Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer
Neveen Said, … , Marta Sanchez-Carbayo, Dan Theodorescu
Neveen Said, … , Marta Sanchez-Carbayo, Dan Theodorescu
Published December 22, 2010
Citation Information: J Clin Invest. 2011;121(1):132-147. https://doi.org/10.1172/JCI42912.
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Research Article Oncology

Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer

Abstract

Many patients with advanced bladder cancer develop lethal metastases to the lung. The vasoconstricting protein endothelin-1 (ET-1) has been implicated in this process, although the mechanism(s) by which it promotes metastasis remains unclear. Here, we have evaluated whether tumor ET-1 expression can serve as a biomarker for lung metastasis and whether it is required for metastatic disease. Evaluation of ET-1 mRNA and protein expression in four patient cohorts revealed that levels of ET-1 are higher in patients with muscle-invasive bladder cancers, which are associated with higher incidence of metastasis, and that high ET-1 levels are associated with decreased disease-specific survival. Consistent with its proinflammatory activity, we found that tumor-derived ET-1 acts through endothelin-1 receptor A (ETAR) to enhance migration and invasion of both tumor cells and macrophages and induces expression of inflammatory cytokines and proteases. Using human and mouse cancer cells depleted of ET-1 and pharmacologic blockade of ET receptors in lung metastasis models, we found that tumor ET-1 expression and ETAR activity are necessary for metastatic lung colonization and that this process is preceded by and dependent on macrophage infiltration of the lung. In contrast, tumor ET-1 expression and ETAR activity appeared less important in established primary or metastatic tumor growth. These findings strongly suggest that ETAR inhibitors might be more effective as adjuvant therapeutic agents than as initial treatment for advanced primary or metastatic disease.

Authors

Neveen Said, Steven Smith, Marta Sanchez-Carbayo, Dan Theodorescu

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Figure 3

ET-1 induces bladder cancer cell invasion, chemotaxis, transendothelial migration, and proteolytic activity.

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ET-1 induces bladder cancer cell invasion, chemotaxis, transendothelial ...
(A) UMUC3 cells (1 × 105 cells/100 μl, MEM-0.4% BSA) were added to the upper chamber of Matrigel-coated, 8-μm-pore-size polycarbonate inserts. Cells were allowed to invade matrix-coated inserts to the bottom chamber containing CGM or ET-1 (10–200 nM) in SFM. Invasion (black bars)/migration (white bars) assays were carried out for 5 hours. Cells attached to the bottom surface of the inserts were stained with DiffQuick and counted. UMUC3 cells were treated with ETAR inhibitors (BQ123, ABT-627, and ZD4054) or ETBR inhibitors (BQ788 and A-192621) (B) or transfected with siRNA targeting ET-1, ECE-1 (100 nM) or siETAR, siETBR (200 nM) or mock vector control siRNA (VC). Effective knockdown was determined in harvested cells 48 hours after transfection by Western blot (C, inset). Cells were then used for Matrigel invasion (C) and chemotaxis (D) assays as described above. *P < 0.05, compared with nonstimulated (NS) VC; **P < 0.05, compared with ET-1 stimulation, Student’s t test. (E) UMUC3 cells knocked down or not with siET-1/siETRs or treated with ZD4045 and BQ788 were allowed to traverse PMVECs grown on 8-μm inserts. Assays were carried out for 6 hours, after which cells on the undersurface of the inserts were counted. Bars represent mean ± SEM of 3 independent experiments performed in triplicate. *P < 0.05, Student’s t test. (F and G) UMUC3 cells transfected or not with siET-1, siETAR, and siETBR were stimulated with ET-1 (100 nM) in SFM for 72 hours. CM was collected, and MMP2 and MMP9 activity was detected in 10× CM. Cell lysates were probed with α-tubulin as loading control.