Abstract 1776: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy due to the selective proteolytic processing of E-cadherin
Authors: Ye Yin , Xiao Yi , Yearsley Kurtis , Barsky Sanford H. 
Published: February 2011

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. In vitro, cells in MARY-X spheroids grow very slowly and can not be cultured as attached monolayers. Our previous data showed that exogenous growth factors could not stimulate the growth of the spheroids in vitro. Whereas treatment with EGF, IGF-1 or TGF-β increases the level of phosphorylated Akt in numerous cell lines which grow as monolayers, treatment of intact MARY-X spheroids with these growth factors actually decreases the levels of phosphorylated Akt. With EGF treatment, while the levels of phosphorylated EGFR increased in the spheroids, indicating that the treatment with EGF results in phosphorylation of its receptor, there is no downstream signaling through Akt. However when the cells within the MARY-X spheroids are dissociated into single cells by a number of different maneuvers, EGF treatment both phosphorylates its receptor and increases the levels of phosphorylated Akt. The results with the other growth factors are similar. It had been reported that E-cadherin-mediated adhesion may inhibit ligand-dependent activation of diverse growth factor receptor tyrosine kinases by decreasing receptor mobility and / or ligand-binding affinity. Because of the uniqueness of the MARY-X model and the role of E-cadherin in possibly regulating this growth factor signaling, 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. Both of these manipulations converted the cells from being growth factor refractory to growth factor sensitive. The selective proteolytic processing of E-cadherin ultimately then is responsible for the state of quiescence exhibited by the MARY-X spheroids and the associated lymphovascular emboli.

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 1776.