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[Cancer Research 61, 4978-4981, July 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Loss of Focal Adhesion Kinase (FAK) Inhibits Epidermal Growth Factor Receptor-dependent Migration and Induces Aggregation of NH2-Terminal FAK in the Nuclei of Apoptotic Glioblastoma Cells1

Graham Jones, Joel Machado, Jr. and Adrian Merlo2

Molecular Neuro-Oncology Laboratory, Department of Clinical and Biological Sciences, University of Basel, 4031 Basel, Switzerland


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
In glioblastoma cells, inhibition of focal adhesion kinase (FAK) by the focal adhesion targeting domain attenuated epidermal growth factor receptor (EGFR) signaling, inhibiting epidermal growth factor-dependent migration. Although the EGFR-specific antagonist PD153035 increased caspase-3 activity, this was independent of FAK activity. Instead, the increase in apoptosis upon inhibition of FAK induced the aggregation of an NH2-terminal FAK fragment normally present in the nucleus. A recombinant NH2-terminal FAK construct was also targeted to the nucleus and aggregated in apoptotic cells upon coexpression with the focal adhesion targeting domain. Therefore, loss of FAK from the focal adhesions inhibits EGFR signaling at the cell membrane and transmits a proapoptotic signal to an NH2-terminal variant of FAK present in the nucleus.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Astrocytic brain tumors, of which glioblastoma multiforme is the most malignant, are highly invasive and resistant to chemotherapy and express high levels of FAK.3 Inhibition of FAK in glioblastoma cells by the FAT domain was sufficient to reduce invasion and increase apoptosis.4 The COOH-terminal region of FAK, which includes the FAT domain, is essential for targeting and activating FAK at the focal adhesions (1 , 2) . The NH2-terminal half of FAK shares homology to the band 4.1 family of proteins, which interact with membrane proteins and also play a role in cytoskeleton architecture (3) . The NH2-terminal domain of FAK also recognizes the cytoplasmic domain of the integrin-ß1 subunit in vitro (4) and is required for the regulation by FAK of EGF-dependent migration (5) . The role of FAK in cell survival is complex, responding to serum growth factors or specific extracellular matrix substrates (6) , and can involve p53-dependent pathways (7) . However, inactivation of the tumor suppressor genes p53 and PTEN (8) in glioblastoma cells shows that other cell death pathways are activated during apoptosis induced by expression of the FAT domain. In addition to the overexpression of FAK, the EGFR often is amplified in astrocytic tumors (9 , 10) . The recent finding linking FAK with EGFR signaling (5) and the requirement for EGFR activation in tumor survival (11) led us to examine in more detail the requirement for FAK and the EGFR in glioblastoma cell migration and survival.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Western Blots and Immunochemistry.
All methods were done according to Jones et al.4 Briefly, EGFR phosphorylation assays were done on a fibronectin substrate (0.2 µg/cm2; Sigma Chemical Co., St. Louis, MO) with cells maintained in 0.5% FCS for 16 h prior to stimulation with EGF (Fluka Chemie AG, Buchs, Switzerland). Western blots were developed using one of the following antibodies; antiphosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY), rabbit EGFR (sc-03; Upstate Biotechnology), actin (Sigma), or rabbit polyclonal anti-FAK A-17, which recognizes an epitope within the NH2-terminal half of FAK (Upstate Biotechnology). For immunochemistry, cells were stained using rabbit anti-FAK (A-17) or an anti-myc tag monoclonal antibody (residues 409–420; Upstate Biotechnology). Nuclei were visualized by incubating the cells for 15 min in 1 µg/ml propidium iodide, and secondary antibodies were conjugated with either BODIPY FL or Cy 3 (Molecular Probes, Eugene, OR).

