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Distinct FAK-Src activation events promote α5β1 and α4β1 integrin-stimulated neuroblastoma cell motility

Oncogene volume 27, pages 1439–1448 (2008)Cite this article

Abstract

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that α4 and α5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either α4β1- or α5β1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that α5β1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for α5β1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, α4β1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-α overexpression inhibited α4β1-stimulated NB motility and Src activation consistent with α4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In α4 shRNA-expressing NB cells, α4β1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated α4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that α4β1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during α5β1-mediated NB migration and support the evaluation of inhibitors to α4, Src and FAK in the control of NB tumor progression.

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Abbreviations

Ad:

adenovirus

BSA:

bovine serum albumin

cFN:

cellular fibronectin

CS-1:

connecting segment-1

DM:

double mutant

FACS:

fluorescence activated cell sorting

FAK:

focal adhesion kinase

FBS:

fetal bovine serum

FN:

fibronectin

GFP:

green fluorescent protein

GST:

glutathione-S-transferase

IP:

immunoprecipitation

KD:

kinase dead

LacZ:

β-galactosidase

mAb:

monoclonal antibody

MEFs:

mouse embryonic fibroblasts

moi:

multiplicity of infection

NB:

neuroblastoma

PTK:

protein-tyrosine kinase

PTPα:

receptor protein-tyrosine phosphatase-α

pTyr:

phosphotyrosine

VCAM-1:

vascular cell adhesion molecular 1

WCL:

whole cell lysate

WT:

wild type

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Acknowledgements

We thank the laboratory of Mark Ginsberg for various α4 fusion proteins and we greatly appreciate the administrative assistance provided by Theresa Villalpando. Y Lim was supported in part by Korea Research Foundation Grant (M01-2005-000-10071-0). This work was supported by grants from the NIH to David Schlaepfer (CA75240, CA87038, CA102310), to Dwayne Stupack (CA107263) and from the Canadian Institutes of Health Research to Catherine Pallen (MOP-49410). Nai-Kong Cheung is supported by CA106450 and the Robert Steel Foundation. D Schlaepfer is an American Heart Association Established Investigator (0540115N). This manuscript is dedicated to the memory of Jaewon Han Ph.D. whose work stimulated our interest into the novel aspects of α4 integrin signaling.

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Author notes

  1. L Wu, S-T Lim, S Uryu & D D Schlaepfer

    Present address: 6Current Address: Moores UCSD Cancer Center, Department of Reproductive Medicine, La Jolla, CA, USA.,

  2. J A Bernard-Trifilo

    Present address: 7Current Address: Chemicon-Millipore, Temecula, CA, USA.,

  3. L Wu and J A Bernard-Trifilo: These authors contributed equally to this study.

Authors and Affiliations

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

    L Wu, J A Bernard-Trifilo, Y Lim, S-T Lim, S K Mitra, S Uryu & D D Schlaepfer

  2. Department of Pediatrics and the Child and Family Research Institute, University of British Columbia, Vancouver, Canada

    M Chen & C J Pallen

  3. Department of Pediatric Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    N-Kv Cheung

  4. Department of Pathology, Moores UCSD Cancer Center, University of California, San Diego, CA, USA

    D Mikolon, A Mielgo & D G Stupack

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Correspondence to D D Schlaepfer.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Wu, L., Bernard-Trifilo, J., Lim, Y. et al. Distinct FAK-Src activation events promote α5β1 and α4β1 integrin-stimulated neuroblastoma cell motility. Oncogene 27, 1439–1448 (2008). https://doi.org/10.1038/sj.onc.1210770

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