Early to intermediate steps of tumor embolic formation involve specific proteolytic processing of E-cadherin regulated by Rab7.
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.
Department of Pathology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Yin
Y
Ye
Jian-Xin
JX
Gao
Honglin
H
Tian
Kurtis
K
Yearsley
Alicia R
AR
Lange
Fredika M
FM
Robertson
Sanford H
SH
Barsky
D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D034622:RNA Interference; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D014183:Transplantation, Heterologous; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D015820:Cadherins; D056827:Endosomal Sorting Complexes Required for Transport; C499374:NOTCH3 protein, human; D010750:Phosphoproteins; D000071656:Receptor, Notch3; D051880:Receptors, Notch; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C000716465:rab7 GTP-binding proteins, mouse; D053829:Amyloid Precursor Protein Secretases; D002154:Calpain; D020691:rab GTP-Binding Proteins