A Role for Endothelin-2 and Its Receptors in Breast Tumor Cell Invasion

Abstract

We have studied the role of endothelins (ET-1, ET-2 and ET-3) and ET receptors (ET-RA and ET-RB) in the invasive capacity of breast tumor cells, which express ET-1 and ET-2 as well as ET-RA and ET-RB. Of five human breast tumor cell lines tested, all expressed mRNAs for ET-1, ET-2, and ET-RB. ET-RA mRNA was expressed by four of five tumor cell lines. Breast tumor cells migrated toward ET-1 and ET-2 but not toward ET-3. Chemotaxis involved signaling via both receptors, and a pertussis toxin-sensitive p42/p44 mitogen-activated protein kinase (MAPK)-mediated pathway that could be inhibited by MAPK kinase (MEK)1/2 antagonists. Chemotaxis toward ETs did not involve p38 or stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) and was not inhibited by hypoxia. Incubation of tumor cells with ET-2 also increased chemotaxis toward the chemokines CXCL12 and CCL21. As well as inducing chemotaxis of tumor cells, ET-1 and ET-2 increased tumor cell invasion through Matrigel. Furthermore, stimulation of macrophage/tumor cell cocultures with ETs led to increased matrix metalloproteinase (MMP)-2 and -9 production by macrophages and a marked increase in invasion of tumor cells. Antagonism of either ET-RA or ET-RB decreased the invasion seen in ET-stimulated cocultures, as did a broad-spectrum MMP inhibitor. Immunohistochemical staining of human breast tumor sections showed increased ET and ET receptor protein expression by tumor cells in invasive ductal carcinoma compared with normal breast tissue or ductal carcinoma in situ. Furthermore, tumor cell ET and receptor expression was stronger at the invasive margin of invasive ductal carcinomas, in the lymphovascular space, and in lymph node metastases. ET expression often colocalized with ET-RB expression in all neoplastic tissue indicating a possible autocrine action of ETs. We suggest that expression of ETs and their receptors by human breast tumors, particularly in conjunction with a high macrophage infiltrate, may have a role in the progression of breast cancer and the invasion of tumor cells.

Macrophages Promote the Invasion of Breast Carcinoma Cells via a Colony-Stimulating Factor-1/Epidermal Growth Factor Paracrine Loop

“…Consistent with this idea, EGFR-positive breast tumors recruit higher numbers of macrophages (19). Other studies have suggested that matrix metalloproteases (MMP) produced by macrophages may increase the invasion of tumor cells (20,21). However, inhibition of MMPs in our assay did not significantly reduce the amount of invasion.…”

The endothelin system in breast tumour–endothelial cell interactions

“…study are consistent with the previously proposed role for ET in tumour invasion. 3,5,15 Our concentrations of 11.6 pg/mL ± 0.2 pg/mL ET-1 produced by MCF-7 after 24-h incubation are comparable with previous findings where cultured MCF-7 cells, a moderately invasive cell line, produced less than 4 pg/mL after a similar incubation period. 16 Hagemann et al 16 found that the amount of ET-1 produced by different breast tumours correlated with their invasiveness.…”

“…However, it is now recognised that there is a complex interaction between ET-1, tumour cells and other associated cells, such that various cell types not only both produce and are affected by ET, but also influence one another's production of ET. 15 Co-culture of tumour cells with macrophages, for example, leads to increased ET production. 15 Of particular interest in our study was the finding that ET-1 production by MCF-7 increased progressively and significantly when these cells were exposed to increasing amounts of DMVEC metabolites.…”

“…15 Co-culture of tumour cells with macrophages, for example, leads to increased ET production. 15 Of particular interest in our study was the finding that ET-1 production by MCF-7 increased progressively and significantly when these cells were exposed to increasing amounts of DMVEC metabolites. Considering the interplay between different cell types in the tumour environment, and the autocrine and paracrine effects of ET-1, we postulate that DMVECs promote MCF-7 to produce more ET-1, which may then play a paracrine role on the endothelial cells.…”

Endothelins and hypoxia-inducible factor in cancer

“…The function of the ET-RB receptor in tumours is particularly enigmatic; in some cases, such as breast cancer, ligand-binding to ET-RB initiates several pro-tumour actions, such as promoting invasion (Grimshaw et al 2004), yet in prostate cancer, the loss of ET-RB expression is postulated to increase ET-1 peptide in the tumour due to the loss of endothelin clearance function of ET-RB (Nelson et al 1997). Several different cell types within solid tumours may contribute to endothelin expression including not only the tumour cells but also fibroblasts, tumour-associated macrophages (TAMs) and endothelial cells.…”