Abstract P5-05-04: E-Cadherin and Its Proteolytic Fragments Trump Epithelial-Mesenchymal Transition and Function as Dominant Oncogenes in Inflammatory Breast Cancer

Abstract

Abstract Background: Inflammatory breast cancer (IBC) and its lymphovascular embolic signature seem to challenge the dogma of prerequisite epithelialmesenchymal transition (EMT) in human breast cancer progression by the apparent maintenance of an epithelial state in all stages of IBC. 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. Although slug, snail and twist are increased in MARY-X spheroids and may decrease E-cadherin transcription to some extent, their effects are neutralized and frankly overridden by the post-transcriptional regulation of E-cadherin which includes its altered degradation and trafficking which, in turn, maintain the epithelial state. Materials and Methods: Because of the uniqueness of the MARY-X model and the observation that E-cadherin was not functioning as a tumor suppressor gene whose loss was triggering EMT but rather whose overexpression was mediating increased survival and resistance to apoptosis, we decided to examine the fate and role of E-cadherin more closely. Results: We discovered that E-cadherin underwent extensive proteolytic processing which resulted in fragments also mediating increased survival and resistance to apoptosis. 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). Corresponding E-cad/CTF1-4 fragments were also generated. 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 generatedby 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. Identical proteolytic fragments of E-cadherin were observed in 3 other human IBC cell lines as well as in lymphovascular emboli of human IBC cases obtained by laser capture microdissection. Discussion: The selective proteolytic processing of E-cadherin ultimately then is responsible for the increased survival of the lymphovascular embolus of IBC and its resistance to chemotherapy-induced apoptosis. Select NT proteolytic fragments may function as adhesion molecules whereas select CT proteolytic fragments may function as transcription factors which confer this survival advantage. E-cadherin and its proteolytic fragments then both trump EMT as well as function as dominant oncogenes in IBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-04.