Abstract
Activation of focal adhesion kinase (FAK), overexpressed in several human cancers, induces survival, proliferation and motility of cells in culture, but its functional importance in human tumor growth in vivo has not been elucidated. I explored the role of FAK in regulating tumorigenicity of human carcinoma cells, HEp3. These cells overexpress urokinase receptor (uPAR) which, by activating α5β1 integrin, initiates an intracellular signal through FAK and Src leading to ERK activation and tumorigenicity in vivo. Down regulation of uPAR in these cells led to a ∼3–5-fold reduction in FAK phosphorylation and association with Src and dormancy in vivo. Both FAK phosphorylation and ability to grow in vivo were restored by re-expression of uPAR. The FAK signaling pathway in T-HEp3 cells, measured by FAK phosphorylation, GTP-loaded Ras and ERK activation, was inhibited by transient or stable transfection of FAK related non-kinase (FRNK), known to have a dominant negative function, but not by a FRNK mutant version (S1034-FRNK). Most importantly, while vector- and mutant-S1034-FRNK transfected cells inoculated onto chicken embryo CAMs formed progressively growing tumors, the HA-FRNK-expressing T-HEp3 cells did not proliferate in vivo and remained dormant for at least 6 weeks. Both cell types had similar rate of apoptosis in vivo and the p38SAPK or PI3K-Akt signaling pathways were unaffected by FRNK. FRNK induced dormancy could be reverted by expression of an active-R4F-Mek1 mutant. These results show that active FAK is an important mediator of uPAR-regulated tumorigenicity of HEp3 cells and that interruption of FAK mitogenic signaling either through down-regulation of uPAR or by expression of FRNK can force human carcinoma cells into dormancy.
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Abbreviations
- Ab:
-
antibody
- CAM:
-
chorioallantoic membrane
- co-IP:
-
coimmunoprecipitate
- DMSO:
-
dimethylsulfoxide
- ECL:
-
enhanced chemiluminescence ERK, extracellular regulated kinase
- FAK:
-
focal adhesion kinase
- FBS:
-
fetal bovine serum
- FN:
-
fibronectin
- GPI:
-
glycosyl-phosphatidylinositol
- GST:
-
glutathione-S-transferase, HA-ERK hemaglutinin-tagged ERK
- HA-FRNK:
-
hemaglutinin tagged FAK related non kinase
- IF:
-
immunofluorescence
- IP:
-
immunoprecipitation
- MAPK:
-
mitogen-activated protein kinase
- p38:
-
p38-MAPK/stress activated protein kinase 2
- PBS:
-
phosphate buffered saline
- PKB:
-
protein kinase B
- PI3K:
-
phosphatidylinositol-3′-kinase, uPA, urokinase-type plasminogen activator
- uPAR:
-
uPA receptor
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Acknowledgements
I am deeply grateful to Dr Liliana Ossowski (Mount Sinai School of Medicine) for continuous encouragement, support and critical reading of the manuscript. I would also like to thank Dr Rafael Mira y Lopez (Mount Sinai School of Medicine) for helpful discussion and critical reading of the manuscript and Dr Dusko Ilic (UCSF) for his helpful and expert advice. The help of Mr Yeriel Estrada and rotating PhD student Lic. Luciana Giono with some of the experiments, is greatly acknowledged. I also thank Dr David Schlaepfer (Scripps Institute, CA, USA) for providing the HA-FRNK and HA-FRNK-S1034 constructs, Dr Fillipo Giancotti (Memorial Sloan Kettering Cancer Center, NY, USA) for the HA-ERK2 construct, Dr Michael Ploug (Finsen Laboratory, Copenhagen, Denmark) for the gift of R2 antibodies, Dr Irwin Gellman (Mount Sinai School of Medicine) for the anti-Src mAb and helpful discussion and Dr Natalie Ahn for the R4F-Mek construct. Confocal laser scanning microscopy was supported with funding from an NSF Major Research Instrumentation grant (DBI-9724504) of the Mount Sinai School of Medicine Microscopy Center. This work was supported by US Public Health Research grant CA-40758 to Dr Liliana Ossowski and by a Charles H Revson Foundation Postdoctoral Fellowship to Dr Julio Aguirre-Ghiso.
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Aguirre Ghiso, J. Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo. Oncogene 21, 2513–2524 (2002). https://doi.org/10.1038/sj.onc.1205342
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DOI: https://doi.org/10.1038/sj.onc.1205342
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