Extracellular matrix degradation is one of the crucial steps in cancer cell invasion and spreading. A number of proteases, including plasmin, mediate disruption of stromal barriers and basement membrane and thus facilitate tumor cell movement. Formation of plasmin is a result of the plasminogen (PLG) activation cascade, which involves PLG activators and receptors. Enolase-1 (ENO-1) is one of the plasminogen receptors (PLG-R). It belongs to the so called moonlighting protein group , which exhibits various functions at distinct cellular and extracellular sites of the cell. This primary glycolytic enzyme was found to be overexpressed in more than 20 types of human cancer and accounts for enhanced cancer progression and poor clinical outcome. Although numerous studies provide evidence for pro-tumorigenic properties of cytoplasmic ENO-1, the contribution of cell surface bound ENO-1 to cancer progression has not yet been described. Here, we demonstrate increased expression of ENO-1 in different types of human cancer, in particular, in breast ductal carcinoma. Cell fractionation of the breast cancer cells (MDA-MB-231) revealed elevated ENO-1 cell surface levels, which correlated with enhanced migratory and invasive properties of these cells. Overexpression of wild-type ENO-1 increased invasion of MDA-MB-231 cells. This effect was not observed when ENO-1 mutant bearing the mutation in a PLG binding site was overexpressed. Exposure of MDA-MB-231 cells to LPS further potentiated ENO-1 cell surface expression and simultaneously increased release of ENO-1 to the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was diminished upon pretreatment of MDA-MB-231 cells with the Ca2+ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca2+-ATPase pump, cyclopiazonic acid. In line with this observation, STIM1 and ORAI1 were found to regulate LPS-induced ENO-1 cell surface expression and release. Accordingly, pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of breast cancer cells. Collectively, these data reveal the functional consequence of extracellulary localized ENO-1 in cancer cell behaviour and the mechanism which drives ENO-1 exteriorization. Thus, targeting cell surface bound ENO-1 may offer a novel therapeutic strategy in patients suffering from cancer.
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