Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M407953200 on August 24, 2004

J. Biol. Chem., Vol. 279, Issue 46, 48342-48349, November 12, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/46/48342    most recent
M407953200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Y.
Right arrow Articles by Kapila, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Y.
Right arrow Articles by Kapila, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Squamous Cell Carcinoma Cell Aggregates Escape Suspension-induced, p53-mediated Anoikis

FIBRONECTIN AND INTEGRIN {alpha}v MEDIATE SURVIVAL SIGNALS THROUGH FOCAL ADHESION KINASE*

Yan Zhang{ddagger}§, Hai Lu{ddagger}§, Paul Dazin¶, and Yvonne Kapila{ddagger}||

From the {ddagger}Department of Stomatology, School of Dentistry, and Howard Hughes Medical Institute, School of Medicine, University of California, San Francisco, California 94143

Received for publication, July 14, 2004 , and in revised form, August 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Resistance to anoikis, or apoptosis triggered by detachment from the extracellular matrix (ECM), lengthens the survival of malignant cells, facilitating reattachment and colonization of secondary sites. To examine the molecular mechanisms underlying resistance to anoikis in human oral squamous cell carcinoma (SCC) cells, we cultured human squamous carcinoma (HSC-3) cells in suspension on plates coated with poly-2-hydroxyethyl methacrylate, which blocks access to the ECM. Cells in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. Aggregates, but not single cells, had high levels of fibronectin. Preincubation with a cyclic arginine-glycine-aspartic acid peptide or fibronectin-blocking antibody significantly increased anoikis. Single cells had markedly lower expression of the integrin {alpha}v receptor than aggregates. Blocking {alpha}v function with a blocking antibody or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, phosphorylation of the integrin-associated focal adhesion kinase (FAK) at tyrosine 397 was reduced, and p53 levels were increased. Apoptosis was increased by blocking FAK with an antisense oligonucleotide and reduced by blocking p53. These findings show that SCC cells escape suspension-induced anoikis by forming multicellular aggregates that avail themselves of fibronectin survival signals mediated by integrin {alpha}v. Single cells in suspension that do not form aggregates undergo anoikis because of decreased FAK phosphorylation and increased p53 levels. Thus, SCC cells appear to use neighboring cells and the ECM molecule FN to promote the metastatic phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Disrupting cell adhesion to the extracellular matrix (ECM)1 rapidly induces programmed cell death, a response that has been termed anoikis (1). Anoikis was first documented in normal epithelial cells and endothelial cells where it helps maintain a dynamic balance between cell turnover and survival (1, 2). Malignant tumor cells are often resistant to anoikis, enabling them to survive after detachment from the ECM and colonize a secondary site. Resistance to anoikis has been described in many types of human malignancies, including gastric cancer, mammary tumors, colon cancers, osteosarcomas, and lung carcinomas (3-7), but little is known about it in the progression of human oral squamous cell (SCC) carcinoma. Oral cancer is the sixth most common solid tumor, accounting for 5.5% of all malignancies worldwide (8). SCC accounts for 96% of all tumors of the oral cavity (9), and many patients with these tumors die from metastatic disease (10).

What factors promote anchorage-independent survival and spread of tumor cells? One possibility is survival signals mediated through cell-cell contacts. One factor that promotes the survival of ECM-deprived SCC cells by such contact is the cadherin receptor family (11). A second possibility is the integrin receptor family, because integrins play a role in multiple steps in tumorigenesis, including cell spreading, invasion, and survival. Integrins are heterodimeric, cation-dependent cell membrane adhesion molecules that mediate cell-cell and cell-ECM interactions (12). In mammals, at least 16 {alpha} and 8 {beta} subunits have been reported that combine into 22 different heterodimers, each with specific recognition and affinities for various ECM components or other cell-bearing adhesion molecules (13, 14). Integrin expression patterns are often altered during tumor development. Metastasis requires changes in integrin expression. In SCC cells, substantial evidence demonstrates altered integrin expression in tumorigenesis compared with the native state. Alterations in the expression and function of integrins are also associated with anoikis (15, 16).

One likely mechanism for integrin-mediated survival and resistance to anoikis is signal transduction through activation of intracellular tyrosine kinases, such as focal adhesion kinase (FAK). FAK is a non-receptor protein-tyrosine kinase that indirectly localizes to sites of integrin clustering through C-terminal domain-mediated interaction with integrin-associated proteins such as paxillin and talin (17). In vitro, its N-terminal domain binds to sequences in the cytoplasmic domain of {beta} integrin subunits (18). Elevation of the phosphotyrosine content of FAK correlates directly with increased cell adhesion. Focal adhesion complexes formed in mouse fibroblasts plated on fibronectin have 2.5-fold higher FAK phosphorylation activity than cells plated on polylysine, where adherence is integrin-independent and the phosphotyrosine content of FAK is minimal (19). Tyrosine 397 is the major site for phosphorylation and catalytic activity of FAK both in vivo and in vitro (20). FAK activation results in the recruitment of Src homology 2 and Src homology 3 domain-containing proteins that transduce signaling to several downstream effectors and has been implicated in various cellular activities. Cancer cells exhibit profound changes in cytoskeletal organization, adhesion, motility growth regulation, and survival. Increased FAK expression has been correlated with increased cancer cell motility, invasiveness, cell cycle alterations, and proliferation (21, 22). Introducing a constitutively active form of FAK into anchorage-dependent cells renders them anchorage-independent (23).

