Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties.
We have proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens that when aberrantly re-expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP-induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non-malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK-related non-kinase (FRNK) under control of a Tet-On (i.e., doxycycline-sensitive) promoter. Under serum-free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP-R antagonist D-Phe(6)(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone-plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time-lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non-deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability.
Department of Medicine, University of Illinois at Chicago and Chicago Veterans Administration Medical Center (West Side Division), Chicago, Illinois 60612, USA.
Sarah
S
Glover
Melissa
M
Delaney
Cecile
C
Dematte
Lori
L
Kornberg
Melissa
M
Frasco
Roger
R
Tran-Son-Tay
Richard V
RV
Benya
D015153:Blotting, Western; D001839:Bombesin / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019886:Gastrin-Releasing Peptide / Q000494:pharmacology* / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D018004:Receptors, Bombesin / Q000502:physiology*
D018004:Receptors, Bombesin; D019886:Gastrin-Releasing Peptide; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D001839:Bombesin