Role of the endothelin axis in astrocyte- and endothelial cell-mediated chemoprotection of cancer cells

Abstract

Background: Recent evidence suggests that astrocytes protect cancer cells from chemotherapy by stimulating upregulation of anti-apoptotic genes in those cells. We investigated the possibility that activation of the endothelin axis orchestrates survival gene expression and chemoprotection in MDA-MB-231 breast cancer cells and H226 lung cancer cells.

Methods: Cancer cells, murine astrocytes, and murine fibroblasts were grown in isolation, and expression of endothelin (ET) peptides and ET receptors (ETAR and ETBR) compared with expression on cancer cells and astrocytes (or cancer cells and fibroblasts) that were co-incubated for 48 hours. Type-specific endothelin receptor antagonists were used to evaluate the contribution of ETAR and ETBR to astrocyte-induced activation of the protein kinase B (AKT)/mitogen-activated protein kinase (MAPK) signal transduction pathways, anti-apoptotic gene expression, and chemoprotection of cancer cells. We also investigated the chemoprotective potential of brain endothelial cells and microglial cells.

Results: Gap junction signaling between MDA-MB-231 cancer cells and astrocytes stimulates upregulation of interleukin 6 (IL-6) and IL-8 expression in cancer cells, which increases ET-1 production from astrocytes and ET receptor expression on cancer cells. ET-1 signals for activation of AKT/MAPK and upregulation of survival proteins that protect cancer cells from taxol. Brain endothelial cell-mediated chemoprotection of cancer cells also involves endothelin signaling. Dual antagonism of ETAR and ETBR is required to abolish astrocyte- and endothelial cell-mediated chemoprotection.

Conclusions: Bidirectional signaling between astrocytes and cancer cells involves upregulation and activation of the endothelin axis, which protects cancer cells from cytotoxicity induced by chemotherapeutic drugs.

Keywords: astrocytes; brain metastasis; breast cancer; endothelin; therapeutic resistance.

Fig. 1.

Modulation of the endothelin axis in MDA-MB-231 cancer cells and astrocytes. (A) RT-PCR analysis of murine ET-1 expression in murine astrocytes, human MDA-MB-231 cancer cells and astrocytes, and MDA-MB-231 cancer cells that were co-incubated for 48 hours. (B) Schematic cartoon depicting the experimental approach used for parts C–E whereby GFP-labeled astrocytes or fibroblasts, which were co-incubated with human MDA-MB-231 cancer cells, were selectively removed so that analysis could be performed on cancer cells only. (C) RT-PCR analysis demonstrating that ET_AR and ET_BR are upregulated in MDA-MB-231 breast cancer cells cultured with GFP-labeled murine astrocytes, but not NIH 3T3 fibroblasts. (D) Western blot analysis of MDA-MB-231 cancer cells that were cultured alone or with astrocytes or fibroblasts. (E) Co-immunoprecipitation analysis demonstrating that ET_AR and ET_BR are phosphorylated on MDA-MB-231 cancer cells that were co-incubated with astrocytes but not fibroblasts. All figures are representative of 3 independent experiments. *P < .05. ND, not detected.

Fig. 2.

MDA-MB-231 cancer cell-derived IL-6 and IL-8 upregulate endothelin signaling. (A) Co-incubation on astrocytes with cancer cells leads to upregulation of IL-6 and (B) IL-8 gene expression. (C) ELISA showing increased IL-6 and (D) IL-8 secretion when cancer cells are co-incubated for 24 hours with murine astrocytes. Treatment of MDA-MB-231 cells with 100 μM of the gap junction inhibitor CBX for 2 hours prior to co-incubation with astrocytes blocks IL-6 and IL-8 upregulation. (E) Treatment of MDA-MB-231 cancer cells with CBX for 2 hours prior to co-incubation with astrocytes blocks ET-1 secretion from astrocytes. (F) Western blot analysis of ET_AR and ET_BR expression in MDA-MB-231 cancer cells and cancer cells that were stimulated with 100 ng/mL of IL-6 or IL-8 proteins for 24 hours. (G) Cartoon depicting bidirectional signaling between murine astrocytes and MDA-MB-231 cancer cells (see text for details). *P < .05, **P < .01.

Fig. 3.