Downstream of integrin/FAK signals, the tumor suppressor p53 may further regulate SCC anoikis in the absence of an ECM. p53 plays a crucial role in several forms of apoptosis, including that induced by irradiation or certain chemotherapeutic compounds (24). One study suggested that p53 increased the survival of transformed fibroblasts and immortalized mammary epithelial cells in suspension by increasing the frequency of colony formation in semisolid medium (25). Thyroid epithelial cells undergo apoptosis through a p53-dependent pathway when integrin-mediated adhesion to the ECM is denied (26). Thus, p53 signaling pathways may regulate anoikis in SCC cells.

Although anchorage-independent growth is a hallmark of malignancy, the molecular mechanisms involved are not completely understood. In this study, we sought to understand the molecular mechanisms underlying resistance to anoikis in human oral SCC cells under various culture conditions, including growth in suspension to simulate detachment from the ECM. Our findings show SCC cells that form multicellular aggregates are more resistant to anoikis through a mechanism involving fibronectin and the integrin {alpha}v receptor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line, Antibodies, and Other Reagents—The highly invasive human oral SCC cell line HSC-3 was kindly provided by Dr. Randy Kramer (University of California, San Francisco). The cells were maintained in a 5% CO2 atmosphere at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Mouse monoclonal antibodies against human integrins {alpha}3 (P1B5), {alpha}4 (P1H4), {alpha}5 (NK1-SAM-1), and {alpha}v (P3G8) were from Chemicon International (Temecula, CA), as were mouse negative control immunoglobulin G (IgG) 1 (CBL600), functional blocking antibodies to fibronectin (3E3) and vitronectin (MAB1945), fluorescein isothiocyanate-conjugated goat anti-mouse IgG, and rabbit polyclonal antibodies against human integrins {alpha}3 (AB1920), {alpha}4 (AB1924), {alpha}5 (AB1949), and {alpha}v (AB1930). Rabbit anti-human fibronectin was from Sigma. Rabbit anti-FAK antibody and mouse anti-p53 monoclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-FAK (pY397) polyclonal antibody was from BIOSOURCE (Camarillo, TX). Cyclic RGD peptide RGDFV (Arg-Gly-Asp-D-Phe-Val) and its negative control RADFV (Arg-Ala-Asp-D-Phe-Val) were from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

Suspension-induced Anoikis—Six-well plates were coated with 4 ml of poly-2-hydroxyethyl methacrylate (poly-HEMA, 10 mg/ml, Sigma) dissolved in 95% ethanol and allowed to dry overnight under sterile conditions in a laminar flow hood. Confluent adherent HSC-3 cells were detached from regular culture plates with 5 mM EDTA, and single cells were harvested by two passages through a 40-µm nylon cell strainer (Falcon), seeded onto the poly-HEMA coated plates at 5 x 105 cells/cm2, and cultured for various times in DMEM containing 1% bovine serum albumin or 10% fetal bovine serum.

Serum-free Culture—HSC-3 cells were detached with 5 mM EDTA, washed twice with phosphate-buffered saline (PBS), seeded in 2 ml of serum-free DMEM in six-well tissue culture dishes (1 x 106 cells/well), and cultured for 36 h.

TUNEL Detection of Apoptosis—Apoptotic cells were identified by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL, Promega) to detect fragmented DNA, according to the manufacturer's instructions. The cells were then counterstained with propidium iodide (2 µg/ml) for 15 min and examined by fluorescence microscopy.

Apoptosis Assay—The percentage of apoptotic cells under various culture conditions was determined with a flow cytometric assay (27-29). HSC-3 cells were cultured for 36 h in DMEM with or without serum. Adherent cells were detached with enzyme-free cell dissociation buffer (Invitrogen). The cells in suspension cultures were collected and passed twice through a 40-µm cell strainer to separate single cells from aggregated cells. Harvested cells were resuspended in 1 ml of ice-cold PBS containing 2% fetal calf serum, 3% enzyme-free dissociation buffer, and 5 µg/ml propidium iodide (Molecular Probes) and kept on ice for 10-15 min. After equilibration to room temperature, each suspension was stained with Hoechst 33342 (4 µg/ml) for 6 min, and the percentage of apoptotic cells was determined with a dual-laser FACstar+ cell sorter (BD Biosciences). Experiments were performed in triplicate and repeated three times.

Immunocytochemical Staining of Fibronectin—After 36 h of culture in suspension, HSC-3 cells were spun onto glass slides at 800 rpm for 3 min. Adherent cells grown on chamber slides in serum-free DMEM for 36 h were washed twice with cold PBS. After fixation in 3% formaldehyde, the cells were incubated first with a mouse anti-human fibronectin monoclonal antibody (P1H11) and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The slides were then washed with PBS, mounted with the Vectashield system (Vector Laboratories, Burlingame, CA), and photographed with a Nikon Eclipse E400 microscope.

