Macrophage migration inhibitory factor is differentially expressed in normal and choriocarcinoma trophoblast cells.

Trophoblast cells are specific for placenta, the organ necessary for development of the fetus. Trophoblast derived choriocarcinoma is a rare cancer, with high metastatic potential, invading surrounding tissues and distant organs. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in a wide range of biological processes, which is increased in almost all human cancers. Expression of MIF in normal and choriocarcinoma trophoblast cells is investigated here, using normal extravillous trophoblast derived cell line HTR-8/SVneo, and choriocarcinoma cell lines JAR and JEG3. Expression of MIF and its receptors CD74 and CXCR2 was investigated at mRNA level using qPCR. Expression of MIF protein was studied using immunofluorescence and western blot, under reducing and native conditions, in whole cell lysates, subcellular fractions and conditioned media. The expression of MIF mRNA was similar in all three cell lines, while CD74 mRNA was more expressed in choriocarcinoma cells (14-fold for JAR, 12-fold for JEG3, p<0.01). CXCR2 mRNA was higher in JEG3 cell line compared to HTR-8/SVneo cells (6-fold, p<0.01). While the cellular level of MIF was similar, the level of secreted MIF was lower in JAR cell conditioned media compared to media of both HTR-8/SVneo (2.8-fold, p<0.01) and JEG3 cells (4.1-fold, p<0.001). Cellular distribution of MIF was similar between the studied cell types. MIF was predominantly cytoplasmic, but also detected in membrane, nuclear soluble and nuclear chromatin fraction. MIF appeared in high molecular weight complexes of >150 kDa under native conditions. A band of 140-145 kDa was consistently present in JEG3 cell lysates, while it was absent or very weak in other cell types. These results show that MIF/CD74 axis is shifted in choriocarcinoma, as previously shown for other cancers, and further justifies research towards the most effective MIF targeting therapeutics.

Human trophoblast requires galectin-3 for cell migration and invasion.

Invasive extravillous cytotrophoblast of the human placenta expresses galectins-1, -3, and -8 in vivo and in vitro. This study aimed to investigate the potential role of galectin-3 in cell migration and invasion, using recombinant human galectin-3 (rhgalectin-3), small molecule galectin inhibitor I47, and galectin-3 silencing. HTR-8/SVneo cell migration was stimulated by rhgalectin-3 and reduced by I47, which could be neutralised by rhgalectin-3. Inhibitor specificity and selectivity for the galectins expressed in extravillous trophoblast were validated in solid phase assays using recombinant galectin-1, -3, -8, confirming selectivity for galectin-3. HTR-8/SVneo cell migration and invasion, and invasion by isolated trophoblast cells in primary culture were significantly reduced in the presence of I47, which could be restored by rhgalectin-3. Upon HTR-8/SVneo cell treatment with galectin-3 siRNA both LGALS3 and galectin-3 protein were dramatically decreased. Silencing of galectin-3 induced significant reduction in cell migration and invasion, which was restored by rhgalectin-3. The influence on known mediators of cell invasion, MMP2 and -9, and integrins α1, α5, and ß1 was followed in silenced cells, showing lower levels of MMPs and a large reduction in integrin subunit ß1. These results show that galectin-3 acts as a pro-invasive autocrine/paracrine factor in trophoblast in vitro.