad‐LacZ or ad‐EPHA6‐WT and treated or untreated with BMP‐2 and 0.5 μmol/L LDN193189 for 24 h. Cell lysates were subjected to IP with FLAG antibody, followed by phospho‐EPHA or FLAG immunoblotting. D, Immunoblots of TGS‐03 cells infected with ad‐EPHA6‐WT, and treated with or without BMP‐2, DMSO control or 1 μmol/L inhibitors for 24 h as indicated. LDN193, LDN193189; Dasa, dasatinib; Fore, foretinib; pSrc, phospho‐Src. E, Phylogenetic tree of the kinase domain of the EPHA receptors
Figure 3
Figure 3
Kinase activities of bone morphogenetic protein (BMP) type I receptor and erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) are differentially required in apoptosis induction. A, Immunoblot analysis of TGS‐03 and TGS‐04 cells infected with ad‐LacZ, ad‐EPHA6‐WT or adenovirus with kinase‐dead EPHA6 K757R (ad‐EPHA6‐KD) and stimulated with or without BMP‐2 for 2 d as indicated. pEPHA, phospho‐EPHA; p‐p38, phospho‐p38; pSMAD1/5, phospho‐SMAD1/5. B, C, Quantification of apoptosis of TGS‐03 (B) and TGS‐04 (C) cells infected with ad‐LacZ, ad‐EPHA6‐WT and ad‐EPHA6‐KD, stimulated with or without BMP‐2 for 5 d with FACS analysis. Graphs represent mean values from two independent experiments. D, FACS diagrams of TGS‐04 cells infected with ad‐LacZ or ad‐EPHA6‐WT with or without 1 μmol/L LDN193189 and BMP‐2 for 6 d. Percentages represent apoptotic cells. Y‐axis plots propidium iodide signal and X‐axis plots Annexin‐V signal. E, Immunoblots of TGS‐04 cells with the same experimental conditions as in (D) for 4 d
Figure 4
Figure 4
Erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) physically interacts with bone morphogenetic protein (BMP) type I receptor ALK‐2. A, Immunoblots showing coimmunoprecipitation of ALK‐2 with EPHA6. TGS‐03 and TGS‐04 cells overexpressing LacZ, wild‐type EPHA6, and constitutively active ALK‐2 (A2‐CA) were immunoprecipitated (IP) with anti‐FLAG antibody 1 day after adenovirus transduction, followed by HA and FLAG immunoblotting for ALK‐2 and EPHA6, respectively. TCL, total cell lysate; pSMAD1/5, phospho‐SMAD1/5. B, Immunoblots of TGS‐03 and TGS‐04 cells after immunoprecipitation of ALK‐2‐CA protein using anti‐HA antibody and the corresponding TCL with the same experimental setting as in (A). C, Immunoblot analysis of TGS‐03 cells showing coimmunoprecipitation of EPHA6 with HA‐tagged ALK‐2‐CA treated with or without 1 μmol/L LDN193189 for 1 d. LacZ overexpression serves as a negative control for all experiments in Figure 4
Figure 5
Figure 5
Wild‐type and kinase‐dead erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) function similarly to promote bone morphogenetic protein (BMP)‐2‐induced apoptosis in TGS‐01 cells. A, FACS analysis of TGS‐01 cells infected with ad‐LacZ, ad‐EPHA6‐WT and ad‐EPHA6‐KD treated or untreated with BMP‐2 for 7 d. Percentages represent apoptotic cells. Propidium iodide (PI) signal is plotted on Y‐axis and Annexin‐V signal on X‐axis. B‐D, TGS‐01 cells were infected with ad‐LacZ, ad‐EPHA6‐WT, and ad‐EPHA6‐KD in the presence or absence of BMP‐2 for 24 h and RNA‐sequencing analysis was carried out. Genes with fragments per kilobase of exon per million mapped fragments (FPKM) values equal or higher than 3 are selected for gene set enrichment analysis. Plots show enrichment of apoptotic genes in LacZ cells treated with vs without BMP‐2 treatment (B), EPHA6‐WT vs LacZ‐expressing cells (both treated with BMP‐2) (C), and EPHA6‐KD vs LacZ‐expressing cells (both treated with BMP‐2) (D). NES, normalized enrichment score
Figure 6
Figure 6
Erythropoietin‐producing hepatocellular carcinoma receptor A6 (EPHA6) gain‐of‐function impedes glioblastoma multiforme tumor growth in a mouse orthotopic model. A, Survival curve of mice inoculated with TGS‐01‐luc (luciferase) cells infected with ad‐LacZ or ad‐EPHA6‐KD and pretreated or not pretreated with 200 ng/mL BMP‐2 for 2 d. n = 4 for all groups except the EPHA6‐KD group treated with BMP‐2 (n = 3). LacZ + BMP‐2 vs EPHA6‐KD + BMP‐2 (*P < .05); LacZ vs EPHA6‐KD (**P < .01). B, Tumor formation shown by luciferase signals in the groups of mice as in experiment (A) at 3 wk‐time point. cps, count per second. C, Survival curve of mice inoculated with TGS‐01‐luc cells infected with ad‐LacZ, ad‐EPHA6‐WT, or ad‐EPHA6‐KD pretreated with 200 ng/mL BMP‐2 for 2 d. n = 5 for all groups. LacZ + BMP‐2 vs EPHA6‐WT + BMP‐2 (**P < .01); LacZ + BMP‐2 vs EPHA6‐KD + BMP‐2 (**P < .01). (D) Tumor formation shown by luciferase signals in the groups of mice as in experiment (C) at the 3‐wk time point. One mouse from LacZ + BMP‐2 group was omitted in the survival analysis (C) due to premature death during anesthesia.

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