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Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model

Oncogene volume 25, pages 4429–4440 (2006)Cite this article

Abstract

Expression of focal adhesion kinase (FAK) is elevated in malignant breast cancer, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. Here, we have inhibited FAK activity or expression in murine 4T1 breast carcinoma cells via dominant-negative focal adhesion kinase-related non-kinase (FRNK) or anti-FAK short hairpin RNA (shRNA) expression, respectively. Neither FRNK nor FAK shRNA (∼80% reduced FAK levels) affected 4T1 proliferation in culture, whereas reduced FAK activity or expression blocked 4T1 cell invasion through Matrigel and resulted in 2–3-fold lower urokinase plasminogen activator (uPA) expression. Control 4T1 cells implanted into mammary fat pads of BALB/c mice exhibited spontaneous metastasis to the lungs, to the peritoneal cavity, and resulted in 90% lethality within 21 days. Whereas FAK shRNA-expressing 4T1 cells formed tumors in mice with low levels of apoptosis, when mammary-injected, these cells did not exhibit lung metastasis after 21 days and caused only 40% lethality up to 60 days. Transient re-expression of wild-type but not kinase-dead FAK in 4T1 FAK shRNA cells promoted uPA production and mammary to lung metastasis within 7 days. In fact, stable human uPA overexpression in 4T1 FAK shRNA cells promoted Matrigel invasion and lung metastasis equal to 4T1 controls. Conversely, treatment with plasminogen activator inhibitor-1 or neutralizing antibody to uPA blocked Matrigel invasion of 4T1 control cells. These studies provide the first direct proof that FAK catalytic activity can facilitate metastatic breast cancer progression by regulating uPA expression.

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Abbreviations

7-AAD:

7-amino-actinomycin D

Ad:

adenoviral

CMV:

cytomegalovirus

FACS:

fluorescence-activated cell sorting

FAK:

focal adhesion kinase

FRNK:

FAK-related non-kinase

GFP:

green fluorescent protein

HA:

hemagglutinin

IPs:

immunoprecipitates

kDa:

kilodalton

MMP:

matrix metalloproteinase

shRNA:

short hairpin RNA

Tyr:

tyrosine

uPA:

urokinase plasminogen activator

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Acknowledgements

We thank Theresa Villalpando for administrative help and S Huang for advice on uPA antibodies and analyses. S Mitra was supported in part by a fellowship (12FT-0122) from the California Tobacco-Related Disease Research Program. D Schlaepfer is an Established Investigator of the American Heart Association (0540115N) and these studies were supported by grants CA75240, CA87038 and CA102310. This is manuscript 17904-IMM from The Scripps Research Institute.

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  1. Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA

    S K Mitra, S-T Lim, A Chi & D D Schlaepfer

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Correspondence to D D Schlaepfer.

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Mitra, S., Lim, ST., Chi, A. et al. Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model. Oncogene 25, 4429–4440 (2006). https://doi.org/10.1038/sj.onc.1209482

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