Construction of pFATmycZeo and {Delta}NFAKR361.
The FAT domain of FAK was constructed as described.4 Recombinant {Delta}NFAKR361 extends from the +2 Ala to the +361 Arg in human FAK and was amplified using the following PCR primers: FAK.A2_s, 5'-ggcccagatctgcagctgcttaccttgac-3' and FAK.R361_as, 5'-gctacgtcgactctgatgataaatgactgcgagg-3'. The resulting PCR fragment was digested with BglII and SalI (underlined nucleotides) and cloned into pEGFP-C1 (Clontech, Palo Alto, CA). The resulting constructs were sequenced and verified by Western blot analysis.

Cell Culture, Transfections, and Cell Extracts.
All cells were transfected with GenePORTER 2 (Gene Therapy Systems, San Diego, CA). To generate stably transfected LN-401 (p53-/-; PTEN TAG(stop) codon 27; p16INK4a/p14ARF deleted) and LN-229 cells (p53-/-; PTEN WT; p16INK4a/p14ARF deleted; Ref. 8 ), cells were transfected and selected for 2 weeks in 800 µg/ml Zeocin (Invitrogen, Groningen, the Netherlands). Single clones were analyzed for FAT domain expression using a polyclonal anti-myc antibody (Upstate Biotechnology) and maintained in the presence of 250 µg/ml Zeocin. No apparent differences were noted between five independent clones of each cell line in terms of invasive behavior, sensitivity to apoptosis, and expression of the FAT domain. Adherent cells were cultured on 6-cm FN-coated dishes (0.2 µg/cm2; Sigma). The BS-153 cell line was generated by digestion of tumor samples with hyaluronidase and collagen at 37°C for 2 h and plating the cell suspension at limiting dilutions in growth medium. Single clones were selected and analyzed for loss of heterozygosity at chromosomes 9 and 10, which produced a pattern identical to the original tumor, including a homozygous deletion spanning the p16INK4a/p14ARF locus for both cell lines and a loss of heterozygosity covering PTEN. Cytoplasmic extracts were prepared by lysing cells in 100 µl of 10 mM HEPES (pH 7.9), 60 mM KCl, 1 mM EDTA, 10% NP-40 on ice for 5 min. For nuclear extracts, nuclei were collected, washed, and lysed in 50 ml of 250 mM Tris (pH 7.8), 60 mM KCl, 1 mM EDTA, 10% NP-40 by freezing and thawing three times in a dry ice-methanol bath.

Cell Migration and Caspase-3 Assays.
Migration assays were performed (5) by precoating cell chamber well inserts with a 8.0 µm pore size (Becton Dickinson, Franklin Lakes, NJ) with 5 µg/ml collagen (Roche Diagnostics, Rotkreuz, Switzerland) for 2 h and washing in PBS. Cells were starved for 16 h in 0.5% FCS and harvested by treatment with trypsin, which was subsequently inactivated by soybean trypsin inhibitor (Fluka). Cells were resuspended at a concentration of 1 x 106 cells/ml in 1% BSA/DMEM, and 100 µl were added to the upper chamber of the well insert, whereas 500 µl of DMEM containing the appropriate concentration of EGF were added to the lower chamber. Cells were allowed to migrate for 2 h, after which time they were fixed in methanol and stained using Hematoxylin (Fluka). Cells remaining in the upper chamber were removed by wiping, and remaining cells adhering to the underside of the membrane were counted. Caspase-3 activity was determined by harvesting attached and nonattached cells and assaying in a specific ELISA-based caspase-3 fluorometric assay according to the manufacturer’s instructions (Roche Diagnostics).


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
To test whether FAK influenced EGFR signaling in glioblastoma cells, we initially made use of the LN-401 glioblastoma cell line, which is mutant for both p53 and PTEN (8) . In mock-transfected LN-401 cells (LN-401/mock), EGF induced phosphorylation of EGFR for up to 60 min, after which phosphorylation of EGFR declined (Fig. 1ACitation , PY, *, LN-401/mock). In contrast, in LN-401 cells that stably expressed the FAT domain (LN-401/FAT), only weak EGFR phosphorylation was seen at 15 min and was almost absent at 60 min (Fig. 1ACitation , PY, LN-401/FAT). This was accompanied by a decrease in total EGFR (Fig. 1ACitation , EGFR), although similar levels of actin were detected in each cell line (Fig. 1ACitation , actin).



