Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer
Neveen Said, Steven Smith, Marta Sanchez-Carbayo, Dan Theodorescu
Neveen Said, Steven Smith, Marta Sanchez-Carbayo, Dan Theodorescu
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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 8

The effect of ZD4054 on primary tumor development and spontaneous lung metastasis in syngeneic immunocompetent C57BL/6 mice inoculated with MB49 murine bladder cancer cells.

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The effect of ZD4054 on primary tumor development and spontaneous lung m...
(A–C) MB49 cells (5 × 104 cells/100 μl) were injected s.c. in mice. Animals were assigned to 3 groups that received ZD4054 24 hours before MB49 injection (ZD-pre); vehicle control (VC); or ZD4054 1 week after tumor cell injection (ZD-post). (A) Kinetics of s.c. growth in 3 cohorts Typical example of macrophage immunostaining (total magnification, ×100) and quantification in s.c. tumors. Bars represent the mean ± SEM of macrophages/HPF as described above; *P < 0.05, Student’s t test, compared with VC-treated mice. (B) Incidence and number of visible metastases (mets) per lung in each cohort (left) following s.c. inoculation. †P < 0.0001, χ2 test; #P < 0.01, Student’s t test. Photomicrographs show infiltrating macrophages in lungs. Bars in the right panel represent mean ± SEM of the number macrophages within metastatic foci and in the surrounding lung parenchyma/HPF. *P < 0.05, as compared with VC-treated mice. (C) ET-1, MCP-1, and IL-6 detected in lungs and s.c. tumors. Bars represent mean ± SEM (n = 5/group). *P < 0.05, Student’s t test, compared with VC-treated mice. (D) Representative images of CD31 immunostaining of s.c. MB49 tumors showing MVD. Bars represent MVD ± SEM counted in 6 HPFs/sample. (E) Mice injected with MB49 cells (104 cells/100 μl) via tail vein were treated with ETR inhibitors (ZD4054, top, and BQ788, bottom) and were assigned to 3 cohorts for each inhibitor as described above. Scatter plot of incidence/number of visible lung metastases determined 3 weeks after injection. †P < 0.01, χ2 test; #P < 0.05, Student’s t test.