Analysis of Integrin Expression by Flow Cytometry—Integrin expression was quantitated by flow cytometry. The cells were detached with 5 mM EDTA, washed with cold PBS, resuspended in PBS (1 x 106 cells/50 µl), and incubated with primary antibodies on ice for 45 min. All antibodies were diluted in PBS containing 0.2% bovine serum albumin and 5% cell dissociation buffer (Invitrogen). Negative controls were incubated with nonimmune mouse IgG (CBL600) as the primary antibody. The cells were then washed twice with PBS, incubated with 50 µl of fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 45 min on ice, washed twice with PBS, resuspended in PBS containing 1.0% bovine serum albumin and 5% cell dissociation buffer, and counterstained with propidium iodide (Sigma; final concentration 1 µg/ml) on ice for 10 min. Fluorescein isothiocyanate-labeled cells were then analyzed with a FACScan (BD Biosciences) gated on forward- and side-scatter intensities. All experiments were performed in triplicate.

Induction of Anoikis—Functional blocking studies with blocking antibodies and peptides were performed as described (30). Briefly, HSC-3 cells were detached with 5 mM EDTA, and single cells were harvested by passing the cell suspension through a 40-µm nylon cell strainer twice. Single cells were incubated with cyclic RGD peptide (RGDFV) with a negative control RADFV (final concentration, 400 µg/ml) or with blocking antibodies to fibronectin or vitronectin (final concentration, 10 µg/ml in serum-free medium) for 30 min at 37 °C and plated on poly-HEMA-coated culture dishes.

Western Blotting—Cells were washed twice with cold PBS and lysed in TNE buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate) containing protease inhibitor mixtures I and II (Sigma) for 15 min at 4 °C. The lysates were pelleted by centrifugation for 15 min at 4 °C, and the protein concentration was determined with a bicinchoninic acid assay (Pierce). Equal amounts of protein (20 µg of total protein) from each sample were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp., Billerica, MA). The membranes were probed with primary antibodies overnight at 4 °C. A control blot with anti-human actin antibody demonstrated equal protein in the samples. The membranes were then washed and probed with a species-specific horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature, followed by ECL detection (Amersham Biosciences). The intensities of the immunoreactive bands were measured with NIH Image, version 1.30.

Antisense Experiments—Antisense oligonucleotides for integrin {alpha}v (31), FAK (32), and p53 (33) were used to target and suppress the region of the translation start site of each protein for antisense analysis. The sequences of antisense, sense, and mismatched oligonucleotides are listed in Table I. The oligonucleotides were synthesized by Oligos Etc. (Wilsonville, OR) and they received a phosphorothioated modification at all positions to minimize intracellular cleavage by degradative enzymes and to increase stability (34). Upon arrival, all oligonucleotides were dissolved in double-distilled water and stored in aliquots at -20 °C. Oligonucleotides (final concentration, 2 µM) were mixed with 8 µl of oligofectamine (Invitrogen), incubated for 20 min, and transfected into HSC-3 cells at 60% confluence. HSC-3 cells were typically cultured with oligonucleotides in serum-free medium for 4 h at 37 °C; fetal bovine serum was then added (final concentration, 10%). Cells were harvested at various times, and total cell lysates were analyzed by Western blot analysis to evaluate the efficiency of antisense treatment.


View this table:
[in this window]
[in a new window]
  		TABLE I
Oligonucleotide sequences for integrin {alpha}v; FAK, p53, and their controls

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Resistance to Anoikis Is Greater in Multicellular Aggregates than Single Cells—HSC-3 cells cultured under different culture conditions demonstrated different phenotypes. Single cells allowed to attach to tissue culture plates but incubated in serum-free medium underwent apoptosis, as indicated by positive TUNEL (green/yellow) staining, whereas those that form multicellular aggregates survived, as indicated by positive propidium iodine (red) staining. Similarly, of the cells that are cultured in suspension, single isolated cells had higher levels of apoptosis than cells that formed multicellular aggregates (Fig. 1A). To quantify apoptosis, we used a FACS-based assay. In suspension cultures, the level of apoptosis was higher in single cells than in aggregated cells in both the absence (30% versus 15%) and presence (20% versus 10%) of serum; in attached cells, the level was only 2-3% (Fig. 1, B and C).



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 1.
HSC-3 cells that form multicellular aggregates have lower levels of anoikis than single cells. A, nuclear staining of cells in suspension cultures and attached cells, grown for 36 h in serum-free medium. Cells in suspension were spun onto glass slides and fixed before staining. All cells were stained with the TUNEL method, counterstained with propidium iodine, and visualized by fluorescence microscopy. Green and yellow indicate apoptotic cells; red indicates non-apoptotic cells. B, percentage of apoptotic HSC-3 cells after 36 h of culture in serum-free medium (SFM) or with 10% fetal bovine serum (FBS). Apoptosis was assessed with a FACS-based assay. Values are the mean ± S.E. of three experiments. C, representative FACS plots from the apoptosis assay. The number in each panel indicates the percentage of apoptotic cells.

