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

Approximately 200 000 cases of brain metastases occur in the United States each year.1 Brain metastasis is associated with poor prognosis, neurological deterioration, diminished quality of life, and extremely short survival.2 Indeed, the median survival time for untreated patients is 1–2 months3 and improves to only 4–6 months for patients treated with chemotherapy and conventional radiotherapy.4 The limited response of brain metastases to chemotherapy has been attributed to structural (ie, tight junctions) and functional (eg, P-glycoprotein) properties of the blood-brain barrier that prevent cytotoxic drugs from entering into the central nervous system (CNS).5,6 However, experimental studies have shown that the blood-brain barrier is breached once metastases exceed 0.2 mm in diameter,7,8 suggesting that additional mechanisms may contribute to the chemoresistant phenotype of brain metastases. An increased understanding of the mechanisms that mediate therapeutic resistance of brain metastases is critical for developing new treatment strategies that improve clinical outcomes. Reactive astrogliosis is widely regarded as the most important histopathological indicator of diseased CNS tissue, regardless of etiology.9 Reactive astrocytes encircle and infiltrate cancer cells residing in the brain.10,11 Recently, we patterned reactive astrogliosis in vitro by co-incubating melanoma cells with astrocytes and discovered that gap junction-mediated communication protected melanoma cells from chemotherapeutic agents.12 A more intensive examination, which employed cross-species hybridization of microarrays on human cancer cells that were co-incubated with murine astrocytes, revealed that direct contact between astrocytes and cancer cells leads to marked alterations in the cancer cell transcriptome including upregulation of the survival-related genes, glutathione S transferase alpha 5 (GSTA5), BCL2-like 1 (BCL2L1), and TWIST-related protein 1 (TWIST1).13 Functional studies demonstrated a role for this subset of genes in protecting cancer cells from chemotherapy, and the gene products were localized to cancer cells in clinical cases of brain metastases.13 However, the astrocyte signal that stimulates survival gene expression in cancer cells remained unknown. Several lines of evidence have suggested a potential role for the small (21-amino acid) endothelin peptides in mediating anti-apoptotic gene expression and chemoprotection in cancer cells. The endothelin signaling pathway includes 3 peptides (ET-1, ET-2, and ET-3), which mediate their activity by binding to 2 high-affinity G-protein–coupled receptors, ETAR and ETBR.14 Immunohistochemical analysis of a large series of human brain metastasis cases revealed that peritumoral astrocytes overexpress endothelin in 85% of metastases.15 Reactive astrocytes are also reported to overexpress endothelin in several other CNS pathologies.16 A comparison of gene expression profiles between melanoma cell variants, which spontaneously metastasized to the brain and parental cells with low metastatic potential, led to the identification of ETBR as a critical determinant in the stepwise progression of melanoma to the brain metastatic phenotype.17 ET-1 has also been shown to signal for activation of survival programs in several types of cancer cells.18–20 The above-mentioned studies prompted us to hypothesize that endothelin-mediated signaling between cancer cells and astrocytes and/or endothelial cells leads to the upregulation of survival genes in cancer cells and thus protection from chemotherapy. Herein, we examined how astrocytes and endothelial cells interact with MDA-MB-231 breast cancer cells and H226 non–small cell lung cancer (NSCLC) cells to modulate different components of the endothelin signaling axis and describe the impact on cancer cell survival.