Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis.
BACKGROUND
Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other.


METHODS
To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior.


RESULTS
All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells.


CONCLUSIONS
The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.
Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, 1151 Richmond Street, London, Ontario, Canada.
Michel S
MS
Beausoleil
Erika B
EB
Schulze
David
D
Goodale
Carl O
CO
Postenka
Alison L
AL
Allan
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D053495:Osteopontin / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D015843:Serpins / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
D016023:Integrins; C085344:SERPIN-B5; D015843:Serpins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D053495:Osteopontin; D013925:Thromboplastin; D013917:Thrombin
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