Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation
© Crowe and Ohannessian; licensee BioMed Central Ltd. 2004
Received: 15 December 2003
Accepted: 07 May 2004
Published: 07 May 2004
Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines.
Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays.
In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity.
We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.
Integrin extracellular domains bind extracellular matrix molecules such as type IV collagen and laminin while the cytoplasmic domains bind specific components of the actin cytoskeleton . Integrin-extracellular matrix interactions also activate signaling cascades such as mitogen activated protein kinases . Integrin binding to extracellular matrix or integrin crosslinking increases tyrosine phosphorylation of focal adhesion kinase (FAK) . FAK consists of a central catalytic domain and amino and carboxyl terminal non- catalytic domains. A focal adhesion targeting sequence within the carboxyl terminus is required for localization to focal adhesions . FAK undergoes autophosphorylation on a single tyrosine residue which creates a binding site for SH2 containing proteins. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration [5, 6].
Paxillin is a focal adhesion protein which is also phosphorylated by a number of stimuli on tyrosine . Paxillin interacts with FAK at two sites in the amino and carboxyl termini which are not required for targeting paxillin to focal adhesions . A mutant paxillin protein lacking both FAK binding sites exhibits reduced tyrosine phosphorylation . FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of the MAPKs ERK and JNK . However JNK phosphorylation is not dependent on FAK nor on phosphatidylinositol 3-kinase (PI3K) activity.
Transformation of FAK deficient fibroblasts with the v-Src oncogene promotes cellular motility equal to that of FAK re-expression . However, these cells are not invasive and required intact FAK expression to produce this phenotype. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model . These studies indicate that FAK has a key role in tumorigenesis and invasion. We previously demonstrated that PI3K, ERK, and JNK activation were required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines [12, 13]. These studies led us to hypothesize that FAK and paxillin may mediate signals from the substratum via ERK in order to regulate cell migration. In the present study, we show that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. This recruitment correlated with increased cellular migration on this substratum. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and migration through reconstituted basement membrane which was dependent on ERK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration and invasion via the mitogen activated protein kinase pathway.
Cell culture and stable transfection
SCC4 and SCC25 cells were cultured in Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum, 40 μg/ml gentamicin at 37°C in a humidified atmosphere of 5% CO2. Cells were plated on tissue culture plastic dishes or commercially available plates coated with type IV collagen (Becton Dickinson). Some cells were pretreated with blocking antibodies to β1 or β4 integrins (Invitrogen) for 1 hour prior to plating [14, 15]. Other cultures were deprived of attachment by plating on uncoated petri dishes in media containing 1.6% methylcellulose. Cells were transfected with 5 μg expression vector for FAK or paxillin (kindly provided by Dr. Thomas Parsons) or neomycin resistance plasmid alone using Lipofectamine reagent according to manufacturer's recommendations (Invitrogen). Cells were selected in 400 μg/ml G418 for 14 days. Resistant clones were picked for expansion and characterization. FAK and paxillin expression was determined by western blot.
Cultures were lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 1% Nonidet P-40, 10% glycerol, 1 mM NaF, 0.1 mM sodium orthovanadate, and protease inhibitors for 30 minutes at 4°C. Lysates were centrifuged at 10,000 × g for 10 minutes and anti-β1 integrin antibody (Invitrogen) was incubated with the supernatants for 1 hour at 4°C. Antigen-antibody complexes were precipitated with protein A/G agarose beads for 1 hour at 4°C. Samples were boiled in Laemmli buffer for 3 minutes, separated by SDS-PAGE, and blotted to polyvinylidene difluoride membranes. Blots were incubated with anti-FAK, paxillin (Transduction Laboratories), Shc, and Grb2 (Santa Cruz Biotechnology) antibodies to determine relative association with β1 integrin. Blots were then incubated with anti-β1 integrin antibody to ensure equal amounts of immunoprecipitated protein in each lane. Bands were analyzed by laser densitometry and the numerical data subjected to t test.
75 μg total cellular protein was separated by SDS-PAGE on 10% resolving gels under denaturing and reducing conditions. Separated proteins were electroblotted to PVDF membranes according to manufacturer's recommendations (Roche Molecular Biochemicals). Blots were incubated with antibodies to human FAK, paxillin, ERK1, activated ERK1 (Transduction Laboratories), or β1 integrin (Invitrogen) for 16 hours at 4°C. After washing in Tris buffered saline containing 0.1% Tween 20 (TBST, pH 7.4), blots were incubated for 30 minutes at room temperature with anti-IgG secondary antibody conjugated to horseradish peroxidase. Following extensive washing in TBST, bands were visualized by the enhanced chemiluminescence method (Roche Molecular Biochemicals). Bands were analyzed by laser densitometry and the numerical data subjected to t test.
