Investigating functions of ERp29 in mesenchymal to epithelial transition (MET) and epithelial plasticity in breast cancer cells

Abstract

Endoplasmic Reticulum protein-29 (ERp29) is a chaperone protein that functions in the unfolding and escort of secretory proteins. Like other reticuloplasmins, ERp29 is believed to be involved in carcinogenesis. In breast cancer, expression of ERp29 is downregulated and there exists a negative association between level of ERp29 and breast cancer stage/grade. To elucidate the role of ERp29 in breast cancer progression, aggressive breast cancer cells - MDA-MB231 and BT549 - were stably transfected with ERp29-expressing vectors. Upon isolation of single stable clones, morphological change from a spindle-like fibroblastic to a typical cobble-stone-like epithelial phenotype was observed in both ERp29-overexpressing MDA-MB231 and BT549 clones. This phenomenon is reminiscence of mesenchymal to epithelial transition (MET).

In malignancy, epithelial to mesenchymal transition (EMT) is believed to facilitate metastasis by medicating cells¿ escape from primary tumors. Its reverse, MET, has been considered both as counteract of EMT, thus preventing metastasis, as well as a mechanism employed by escaped cells to establish metastatic tumors at secondary sites, thus supporting metastasis. EMT/MET is characterized by morphological, molecular or behavioral changes in cells. In addition to the morphological change mentioned above, overexpression of ERp29 in MDA-MB231 cells induced behavioral changes typified by decrease in expression of mesenchymal cell markers (vimentin and fibronectin) and increase in expression of epithelial cell markers (E-cadherin, ß-catenin, and cytokeratin-19). These changes were believed to be brought upon downregulation of E-cadherin repressors (SNAI1, SNAI2, ZEB2, and Twist). Furthermore, ERp29-overexpressing MDA-MB231 clones exhibited lower migration and invasion capacity, indication of behavioral MET. In contrast, overexpression of ERp29 in BT549 cells only reduced the expression of fibronectin without changes in other markers and transcriptional repressors, as well as in cells¿ behavior.

Further investigation into the morphologic MET revealed that the morphological alterations observed in both cell lines were characterized by rearrangement of actin cytoskeleton, from stress fiber to cortical actin formation. In addition, mechanistic studies demonstrated that the levels of tight junction protein, ZO-1, and apical-basal polarity proteins, Par3 and Scribble, were markedly increased by ERp29 and mainly localized at the membrane to enhance cell-cell contact and polarization. However, other polarity proteins, including CDC42, Par6 and aPKC, did not seem to be involved in the ERp29-induced epithelial morphogenesis.

These findings demonstrated a novel function and mechanism of ERp29 in regulating epithelial plasticity. Though the consequences varied between cell lines (complete MET in MDA-MB231 cells and incomplete MET in BT549 cells), several common features were observed upon ERp29 overexpression; including rearrangement of actin cytoskeleton, regulation of cell-cell junctions, as well as cell polarization. Taken together, overexpression of ERp29 could reprogram aggressive breast cancer cells to induce MET and thus regulate metastasis.