Abstract
LAM is a rare low-grade metastasizing lung neoplasm. Inhibitors of mTOR improve clinical outcome of LAM patients by preventing loss of lung function. Nevertheless, other cell targets may be of interest for drug development. Therefore, we explored the potential role of EDN1 (endothelin) in LAM. We report an increased endothelin blood level in LAM patients as well as EDN1 overexpression and EDN1 receptor downregulation in LAM-derived primary cells and in TSC2NEG cells mutated in TSC2. We evidenced EDN pathway dysregulation based on EDN1, EDNRA, EDNRB and ARRB1 mRNA expression in LAM-derived primary cells. We showed overexpression of EDN1 and ARRB1 mRNAs in TSC2NEG cells; these cells lost their ability to respond to stimulation by endothelin. We analyzed the effects of endothelin receptor antagonists alone or in combination with rapamycin, an mTOR inhibitor, on proliferation and migration of LAM cells. Rapamycin treatment of TSC2NEG cells significantly reduced cell proliferation or migration, while none of the tested inhibitors of EDN receptors impaired these functions. We showed that TSC2NEG cells have acquired a transformed phenotype as showed by their ability to grow as spheroids in semi-solid medium and that unlike endothelin receptors antagonists, rapamycin reduced anchorage-independent cell growth and prevented expansion of TSC2NEG spheroids.
Highlights
Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease mainly affecting young women[1]
Primary LAM cells expressed α-smooth muscle actin (α-SMA), vimentin and rarely desmin (Fig. 2A) and they were hardly positive for the melanocytic markers HMB45 and PNL2
LAM cells are characterized by increased proliferation associated to TSC1 and TSC2 mutations, leading to the activation of the mTOR pathway[30]
Summary
Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease mainly affecting young women[1]. High expression levels of EDN1 (Endothelin 1) and of endothelin receptors A and B (EDNRA and EDNRB) are associated with the increase of circulating VEGF and of microvessel density[16,17,18,19]. The EDN1/EDNR/ARRB1 (β Arrestin 1) pathway is implicated in cell proliferation, migration, invasion, survival and angiogenesis in several diseases, among them lung, ovary, prostate and breast cancers[20,21]. We explored the role of EDN1 and of its receptors in LAM-derived primary cells and in angiomyolipoma-derived cells lines. We report an increased blood level of endothelin in LAM patients as compared to controls, and the overexpression of EDN1 and downregulation of its receptors in LAM-derived primary cells as well as in TSC2NEG cell lines. We analyzed the effects of ERAs, alone or in combination with rapamycin, on LAM cell proliferation and migration
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