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  		Fig. 1. A, the FAT domain attenuates EGFR phosphorylation and increases EGFR degradation in LN-401 cells. Cells were stimulated with 40 ng/ml EGF for 15, 60, or 240 min and examined for total phosphotyrosine (PY), total EGFR (EGFR), or actin (actin). Results are representative of three similar experiments. B, top, the BS-153 cell line highly expresses normal (EGFR) and mutant (EGFRvIII) receptors. Western blot shows levels of EGFR and EGFRvIII and prolonged phosphorylation of EGFR (EGFR PY) for up to 240 min by EGF in nontransfected cells. Bottom, surface expression of EGFR (EGFR) is lost on BS-153 cells transfected to express the FAT domain (FAT, arrowheads) following stimulation with 40 ng/ml EGF. FAT was visualized using an anti-myc antibody, whereas EGFR and EGFRvIII were recognized by an antibody against the COOH terminus of EGFR.

 
The duration of EGFR signaling is regulated by the internalization and degradation of the receptor (12) ; this led us to directly examine the distribution of EGFR throughout the cell. The BS-153 glioblastoma cell line overexpresses normal EGFR as well as a mutant, EGFRvIII (Fig. 1BCitation , top), which is constitutively activated and poorly internalized (13) . Stimulation with EGF induced sustained phosphorylation of the normal EGFR in BS-153 cells for at least 240 min (Fig. 1BCitation , top). To determine whether FAK influenced the cellular distribution of EGFR, BS-153 cells were transfected to express the FAT domain, stimulated with EGF, and visualized using an EGFR antibody that recognized both normal and mutant EGFR. In nontransfected cells, stimulation with EGF induced strong EGFR immunoreactivity at the membrane and cell surface (Fig. 1BCitation , bottom, EGFR), which was inhibited in cells expressing the FAT domain (Fig. 1BCitation , FAT and EGFR, arrowheads). Although EGFR was absent from the surface of transfected cells, receptor immunoreactivity was seen in the perinuclear region (Fig. 1BCitation , EGFR, arrowheads), which is indicative of internalized receptor (12) . Taken together, these data indicate that expression of the FAT domain attenuated EGFR signaling in glioblastoma cells by increasing internalization and degradation of this receptor.

In a cell migration assay, addition of as little as 2 ng/ml EGF to LN-401/mock cells increased migration 10-fold (Fig. 2ACitation , mock), whereas migration of LN-401/FAT cells was increased only 2-fold (Fig. 2ACitation , FAT). Activation of EGFR is specifically inhibited by the antagonist PD153035 (14) in a receptor concentration-dependent manner (15) . EGFR is a potent survival signal for tumor cells (11) , and PD153035 is cytotoxic for glioblastoma cells (data not shown). If the proapoptotic effect imparted by expression of the FAT domain involves EGFR, it would be predicted that the FAT domain would enhance the cytotoxic effects of the EGFR-specific antagonist PD153035. Because EGFR induces phosphorylation of PKB/Akt (11) and the PTEN phosphatase counters this effect (16 , 17) , we examined the LN-229 and LN-401 glioblastoma cell lines, which are wild type and mutant for PTEN, respectively (8) . As in LN-401/FAT cells, expression of the FAT domain in the LN-229 cell line (LN-229/FAT) also inhibited EGFR phosphorylation (data not shown). In reduced serum, PD153035 increased caspase-3 activity in LN-229 cells 2-fold (Fig. 2BCitation , LN-229, mock). In LN-229/FAT cells, caspase-3 activity increased 2-fold in reduced serum (0.5% FCS) but was not further enhanced by incubation with PD153035 (Fig. 2BCitation , LN-229, FAT). PD153035 also increased caspase-3 activity in the LN-401/mock and LN-401/FAT cell lines, and although the absolute increase in caspase-3 activity was greater in LN-401/FAT cells (Fig. 2BCitation , LN-401, mock and FAT), the magnitude of the increase afforded by PD153035 was the same in both cell lines. Therefore, although the attenuation of EGFR signaling by the FAT domain inhibited EGF-dependent migration, we could find no evidence of a similar sensitization of possible EGFR-dependent mechanisms of cell survival, and this led us to consider other apoptotic pathways.



