Multiple Post-Translational Modifications Of E-Cadherin During Er Stress And Apoptosis

188 As a vital cell adhesion molecule, E-cadherin plays an important role in the suppression of tumorigenesis, tumor invasion and metastasis. Because stress and apoptosis are common events in tumors, we examined the regulation of E-cadherin expression during endoplasmic reticulum (ER) stress and apoptosis in the MCF-7 human breast cancer cell line. We used different agents to elicit ER stress and analyzed alterations of E-cadherin in MCF-7 cells by western blotting. To assay glycosylation, wheat germ agglutinin (WGA)-agarose was used to precipitate glycoproteins from cell lysates. To differentiate O-linked and N-linked glycosylation, N-linked carbohydrate was removed from samples by digestion with PNGase. Digestion with the proprotein convertase furin was used to examine pro-protein processing. Here we report that after the induction of ER stress by either thapsigargin or brefeldin A, as well as after induction of apoptosis by a cell-permeable ceramide derivative, both the mature form of E-cadherin (120 kDa) and a multiply modified form of E-cadherin (135 kDa) were detected in MCF-7 cells. PNGase digestion revealed that both forms are N-glycosylated. However, the 135 kDa form bound WGA even after PNGase digestion. Furthermore, pretreatment with tunicamycin to inhibit N-glycosylation did not reduce binding of the 135 kDa form to WGA confirming this form is also O-glycosylated. This form of E-cadherin was cleaved in vitro by furin into a mature form (120 KDa) suggesting it contains an unprocessed pro-region. Moreover, inhibition of furin generated a 135 kDa form of E-cadherin in unstressed cells. Therefore, upregulation of O-glycosylation and inhibition of proprotein processing of E-cadherin occur in response to some ER stress and apoptosis pathways. These modifications block E-cadherin cell surface transport suggesting they might lead to increased metastasis.