 
Resistance to Anoikis Involves Fibronectin/RGD-mediated Survival Signals—To assess the importance of ECM components in promoting survival, we stained HSC-3 cells for fibronectin. Staining was intense in multicellular aggregates in suspension culture and in attached cells; single cells in suspension had little or no staining (Fig. 2A). Western blot analysis showed lower levels of fibronectin expression in single cells than in multicellular aggregates or attached cells (Fig. 2B).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 2.
HSC-3 cell aggregates in suspension escape anoikis by receiving fibronectin/RGD-mediated survival signals. A, immunofluorescence images showing fibronectin expression (green) in cells cultured in suspension and in attached cells. Cells were counterstained with propidium iodine (red). B, Western blot analysis shows higher levels of fibronectin in adherent cells and aggregated cells than in single cells in suspension after 36 h of culture. Actin served as a loading control. C, percentage of apoptotic cells after pretreatment with RGDFV or control peptide (RADFV) for 30 min. Cells were cultured in suspension for 36 h and analyzed with a FACS-based assay. Values are the mean ± S.E. of three experiments. D, percentage of apoptotic cells after pretreatment for 30 min with function-blocking antibodies against fibronectin (FN) or vitronectin (VN) or with nonspecific IgG. Cells were cultured in suspension for 36 h and analyzed with a FACS-based assay. Values are the mean ± S.E. of three experiments. *, p < 0.05 versus control and anti-VN, determined by an ANOVA followed by Fisher's post-hoc test.

 
Functional analyses with a blocking RGD peptide (Fig. 2C) or an anti-fibronectin antibody (Fig. 2D) confirmed that fibronectin/RGD-mediated survival signals were critical to promoting survival in the multicellular aggregates because blocking their function increased apoptosis compared with the controls. A vitronectin-blocking antibody was used as a control because vitronectin is an ECM protein that contains RGD-like motifs. As shown by FACS-based apoptosis assays, pretreatment of suspended cells with the cyclic RGD peptide induced higher levels of anoikis than the control peptide (32 versus 12%), as did the fibronectin blocking antibody (27.1 versus 11.3%) (Fig. 2D).

Fibronectin/RGD-mediated Survival Signals Are Transmitted via the Integrin {alpha}v Receptor—To investigate whether integrin receptors were transducing the survival signals mediated by fibronectin/RGD, we examined cell-surface expression of fibronectin- and RGD-binding integrins by flow cytometry. HSC-3 cells expressed the {alpha}3, {alpha}4, {alpha}5, and {alpha}v integrin subunits, although {alpha}4 was expressed at very low levels (Fig. 3A, left panel). Western blot analyses showed that integrin {alpha}v levels decreased steadily in single cells in suspension cultures but not in the multicellular aggregates or in attached cells (Fig. 3A, right panel). All other integrins examined remained essentially unchanged under all the conditions tested (Fig. 3A, right panel), and integrin {alpha}4 was undetectable by Western blot (data not shown).



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 3.
Resistance to anoikis in HSC-3 cell aggregates in suspension involves the {alpha}v integrin. A, left, FACS analysis showing cell-surface expression of integrins {alpha}3, {alpha}4, {alpha}5, and {alpha}v by adherent HSC-3 cells. A, right, Western blots showing expression of integrins {alpha}3, {alpha}5, and {alpha}v in adherent cells and in single and aggregated cells in suspension at 8, 16, and 36 h. Actin served as a loading control. B, percentage of apoptotic cells after pretreatment with function-blocking antibodies to integrins {alpha}3, {alpha}4, {alpha}5, and {alpha}v (10 µg/ml) for 30 min. Cells were cultured in poly-HEMA-coated plates for 36 h and analyzed with a FACS-based assay. Nonspecific mouse IgG (CBL600) served as a negative control. Values are the mean ± S.E. of three experiments. *, p < 0.05 versus other test conditions, determined by an ANOVA followed by Fisher's post-hoc test. C, Western blots (left) showing integrin {alpha}v expression in HSC-3 cells after control transfection or transfection with {alpha}v antisense or sense oligonucleotides. Graph below the blots shows band intensity determined by densitometry. Photomicrographs (right) show images of hematoxylin-stained cells transfected with control or {alpha}v antisense or sense oligonucleotides and grown in suspension culture for 36 h. Graph below images shows the percentages of apoptotic cells, determined with a FACS-based assay. Values are the mean ± S.E. of three experiments. *, p < 0.05 versus control and sense, determined by an ANOVA followed by Fisher's post-hoc test.

 
In functional studies, an integrin {alpha}v blocking antibody increased apoptosis nearly 3-fold in cells in suspension cultures (28.9 versus 10% in controls), indicating that integrin {alpha}v mediates a critical survival signal (Fig. 3B). Successful suppression of integrin {alpha}v with an antisense oligonucleotide (58%) confirmed this role. Antisense-treated cells had higher levels of apoptosis (18.9%) and formed fewer multicellular aggregates than control cultures (Fig. 3C).