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  		Fig. 2. A, the FAT domain inhibits EGF-dependent migration. Cells were allowed to migrate for 2 h in the presence of the indicated amounts of EGF. Migration is normalized to untreated LN-401/mock cells. B, the EGFR antagonist PD153035 increases caspase-3 activity in LN-229 and LN-401 glioblastoma cells. Cells were incubated with 2.5 µM PD153035 for 48 h in 0.5% serum, after which samples were assayed for caspase-3 activity. Values are mean ± SE (bars) of triplicate values from three independent experiments normalized to untreated mock in each case.

 
During apoptosis, FAK is cleaved by caspases to generate the COOH-terminal FRNK fragment (18) . Under serum-free conditions, caspase-3 activity was increased 2.5-fold in LN-401/FAT cells (Fig. 2BCitation , LN-401, FAT), and FAT was targeted to the focal adhesions (Fig. 3ACitation , FAT). In contrast, FAK, visualized using antibody A-12, which recognized the NH2-terminal domain of FAK, was largely absent from the cell periphery. Instead, FAK was localized in the nucleus, where dense aggregates of FAK appeared (Fig. 3ACitation , FAK, arrowheads). Nuclear aggregation of the FAT domain was not observed (Fig. 3ACitation , FAT). This suggested that loss of FAK from the focal adhesions under apoptotic conditions was associated with the appearance of aggregates of FAK in the nucleus. To test this, LN-401/mock (Fig. 3BCitation , mock, top panels) and LN-401/FAT (Fig. 3BCitation , FAT, bottom panels) cells were cultured either in 10% FCS (Fig. 3BCitation , +) or in the absence of serum (Fig. 3BCitation , -), the latter of which favored apoptosis. Nuclei were then visualized using propidium iodide, and FAK was detected using antibody A-12. In LN-401/mock cells, serum starvation increased the frequency of nuclei with FAK aggregates 2.5-fold to ~20% of all cells examined (Fig. 3CCitation , mock). In LN-401/FAT cells in 10% FCS, the number of FAK-containing focal adhesions was reduced (Fig. 3CCitation , FAT, +), and 30% of cells exhibited nuclear N-FAK aggregates. This value increased to nearly 60% after serum starvation for 24 h (Fig. 3CCitation , FAT) and was accompanied by an increase in caspase-3 activity (Fig. 2BCitation , LN-401, FAT).



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  		Fig. 3. Aggregation of FAK in the nuclei of glioblastoma cells is enhanced by expression of the FAT domain. A, nuclear aggregates (arrowheads) of FAK in LN-401/FAT cells were detected using an antibody recognizing the NH2 terminus of FAK (FAK). The FAT domain (FAT) was visualized with a monoclonal antibody against the myc tag epitope. Note that the FAT domain is restricted to the focal adhesions and does not form nuclear aggregates. B, nuclear localization of FAK in LN-401/mock cells (mock) or LN-401/FAT cells (FAT) in the presence of normal serum (+) or under serum-free conditions (-). C, quantitation of the frequency of nuclear FAK aggregates seen under each condition described in B. Nuclei were identified by propidium iodide staining, and 100 cells were scored in each case.

