The use of cystatin C to inhibit epithelial-mesenchymal transition and morphological transformation stimulated by transforming growth factor-beta.
BACKGROUND
Transforming growth factor-beta (TGF-beta) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-beta-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-beta. Recent findings suggest that therapeutics designed to antagonize TGF-beta signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-beta type II receptor antagonist that inhibits TGF-beta binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-beta, particularly its stimulation of mammary epithelial-mesenchymal transition (EMT), can be prevented by CystC.


METHODS
Retroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Delta14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-beta stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-beta stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-beta.


RESULTS
CystC and Delta14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-beta by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-beta-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Delta14CystC also inhibited TGF-beta signaling in two tumorigenic human breast cancer cell lines.


CONCLUSIONS
Our findings show that TGF-beta stimulation of initiating metastatic events, including decreased cell polarization, reduced cell-cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-beta.
Sokol
Jonathan P
JP
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA. jpsokol@uwalumni.com
Neil
Jason R
JR
Schiemann
Barbara J
BJ
Schiemann
William P
WP
D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D055316:Cystatin C; D015891:Cystatins / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005260:Female; D006801:Humans; D007668:Kidney; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C525030:CST3 protein, human; C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D011480:Protease Inhibitors; D016212:Transforming Growth Factor beta