FAK Is Critical for Resistance to Anoikis in HSC-3 Cell Aggregates in Suspension—To study the signaling mechanism by which integrin {alpha}v promoted the survival of SCC multicellular aggregates, we examined the expression of the early integrin-signaling molecule FAK by Western blot analysis. Although total expression levels were similar under different culture conditions, phosphorylation of FAK at tyrosine 397 at 4 h was markedly lower in single cells in suspension than in multicellular aggregates or attached cells (Fig. 4A). In confirmatory studies with FAK antisense strategies in suspension cultures, the level of anoikis was higher in cells transfected with antisense oligonucleotides than in cells transfected with sense oligonucleotides or control-transfected cells (16.9 versus 10.8 and 10.3%) (Fig. 4C).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 4.
Fibronectin/RGD-mediated survival signals are transmitted via the {alpha}v integrin receptor and FAK. A, Western blots showing the levels of FAK and FAK phosphorylated at tyrosine 397 (P-FAK) in adherent cells and in single and aggregated cells cultured in suspension for 4, 8, and 16 h. Actin served as a loading control. B, Western blot analysis of FAK and p-FAK levels in HSC-3 cells after control transfection or transfection with FAK antisense or sense oligonucleotides for 36 h and culturing under suspension conditions for 4 h (left); the graph shows band intensity determined by densitometry (right). C, percentage of apoptotic cells after control transfection or transfection with FAK antisense or sense oligonucleotides. Cells were cultured in suspension for 4 h, and apoptosis was determined with a FACS-based assay. Values are the mean ± S.E. of three experiments. *, p < 0.05 versus control and sense, determined by an ANOVA followed by Fisher's post-hoc test.

 
HSC-3 Cell Aggregates in Suspension Escape Anoikis by Suppressing p53-mediated Signals—Because p53 status has been linked to FAK signaling in survival pathways regulated by the ECM (28, 29, 35, 36), we investigated whether suspension-induced anoikis was mediated by p53. Western blot analyses of HSC-3 cells under different culture conditions showed that p53 levels increased steadily in single cells in suspension but not in multicellular aggregates or attached cells (Fig. 5A). Abrogating p53 function by antisense treatment (~90% suppression) rescued cells from suspension-induced anoikis (Fig. 5B). Levels of apoptosis were lower in antisense-transfected cells than in sense- or control-transfected cells (4 versus 11.4 and 11.8%).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 5.
HSC-3 cell aggregates in suspension escape anoikis by suppressing p53-mediated signals. A, Western blot analysis showing p53 levels in attached cells and in single and aggregated cells cultured in suspension for 8, 16, and 36 h. Actin served as a loading control. B, Western blot analysis showing p53 levels in HSC-3 cells after control transfection or transfection with p53 antisense or sense oligonucleotides for 24 h and culturing under suspension conditions for 8 h (left); the graph shows band intensity determined by densitometry (right); the graph shows the percentage of apoptotic cells after control transfection or transfection with p53 antisense or sense oligonucleotides (bottom). Cells were cultured in suspension for 8 h and evaluated for apoptosis with a FACS-based assay. Values are the mean ± S.E. of three experiments. *, p < 0.05 versus control and sense, determined by an ANOVA followed by Fisher's post-hoc test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that oral SCC cells that formed aggregates in suspension cultures had significantly lower levels of apoptosis than single cells, indicating increased resistance to anoikis. Aggregates had high levels of fibronectin, and single cells had low levels of the integrin {alpha}v receptor. Blocking {alpha}v function with antibodies or by transfection with an antisense oligonucleotide increased apoptosis and inhibited aggregation. In single cells but not aggregates, FAK phosphorylation at tyrosine 397 was reduced and p53 levels were increased. In functional studies with antisense oligonucleotides, apoptosis was increased by blocking FAK and reduced by blocking p53. Thus, fibronectin survival signals mediated by integrin {alpha}v enable SCC cells to escape suspension-induced anoikis by forming multicellular aggregates.

In previous studies, hepatocyte growth factor (37) and cadherins (11) were associated with anchorage-independent growth and resistance to anoikis in SCC cells. We focused on fibronectin and its integrin-mediated signaling pathways. Fibronectin is critical for transducing survival signals in various cell types (29, 35), and blocking fibronectin/integrin-mediated signals by various methods leads to apoptosis or anoikis (38). Thus, the reduced apoptosis of multicellular aggregates in suspension cultures suggested that resistance to anoikis involves survival signals from fibronectin. Fibronectin begins to appear at the cell surface and in the culture medium within 30-40 min after synthesis (39). Western blot analysis of suspension cultures showed that fibronectin was highly expressed by cells that had aggregated rather than single cells, suggesting that bound fibronectin permitted cell-cell adhesion and conferred resistance to anoikis. Blocking fibronectin binding with a potent cyclic RGD peptide (40) or fibronectin function with an antibody increased apoptosis. These findings indicate that fibronectin promotes the survival of aggregated SCC cells through a signal mediated by its RGD site.

