Increased homotypic adhesion and resistance to apoptosis of lymphovascular tumor emboli is due to selective proteolytic processing of E‐cadherin rather than its mere overexpression

Abstract

Previous studies with a human xenograft model of inflammatory breast cancer, MARY‐X, demonstrated that overexpressed E‐cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super‐high density in vitro. Because of the uniqueness of this model we decided to examine E‐cadherin more closely. In both MARY‐X and the MARY‐X spheroids, Western blot revealed 5 E‐cadherin bands: one full length (120 kDa) and four fragments: E‐cad/NFT1 (100 kDa), E‐cad/NTF2 (95 kDa), E‐cad/NTF3 (85 kDa), E‐cad/NTF4 (80kDa). Compared to MARY‐X, NFT1 was dramatically increased in the MARY‐X spheroids whereas NFT2‐4 were significantly decreased. These E‐cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ‐secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY‐X spheroids. NFT1 generated by increased calpain activity retained the p120ctn binding site but lost both the β‐catenin and α‐binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E‐cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. This work was supported by the U.S. Army Breast Cancer Research Program grant W81XWH‐06‐1‐0631.