Early to intermediate steps of tumor embolic formation involve specific proteolytic processing of E-cadherin regulated by Rab7

• Published in: Molecular cancer research 2012-06, Vol.10 (6), p.713-726
• Main Authors: Ye, Yin, Gao, Jian-Xin, Tian, Honglin, Yearsley, Kurtis, Lange, Alicia R, Robertson, Fredika M, Barsky, Sanford H
• Format: Article
• Language: English
• Subjects: Amyloid Precursor Protein Secretases - genetics; Amyloid Precursor Protein Secretases - metabolism; Animals; Blotting, Western; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cadherins - genetics; Cadherins - metabolism; Calpain - genetics; Calpain - metabolism; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic - genetics; Cell Transformation, Neoplastic - metabolism; Endosomal Sorting Complexes Required for Transport - genetics; Endosomal Sorting Complexes Required for Transport - metabolism; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, SCID; Neoplastic Cells, Circulating - metabolism; Oligonucleotide Array Sequence Analysis; Phosphoproteins - genetics; Phosphoproteins - metabolism; Proteolysis; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - metabolism; Receptor, Notch3; Receptors, Notch - genetics; Receptors, Notch - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Spheroids, Cellular - metabolism; Spheroids, Cellular - pathology; Transplantation, Heterologous
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-12-0009

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. Using MARY-X, a human breast cancer xenograft that exhibits florid lymphovascular emboli in mice 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, decreased Hgs and Rab7, 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 γ-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, γ-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 increased N1icd. Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increased N3icd, 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 compete with γ-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of γ-secretase to Notch 3, resulting in increased Notch 3 signaling in the well-formed spheroids.