How might fibronectin convey the survival signal transmitted to the aggregated cells in suspension? One logical explanation is integrin receptors known to interact with fibronectin. Using flow cytometry, we found that HSC-3 cells expressed integrins {alpha}3, {alpha}5, {alpha}v, and, at low levels, {alpha}4. Western blot analyses showed that single cells in suspension expressed lower levels of integrin {alpha}v than aggregated cells in suspension or attached cells. Functional studies with integrin-blocking antibodies and antisense approaches confirmed that integrin {alpha}v was necessary for survival because abrogating its function increased apoptosis of cells in suspension. Consistent with this finding, Bates et al. (41) showed that inhibiting cell-cell contact with anti-{alpha}v antibody resulted in rapid apoptosis of colon carcinoma cells. Thus, cell-matrix-cell interactions mediated by {alpha}v integrin may be important in the regulation of apoptosis in human carcinoma cells.

We hypothesized that the integrin-associated signaling molecule FAK would be part of the signal transduction pathway mediating resistance to anoikis in SCC cells. FAK has been reported to transduce integrin signals, including those regulating survival and migration (42-47). In carcinomas, suppression of FAK promoted anoikis and suppressed metastasis (48), whereas overexpression correlated with tumor invasiveness and metastasis (49). Furthermore, an experimentally activated form of FAK rendered certain epithelial cells resistant to anoikis and conferred a transformed phenotype (23). Finally, studies with FAK-null fibroblasts have shown that FAK is required for cell migration (17).

In agreement with our hypothesis, single SCC cells in suspension had much lower levels of FAK phosphorylation (consistent with their increased apoptosis) than the multicellular SCC aggregates. Moreover, suppressing FAK function increased anoikis. These findings suggest that FAK promotes anchorage-independent growth and migration of oral SCC cells, which are both critical to the metastatic phenotype.

FAK has been linked to the p53 status of cells in regulating ECM survival signals. ECM survival signals transduced by FAK suppress p53-mediated apoptosis (28) by preventing upregulation of p53 by death-associated protein kinase (36). Similarly, we found that suppressing p53 function in SCC cells reduced anoikis in SCC cells in suspension cultures, supporting the link between FAK and p53 status in suspension-induced anoikis in SCC cell aggregates. The characterization of this survival-signaling pathway in SCC cells extends our knowledge of tumor cell biology, and this may be of therapeutic value in preventing tumor cell proliferation and metastasis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01 DE14429 and P01 DE13904 (to Y. K.). 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. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: University of Michigan, School of Dentistry, Dept. of Periodontics/Prevention/Geriatrics, 1011 N. University Ave., Rm. 5213, Ann Arbor, MI 48109-1078. Tel.: 734-615-2295; Fax: 734-763-5503; E-mail: ykapila{at}umich.edu.

