Angiogenesis , Metastasis , and the Cellular Microenvironment Early to Intermediate Steps of Tumor Embolic Formation Involve Speci fi c Proteolytic Processing of E-Cadherin Regulated by Rab 7

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. UsingMARY-X, a human breast cancer xenograft that exhibitsflorid lymphovascular emboli inmice and spheroids in vitro, we established a model where the in vitro transition stages from minced tumoral aggregates to well-formed spheroids served as a surrogate for in vivo emboli formation. MARY-X well-formed spheroids and emboli exhibited strong similarity of expression. The aggregate-to-spheroid transition stages were characterized by increased ExoC5, decreasedHgs andRab7, increased calpains, increased full-length E-cadherin (E-cad/FL), and the transient appearance of E-cad/NTF2, a 95 kDa E-cadherin fragment and increased Notch3icd (N3icd), the latter two fragments produced by increased g-secretase. Both transient and permanent knockdowns of Rab7 in MCF-7 cells increased protein but not transcription of E-cad/FL and resulted in the de novo appearance of E-cad/NTF2, the presence of nuclear E-cad/CTF2, and increased Notch1icd (N1icd). Overexpression of Rab7 conversely decreased E-cad/FL, g-secretase (PS1/NTF), and E-cad/NTF2. Overexpression of calpains did not alter PS1/NTF but decreased E-cad/FL and E-cad/NTF2 and increasedN1icd.Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increasedN3icd, the latter two fragments being the direct and indirect consequences, respectively, of increased calpains (calpain 1 and calpain 2). Inhibition of calpains decreased E-cad/NTF1 but increased E-cad/NTF2 showing that calpains competewith g-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of g-secretase toNotch 3, resulting in increasedNotch 3 signaling in thewell-formed spheroids.MolCancer Res; 10(6);