ET-1 phosphorylates AKT/MAPK signaling pathways in MDA-MB-231 cancer cells. (A) Western blot analysis of total and activated forms of AKT and MAPK in MDA-MB-231 cancer cells incubated with 100 nM ET-1 for the indicated periods (upper panel) or with ET-1 at the indicated concentrations for 30 minutes (lower panel). (B) Immunofluorescence staining of phospho-AKT (upper panel) and phospho-MAPK (bottom panel). Cells were incubated for 30 minutes with vehicle or 100 nM ET-1 and then labeled with antibodies directed against the phosphorylated forms of AKT and MAPK (red). Nuclei were stained with DAPI (blue). (C) MDA-MB-231 cancer cells were incubated for 30 minutes with 100 nM ET-1. Cell lysates were subjected to immunoprecipitation and Western blotting, as described in Figure 2. Control immunoprecipitations were performed with normal mouse or rabbit IgG.

Fig. 4.

ET-1 increases gene expression of BCL2L1, GSTA5, and TWIST1 in cancer cells. (A) MDA-MB-231 cancer cells were stimulated with 100 nM ET-1, and gene expression levels of BCL2L1, GSTA5, and TWIST1 were measured every 24 hours with RT-PCR. Fold increase refers to the ratio of mRNA levels in ET-1 treated cells to that of 24-hour treated cells. (B) Western blot analysis comparing protein expression of Bcl2_l1, Gsta5, and Twist1 in untreated MDA-MB-231 cancer cells with cells that were stimulated with 100 nM ET-1. (C) H226 NSCLC cells were stimulated with 100 nM ET-1, and gene expression levels of BCL2L1, GSTA5, and TWIST1 were measured every 24 hours with RT-PCR. (D) Western blot analysis comparing protein expression of Bcl2_l1, Gsta5, and Twist1 in untreated H226 NSCLC cells with cells that were stimulated with 100 nM ET-1. Protein levels were quantified by densitometry using ImageJ 1.40 software. ß-Actin expression was used as an internal control. Statistical significances are compared with 24-hour treated cells. *P < .05, **P < .01.

Fig. 5.

Dual antagonism of ET_AR and ET_BR block astrocyte-induced activation of AKT/MAPK signaling and anti-apoptotic gene expression in MDA-MB-231 cancer cells. (A) Western blot analysis of MDA-MB-231 cancer cells that were pre-incubated for 2 hours with 1 μM BQ123, 1 µM BQ788, BQ123 plus BQ788, or 100 nM macitentan and then cultured alone (Tc) or co-incubated with murine astrocytes (Tc + astrocytes). GFP-labeled astrocytes were removed by FACS, and Western blot was performed on cancer cells as described in Material and Methods. (B) RT-PCR analysis of BCL2L1, GSTA5, and TWIST1 expression in MDA-MB-231 cancer cells cultured alone or co-incubated with murine astrocytes. Target gene expression levels were normalized to the 18S rRNA level. Fold increase refers to the ratio of mRNA levels in MDA-MB-231 cells cultured alone versus cells co-incubated with GFP-labeled murine astrocytes. (C) Western blot analysis of Bcl2_l1, Gsta5, and Twist1expressed by MDA-MB-231 cancer cells alone or cells that were co-incubated with astrocytes for 48 hours. ß-Actin expression was used as an internal control for Western blotting. Values shown are means ± SD of 3 independent experiments, each performed with triplicate cultures. Statistical significances were compared with cancer cell growing alone. *P < .05, **P < .01.

Fig. 6.

Activation of both ET_AR and ET_BR is essential for astrocyte-mediated chemoprotection of cancer cells. (A) MDA-MB-231 cancer cells were transfected with 100 nM siRNA targeting ET_AR (ET_AR-siRNA), ET_BR (ET_BR-siRNA), or both (ET_AR + ET_BR siRNA). Nonspecific control siRNA (NS-siRNA) was used as a control. (B) Comparison of the apoptotic index of MDA-MB-231 cancer cells and siRNA transfected MDA-MB-231 cancer cells cultured alone in the presence of taxol with cancer cells co-incubated with murine astrocytes in the presence of taxol. (C) Chemoprotection assay in which MDA-MB-231 cancer cells were pre-incubated for 2 hours with 1 μM of BQ123, BQ788, both antagonists, or the dual antagonist, macitentan (100 nM) and then cultured alone (control) or co-incubated with GFP-labeled murine astrocytes in the presence of taxol (15 ng/mL). (D) Chemoprotection assay with siRNA transfected MDA-MB-231 cancer cells and human astrocytes. (E) Chemoprotection assay with MDA-MB-231 cells treated with endothelin antagonists and co-incubated with human astrocytes. *P < .05 versus control cancer cells growing alone in the presence of taxol.