1 The abbreviations used are: ECM, extracellular matrix; SCC, squamous cell carcinoma; HSC, human squamous carcinoma; RGD, arginine-glycine-aspartic acid; FAK, focal adhesion kinase; DMEM, Dulbecco's modified Eagle's medium; poly-HEMA, poly-2-hydroxyethyl methacrylate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; FACS, fluorescence-activated cell sorter; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Randy Kramer for critically reviewing this manuscript and Stephen Ordway for editorial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Frisch, S. M., and Francis, H. (1994) J. Cell Biol. 124, 619-626[Abstract/Free Full Text]
  2. Meredith, J. E., Jr., Winitz, S., Lewis, J. M., Hess, S., Ren, X. D., Renshaw, M. W., and Schwartz, M. A. (1996) Endocr. Rev. 17, 207-220[Abstract/Free Full Text]
  3. Yawata, A., Adachi, M., Okuda, H., Naishiro, Y., Takamura, T., Hareyama, M., Takayama, S., Reed, J. C., and Imai, K. (1999) Oncogene 16, 2681-2686
  4. Streuli, C. H., and Gilmore, A. P. (1999) J. Mammary Gland Biol. Neoplasia 4, 183-191[CrossRef][Medline] [Order article via Infotrieve]
  5. Shanmugathasan, M., and Jothy, S. (2000) Pathol. Int. 50, 273-279[CrossRef][Medline] [Order article via Infotrieve]
  6. Diaz-Montero, C. M., and McIntyre, B. W. (2003) Eur. J. Cancer 39, 2395-2402[CrossRef][Medline] [Order article via Infotrieve]
  7. Wei, L., Yang, Y., and Yu, Q. (2001) Cancer Res. 61, 2439-2444[Abstract/Free Full Text]
  8. Thomas, G. J., Jones, J., and Speight, P. M. (1997) Oral Oncol. 33, 381-388[Medline] [Order article via Infotrieve]
  9. Ramos, D. M., Chen, B. L., Boylen, K., Stern, M., Kramer, R. H., Sheppard, D., Nishimura, S. L., Greenspan, D., Zardi, L., and Pytela, R. (1997) Int. J. Cancer 72, 369-376[CrossRef][Medline] [Order article via Infotrieve]
  10. Silverman, S., Jr. (1998) Oral Cancer, 4th Ed., p. xi, B. C. Decker, Ontario, Canada
  11. Kantak, S. S., and Kramer, R. H. (1998) J. Biol. Chem. 273, 16953-16961[Abstract/Free Full Text]
  12. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002) Molecular Biology of the Cell, 4th Ed., pp. 1065-1126, Garland Science, New York
  13. Hynes, R. O. (1987) Cell 48, 549-554[CrossRef][Medline] [Order article via Infotrieve]
  14. Green, L. J., Mould, A. P., and Humpries, M. J. (1998) Int. J. Biochem. Cell Biol. 30, 179-184[CrossRef][Medline] [Order article via Infotrieve]
  15. Marco, R. A., Diaz-Montero, C. M., Wygant, J. N., Kleinerman, E. S., and Mclntyre, B. W. (2003) J. Cell. Biochem. 88, 1038-1047[CrossRef][Medline] [Order article via Infotrieve]
  16. Frisch, S. M., and Screaton, R. A. (2001) Curr. Opin. Cell Biol. 13, 555-562[CrossRef][Medline] [Order article via Infotrieve]
  17. Sieg, D. J., Hauck, C. R., Ilic, D., Klingbeil, C. K., Schlaepfer, E., Damsky, C. H., and Schaepfer, D. D. (2000) Nat. Cell Biol. 2, 249-256[CrossRef][Medline] [Order article via Infotrieve]
  18. Schaller, M. D., Otey, C. A., Hildebrand, J. D., and Parsons, J. T. (1995) J. Cell Biol. 130, 1181-1187[Abstract/Free Full Text]
  19. Guan, J. L., and Shalloway, D. (1992) Nature 358, 690-692[CrossRef][Medline] [Order article via Infotrieve]
  20. Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 1680-1688[Abstract/Free Full Text]
  21. Owens, L. V., Xu, L., Craven, R. J., Dent, G. A., Weiner, T. M., Kornberg, L., Liu, E. T., and Cance, W. G. (1995) Cancer Res. 55, 2752-2755[Abstract/Free Full Text]
  22. Slack, J. K., Adams, R. B., Rovin, J. D., Bissonette, E. A., Stoker, C. E., and Parsons, J. T. (2001) Oncogene 20, 1152-1163[CrossRef][Medline] [Order article via Infotrieve]
  23. Frisch, S. M., Vuori, K., Ruoslahti, E., and Chan-Hui, P. Y. (1996) J. Cell Biol. 134, 793-799[Abstract/Free Full Text]
  24. Vogelstein, B., and Kinzler, K. M. (1994) Nature 370, 174-175[CrossRef][Medline] [Order article via Infotrieve]
  25. Nikiforov, M. A., Hagen, K., Ossovskaya, V. S., Connor, T. M., Lowe, S. W., Deichman, G. I., and Gudkov, A. V. (1996) Oncogene 13, 1709-1719[Medline] [Order article via Infotrieve]
  26. Vitale, M., Di Matola, T., Bifulco, M., Casamassima, A., Fenzi, G., and Rossi, G. (1999) FEBS Lett. 462, 57-60[CrossRef][Medline] [Order article via Infotrieve]
  27. Hamel, W., Dazin, P., and Israel, M. A. (1996) Cytometry 25, 173-181[CrossRef][Medline] [Order article via Infotrieve]
  28. Ilic, D., Almeida, E. A., Schlaepfer, D. D., Dazin, P., Aizawa, S., and Damsky, C. (1998) J. Cell Biol. 143, 547-560[Abstract/Free Full Text]
  29. Kapila, Y. L., Wang, S., Dazin, P., Tafolla, E., and Mass, M. (2002) J. Biol. Chem. 277, 8482-8491[Abstract/Free Full Text]
  30. Rezaee, M., Penta, K., and Quertermous, T. (2002) Am. J. Physiol. 282, H1924-H1932
  31. Townsend, P. A., Villanova, I., Uhlmann, E., Peyman, A., Knolle, J., Baron, R., Teti, A., and Horton, M. A. (2000) Eur. J. Cancer 36, 397-409[CrossRef][Medline] [Order article via Infotrieve]
