The endothelin pathway plays a critical role in melanoma tumor progression by a variety of mechanisms that enhance tumor cell growth, invasion, and metastasis. Here, we investigate the effect of this pathway on CXC chemokine expression in human melanoma cells and melanocytes. As determined by ELISA, endothelin-1 (ET-1) induces CXCL1 and CXCL8 secretion in three human melanoma cell lines in a concentration-dependent fashion. These responses are mediated by the endothelin-B receptor and are sustained over a 40 h time course. ET-1 does not induce CXCL1 secretion in primary human melanocytes but ET-3, an endothelin isoform, induces a low level of CXCL1 secretion in certain cultures. Neither ET-1 nor ET-3 induces secretion of CXCL8 in primary human melanocytes; thus, this response may be specific for melanocytic cells that have undergone malignant transformation. We have previously demonstrated that ET-1 induces changes in the expression of adhesion molecules in melanoma cells such that invasion and metastasis are favored. This study demonstrates that ET-1 additionally induces secretion of CXC chemokines critical for melanoma metastasis and tumor progression.

Melanoma cell adhesion molecule (MCAM) is a cell-surface adhesion molecule expressed on over 70% of metastatic melanoma cells but not expressed in normal melanocytes invivo. Protein levels of MCAM correlate with aggressive invasive behavior of melanoma cells in vitro and invivo. Here we demonstrate that endothelin-1 (ET-1) upregulates MCAM protein in primary human melanocytes. MCAM upregulation by ET-1 occurs irrespective of degree of melanocyte pigmentation and is dose-responsive. The drug BQ788 is an endothelin-B (ET(B)) receptor antagonist and inhibits upregulation of MCAM by ET-1. In addition, endothelin-3 (ET-3) and N-succinyl-[Glu9, Ala11, 15]-ET-1-1620, both selective ET(B) agonists, are potent upregulators of MCAM. These demonstrate a critical role for the ET(B) receptor in the upregulation of MCAM by ET-1 and related isoforms. MCAM mRNA abundance is also increased by ET-1 stimulation, thus the mechanism of MCAM protein upregulation may occur at the level of transcription. Our previous studies have demonstrated that ET-1 downregulates E-cadherin in melanocytes and melanoma cells. Since E-cadherin is a melanoma invasion suppressor, and MCAM is a melanoma invasion promoter, ET-1 may promote melanoma invasion and metastasis through the regulation of adhesion molecule expression.

In previous studies, GnT-V, the enzyme responsible for producing beta 1,6-branched oligosaccharides, was associated with metastasis in carcinomas of the breast and colon. Beta 1,6-branched oligosaccharides (detected with the lectin, LPHA) were common to 22 types of human cancers, and were particularly evident in metastases. Some of the glycoprotein substrates for GnT-V were identified, and included matriptase, a proteolytic inducer of both the HGF/cmet and plasminogen activator pathways, highly associated with metastasis. A potential prometastatic effect of GnTV was due to stabilization of active matriptase through beta 1-6 branching. Thus, GnT-V might function as an initiator of metastatic pathways through regulation of matriptase. Matriptase has been detected in several types of cancers, however, little is known of its status in melanoma. Here we report studies on matriptase, GnT-V, and beta 1,6-branched oligosaccharides in pathology specimens of malignant melanoma from a small cohort of Japanese patients (n ¼ 10). Sequential sections were stained with anti-GnT-V, anti-matriptase, or biotinylated LPHA. In seven of 10 cases, the stains corresponded to the same regions of the tumors. Patterns varied between tumors, from involving only small regions, to encompassing the entire tumor. Thus, in the majority of cases, matriptase, GnT-V, and beta 1,6-branched oligosaccharides were closely associated, suggesting that metastatic pathways might have been activated in these tumors. To our knowledge, this is the first report of matriptase expression in melanoma, and also the first demonstration of an association between matriptase and beta 1,6-branching in vivo.