Migration and invasion assays
Cells were plated at confluence on plastic in the presence or absence of type IV collagen for 24–48 hours. Some cells were pretreated with blocking antibodies to β1 or β4 integrins for 1 hour prior to plating. Other cultures were treated with 50 μM PD98059, a selective inhibitor of ERK activation. The dishes were scored with a pipet tip across the center of the plate. The number of cells which migrated into the blank area of the dish during the specified time period was determined by counting of 10 random fields using phase contrast microscopy. For invasion assays, 2 × 105 cells were plated in triplicate into commercially available Matrigel invasion chambers (Becton Dickinson). Cells which migrated through the reconstituted basement membrane after 24 hours were fixed in methanol, stained with hematoxylin, and counted.
The results of this study demonstrated that FAK and paxillin were recruited to the β1 integrin subunit when cells were plated on plastic or type IV collagen. However, the 52 kD isoform of the Shc adaptor protein was present in the complex only when cells were maintained under anchorage deprived conditions. Stratified squamous epithelial cells ultimately undergo terminal differentiation when deprived of attachment , and 52 kD Shc overexpression was sufficient in our study to produce terminal differentiation of cells plated on plastic tissue culture dishes. These results suggest that localization of 52 kD Shc to integrin signaling complexes may regulate terminal differentiation of stratified squamous epithelial cells following loss of basement membrane attachment. Shc is known to regulate growth factor receptor induction of the MAPK pathway ; it will be interesting to determine if MAPK inhibition prevents anchorage deprived terminal differentiation of stratified squamous epithelial cells.
In this context, FAK has been shown to suppress anchorage deprived cell death in MDCK cells . The kinase activity and autophosphorylation site of the protein is required for this effect. FAK inhibition by overexpression of the carboxyl terminal domain causes loss of adhesion and cell death in human breast cancer lines . Similarly, anchorage independent breast cancer cells undergo apoptosis when FAK function is disrupted. Interestingly, the FAK carboxyl terminus has no effect on adhesion or viability of normal mammary epithelial cells. FAK has been shown to activate JNK which then colocalizes at focal adhesions in primary fibroblasts . This activation occurs via a Ras/Rac1/Pak1/MKK4 pathway. In the presence of serum, survival signals are also transduced by PI3K and Akt. These studies suggest that FAK is also required for survival signaling from the extracellular matrix. Our future studies will examine this property of FAK in human SCC lines.
Our results demonstrated that FAK and paxillin induced migration and invasion of SCC lines was dependent on ERK activity. In colon cancer cell lines, cell attachment to collagen or laminin stimulates phosphorylation of FAK and paxillin and activates ERK1 . FAK inhibition reduces attachment dependent ERK1 activation. ERK1 inhibition reduced migration of colon cancer cells , and inhibited growth factor induced migration in SCC lines used in the present study. FAK inhibition also disrupted growth factor stimulated migration of human lung cancer cells . These studies highlight an important role for ERK signaling in mediating collagen induced migration and invasion of human cancer cell lines. FAK expression has been correlated with tumor invasion and lymph node metastasis of cancer cells . In this study, FAK overexpression was detected in 59% of esophageal cancer pathology specimens. This overexpression was associated with decreased cellular differentiation, late clinical stage, increased depth of invasion, poor survival rate, and lymph node metastasis. Future clinical studies may examine FAK inhibition as potential antitumor therapy.
FAK and paxillin were recruited to β1 integrin when cells were attached to substratum while Shc was associated with the receptor in anchorage deprived cells. FAK and paxillin recruitment to β1 integrin correlated with increased migration on type IV collagen. FAK and paxillin overexpression promote migration of human SCC lines which is dependent on β1 integrin attachment to its physiologic substratum. MAPK inhibition could decrease FAK and paxillin induced migration and invasion of human SCC cells.
List of abbreviations
extracellular signal regulated kinase
focal adhesion kinase
growth factor receptor bound protein 2
jun N-terminal kinase
squamous cell carcinoma
We thank Dr. Thomas Parsons for expression vectors. This study was supported by National Institutes of Health grant DE10966.
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