 
In the next series of experiments, we examined the nature of FAK in the nucleus and whether this form of FAK was itself apoptotic. Initially, nuclear and cytoplasmic extracts (Fig. 4ACitation , N and C, respectively) were prepared from LN-401 cells cultured in normal serum (10% FCS; Fig. 4ACitation , FCS), serum free (Fig. 4ACitation , SF), or with 20 µM camptothecin, the latter of which increased caspase-3 activity 5–10-fold in these cells (data not shown, and Fig. 4ACitation , CPT). Under each condition and in both LN-401 cell lines, the A-17 FAK antibody detected an NH2-terminal FAK fragment of 40–50 kDa in the nuclear, but not cytoplasmic fractions (Fig. 4ACitation ). Because the FAK A-17 antibody recognizes an epitope within the first 65 amino acids of FAK, the molecular weight of the NH2-terminal FAK variant present in the nucleus would exclude the presence of the kinase domain of FAK, although it would be sufficient to include sequences homologous to the band 4.1 proteins (3) . Furthermore, because a similar amount of this fragment was present in each case, we concluded that the NH2-terminal FAK was constitutively present in the nucleus and was aggregated only upon apoptotic stimulation. Full-length 120-kDa FAK was detected in the cytoplasm of both cell lines and was reduced after either the withdrawal of serum or the addition of camptothecin (Fig. 4ACitation , compare Lanes 2, 4, and 6); a low level of full-length FAK was also detected in the nuclear fraction (Fig. 4ACitation , compare N and C).



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  		Fig. 4. A, an NH2-terminal domain of FAK is constitutively present in the nucleus. Nuclear (N) or cytoplasmic (C) extracts were prepared from either LN-401/mock or LN-401/FAT cells cultured in 10% FCS (FCS), 0.5% FCS (SF), or 20 µM camptothecin (CPT). An equal amount of protein was loaded in each lane. B, the recombinant NH2-terminal {Delta}NFAKR361 construct is localized to the nucleus and forms nuclear aggregates. LN-401 cells were transfected to express {Delta}NFAKR361 and cultured in 10% FCS. {Delta}NFAKR361 was visualized by EGFP autofluorescence (EGFP), and total FAK was visualized using the A-17 polyclonal antibody (FAK). The top and bottom panels show several examples of {Delta}NFAKR361 in which the nuclear localization and aggregation of {Delta}NFAKR361 is visible (EGFP). Also shown is a nontransfected cell in which FAK is present at the focal adhesions and diffusely distributed in the nucleus (FAK, arrowhead) C, association of nuclear aggregates of {Delta}NFAKR361 and apoptotic nuclei upon coexpression with the FAT domain. LN-401 cells were transfected either singly with a plasmid encoding {Delta}NFAKR361 ({Delta}NFAKR361) or in a 5:1 ratio with a plasmid encoding the FAT domain ({Delta}NFAKR361/FAT). Cells were starved in 0.5% FCS for 24 h, stained with Hoechst 33342, and visualized by EGFP. In each case, 100 cells were scored for EGFP expression and nuclear morphology. An example of apoptotic nuclei (DAPI) in transfected cells (EGFP) is shown.

 
To confirm that an NH2-terminal fragment of FAK was normally present in the nucleus and was aggregated during apoptosis, a recombinant EGFP-tagged NH2-terminal FAK variant that extended to Arg361 of FAK ({Delta}NFAKR361) was constructed that included the region homologous to the band 4.1 proteins (3) . In normal serum (10% FCS), {Delta}NFAKR361 was targeted to the nucleus of LN-401 cells and to the cell periphery (Fig. 4BCitation , EGFP, top and bottom). In some cells, nuclear aggregates of {Delta}NFAKR361 were also visible (Fig. 4BCitation , bottom, EGFP). In nontransfected cells, FAK was seen at focal adhesions and also as diffuse staining in the nucleus (Fig. 4BCitation , NFAK, top, arrowhead).

