MUC1 is a transmembrane glycoprotein which is typically expressed at the apical membrane of normal epithelial cells. In cancer cells, the over-expression of MUC1 and its aberrant localization around the cell membrane and in the cytoplasm favours its interaction with different protein partners such as epidermal growth factor receptor (EGFR) and can promote tumour proliferation through the activation of oncogenic signalling pathways. Our aims were to study the mechanisms inducing MUC1 cytoplasmic localization in pancreatic cancer cells, and to decipher their impact on EGFR cellular localization and activation. Our results showed that galectin-3, an endogenous lectin, is coexpressed with MUC1 in human pancreatic ductal adenocarcinoma, and that it favours the endocytosis of MUC1 and EGFR. Depletion of galectin-3 by RNA interference increased the interaction between MUC1 and EGFR, EGFR and ERK-1,2 phosphorylation, and translocation of EGFR to the nucleus. On the contrary, silencing of galectin-3 led to a decrease of cyclin-D1 levels and of cell proliferation. The galectin-3-dependent regulation of MUC1/EGFR functions may represent an interesting mechanism modulating the EGFR-stimulated cell growth of pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is thought to derive from the epithelial ductal cells of which the apical pole is facing the lumen of the pancreatic ducts. In physiological state, the transmembrane mucin MUC1 is expressed at the apical pole where it protects the cell, senses the environment and modulates signal transduction notably by interacting with EGFR. In tumor cells, the localization of MUC1 glycoprotein is modified. MUC1 expression at the membrane becomes circumferential and accumulates in the cytoplasm. This sequestration is thought to deliver oncogenic signals to the cell, and is used by pathologists on fine-needle aspirates of pancreatic lesions as an indicator of malignancy. However, the mechanisms explaining the modified topography of MUC1 expression in cancer cells are still unknown. Since it was previously demonstrated that endogenous lectins from the galectin family, especially galectin-3 and −4, control the apical targeting of glycoproteins in epithelial cells, our aim was to study the role of galectin-3 in the control of MUC1 expression topography in PDAC, and in the cell invasiveness in vitro. To this end we stably and selectively knocked-down (KD) galectin-3 expression by shRNA approach in the pancreatic polarized CAPAN-1 cells. Three different shRNA targeting LGALS3 mRNA were designed (sh1, sh2, sh3) together with a scramble control one (sc). Sh1 and sh3 cells were selected on the basis of the efficiency of the KD evaluated both at RNA and protein levels. Galectin-3 down-regulation is associated with a decrease of both MUC1 and EGFR protein levels. However, sh1 and sh3 cells expressed 2-fold higher levels of phosphoEGFR in response to EGF treatment than sc cells. The localization of MUC1 and EGFR was studied by confocal microscopy. In sc cells, MUC1 staining mainly concerns the apical surface and the cytoplasm as expected. In sh1 cells, invalidation of galectin-3 led to a strong decrease of MUC1 cytoplasmic labeling. To check the clinical relevance of our data, we performed MUC1 and galectin-3 immunostaining on 16 human PDAC surgically removed in our hospital. In all PDAC, 100% of the tumor cells exhibited a positive and strong MUC1 immunostaining that concerned the cell cytoplasm. By contrast, in normal ductal cells MUC1 staining was restricted to the apical side. Galectin-3 staining was weaker and more heterogeneous than that of MUC1. In all PDAC, 5-80% of the tumor cells were galectin-3 positive with a variable intensity of the immunostaining. Finally, sh1, sh3 and sc cell invasiveness was assessed in vitro on collagen type I matrix. Preliminary results showed that sh1 and sh3 cells lost their in vitro invasive phenotype. In conclusion, our data showed that galectin-3 is responsible for MUC1 cytoplasmic retention in pancreatic tumor cells. Silencing of galectin-3 promotes MUC1 localization at the cell membrane, and increases EGF-induced EGFR phosphorylation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1056.