To examine whether expression of {Delta}NFAKR361 induced apoptosis, cells were starved of serum for 24 h. Approximately 20% of transfected cells showed aggregates of {Delta}NFAKR361, although less than one-third of these cells were apoptotic when scored for nuclear condensation (Fig. 4CCitation , {Delta}NFAKR361). In contrast, cotransfection of {Delta}NFAKR361 with a plasmid encoding the FAT domain increased the frequency of nuclear aggregates of {Delta}NFAKR361 >3-fold, with more than half of these cells exhibiting abnormal apoptotic nuclei (Fig. 4CCitation , {Delta}NFAKR361/FAT; right; DAPI and EGFP). Therefore, we concluded that the NH2-terminal fragment of FAK was itself not proapoptotic, but instead aggregated in response to an apoptotic signal that was correlated with the depletion of FAK from the cell membrane.

In summary, the FAT domain, by competing with FAK for localization to the focal adhesions, attenuated EGFR signaling at the cell membrane and thereby inhibited EGF-dependent migration. We could further show that FAT increased the degradation of EGFR after stimulation with EGF. Although an EGFR-specific antagonist increased caspase-3 activity in glioblastoma cell lines, no evidence was found linking loss of FAK activation to enhanced EGFR-dependent apoptosis. Instead, apoptosis was accompanied by aggregation in the nucleus of both an endogenous NH2-terminal variant of FAK and a recombinant {Delta}NFAKR361. Nuclear localization of FAK has been described recently in endothelial cells, although in this case apoptotic stimulation increased the amount of nuclear FAK (19) . Although the aggregation of FAK in the nucleus was highly reminiscent of nuclear bodies that include the proapoptotic protein PML (20) , we could detect only a weak interaction between these two proteins in extracts of glioblastoma cells, and no colocalization of PML with nuclear FAK was seen by confocal microscopy.5

In the absence of a well-defined nuclear localization sequence, the means by which FAK accumulates in the nucleus are not known. Functionally, nuclear FAK was not itself apoptotic. Instead, the redistribution within the nucleus suggested a structural role in the apoptotic process. A number of signaling molecules are associated with FAK at the focal adhesion, including c-Jun NH2-terminal kinase, which is present at both the focal adhesions and in the nucleus (6) . Although c-Jun NH2-terminal kinase is only poorly activated in LN-401 glioblastoma cells,6 it is possible that FAK might associate with a similar signaling protein in the nucleus. Nuclear bodies have been proposed to modulate transcription by recruiting and inactivating transcriptional repressors or activators (21) , and a nuclear isoform of protein 4.1, which has homology to the NH2-terminal domain of FAK, is redistributed in the nucleus in a transcription-dependent manner (22) .

In conclusion, these results establish that FAK acts not only at the cell membrane, but also has a role in the nucleus of glioblastoma cells during apoptosis.


   ACKNOWLEDGMENTS
 
We would like thank Dr. Gertraud Orend for comments on the manuscript and acknowledge the excellent technical assistance of Beatrice Dolder.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Swiss National Science Foundation program in somatic gene therapy (NF374037-055167/1 to A. M.). Back

2 To whom requests for reprints should be addressed, at Brain Tumor Research/Neurosurgery, Spitalstrasse 21, 4031 Basel, Switzerland. Phone: 41 61 265 7456; Fax: 41 61 265 7138; E-mail: amerlo{at}uhbs.ch Back

3 The abbreviations used are: FAK, focal adhesion kinase; FAT, focal adhesion targeting; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EGFP, enhanced green fluorescence protein. Back

4 G. Jones, J. Machado, Jr., M. Tolnay, and A. Merlo. PTEN-independent induction of caspase-mediated cell death and reduced invasion by the focal adhesion targeting domain (FAT) in human astrocytic brain tumors which highly express focal adhesion kinase (FAK), submitted for publication. Back

5 G. Jones and A. Merlo, unpublished observations. Back

6 G. Jones and A. Merlo, unpublished data. Back

Received 2/20/01. Accepted 5/16/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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H. Mao, F. Li, K. Ruchalski, D. D. Mosser, J. H. Schwartz, Y. Wang, and S. C. Borkan
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G. Liu, C. D. Guibao, and J. Zheng
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