  32. Hauck, C. R., Sieg, D. J., Hsia, D. A., Loftus, J. C., Gaarde, W. A., Monia, B. P., and Schlaepfer, D. D. (2001) Cancer Res. 61, 7079-7090[Abstract/Free Full Text]
  33. Seewaldt, V. L., Mrozek, K., Sigle, R., Dietze, E. C., Heine, K., Hockenbery, D. M., Hobbs, K. B., and Caldwell, L. E. (2001) J. Cell Biol. 155, 471-486[Abstract/Free Full Text]
  34. Akhtar, S., Kole, R., and Juliano, R. L. (1991) Life Sci. 49, 1793-1801[CrossRef][Medline] [Order article via Infotrieve]
  35. Kapila, Y. L., Wang, S., and Johnson, P. W. (1999) J. Biol. Chem. 274, 30906-30913[Abstract/Free Full Text]
  36. Wang, W. J., Kuo, J. C., Yao, C. C., and Chen, R. H. (2002) J. Cell Biol. 159, 169-179[Abstract/Free Full Text]
  37. Zeng, Q., McCauley, L. K., and Wang, C. Y. (2002) J. Biol. Chem. 277, 50137-50142[Abstract/Free Full Text]
  38. Fukai, F., Mashimo, M., Akiyama, K., Goto, T., Tanuma, S., and Katayama, T. (1998) Exp. Cell Res. 242, 92-99[CrossRef][Medline] [Order article via Infotrieve]
  39. Hynes, R. O. (1990) Fibronectins, pp. 8-16, 177, Springer-Verlag New York Inc., New York
  40. Aumailley, M., Gurrath, M., Muller, G., Calvete, J., Timpl, R., and Kessler, H. (1991) FEBS Lett. 291, 50-54[CrossRef][Medline] [Order article via Infotrieve]
  41. Bates, R. C., Buret, A., van Helden, D. F., Horton, M. A., and Burns, G. F. (1994) J. Cell Biol. 125, 3-15
  42. Frisch, S. M., and Ruoslahti, E. (1997) Curr. Opin. Cell Biol. 9, 701-706[CrossRef][Medline] [Order article via Infotrieve]
  43. Schlaepfer, D. D., and Hunter, T. (1998) Trends Cell Biol. 8, 151-157[CrossRef][Medline] [Order article via Infotrieve]
  44. Kurenova, E., Xu, L. H., Yang, X., Baldwin, A. S., Jr., Craven, R. J., Hanks, S. K., Liu, Z. G., and Cance, W. G. (2004) Mol. Cell. Biol. 24, 4361-4371[Abstract/Free Full Text]
  45. Golubovskaya, V. M., Gross, S., Kaur, A. S., Wilson, R. I., Xu, L. H., Yang, X. H., and Cance, W. G. (2003) Mol. Cancer Res. 1, 755-764[Abstract/Free Full Text]
  46. Hanks, S. K., Ryzhova, L., Shin, N. Y., and Brabek, J. (2003) Front. Biosci. 8, 982-996
  47. Parsons, J. T. (2003) J. Cell Sci. 116, 1409-1416[Abstract/Free Full Text]
  48. Duxbury, M. S., Ito, H., Zinner, M. J., Ashley, S. W., and Whang, E. E. (2004) Surgery 135, 555-562[CrossRef][Medline] [Order article via Infotrieve]
  49. Miyazaki, T., Kato, H., Nakajima, M., Soda, M., Fukai, Y., Masuda, N., Manda, R., Fukuchi, M., Tsukada, K., and Kuwano, H. (2003) Br. J. Cancer 89, 140-145[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
Y. Wu, J. Zuo, G. Ji, H. Saiyin, X. Liu, F. Yin, N. Cao, Y. Wen, J. J. Li, and L. Yu
Proapoptotic Function of Integrin {beta}3 in Human Hepatocellular Carcinoma Cells
Clin. Cancer Res., January 1, 2009; 15(1): 60 - 69.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Sekine, S. Tsuji, O. Ikeda, K. Sugiyma, K. Oritani, K. Shimoda, R. Muromoto, N. Ohbayashi, A. Yoshimura, and T. Matsuda
Signal-Transducing Adaptor Protein-2 Regulates Integrin-Mediated T Cell Adhesion through Protein Degradation of Focal Adhesion Kinase
J. Immunol., August 15, 2007; 179(4): 2397 - 2407.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Gius, M. C. Funk, E. Y. Chuang, S. Feng, P. C. Huettner, L. Nguyen, C. M. Bradbury, M. Mishra, S. Gao, B. M. Buttin, et al.
Profiling Microdissected Epithelium and Stroma to Model Genomic Signatures for Cervical Carcinogenesis Accommodating for Covariates
Cancer Res., August 1, 2007; 67(15): 7113 - 7123.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
A. Marconi, K. Dallaglio, R. Lotti, C. Vaschieri, F. Truzzi, F. Fantini, and C. Pincelli
Survivin Identifies Keratinocyte Stem Cells and Is Downregulated by Anti-{beta}1 Integrin During Anoikis
Stem Cells, January 1, 2007; 25(1): 149 - 155.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
R. J. B. Francis and C. W. Lo
Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation
Development, September 1, 2006; 133(17): 3451 - 3460.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. M.F. Siesser and S. K. Hanks
The Signaling and Biological Implications of FAK Overexpression in Cancer.
Clin. Cancer Res., June 1, 2006; 12(11): 3233 - 3237.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-M. Yang, P. O'Neill, W. Jin, R. Foty, D. J. Medina, Z. Xu, M. Lomas, G. M. Arndt, Y. Tang, M. Nakada, et al.
Extracellular Matrix Metalloproteinase Inducer (CD147) Confers Resistance of Breast Cancer Cells to Anoikis through Inhibition of Bim
J. Biol. Chem., April 7, 2006; 281(14): 9719 - 9727.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Stylianou, R. B. Clarke, and K. Brennan
Aberrant Activation of Notch Signaling in Human Breast Cancer
Cancer Res., February 1, 2006; 66(3): 1517 - 1525.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. J. van Nimwegen, S. Verkoeijen, L. van Buren, D. Burg, and B. van de Water
Requirement for Focal Adhesion Kinase in the Early Phase of Mammary Adenocarcinoma Lung Metastasis Formation
Cancer Res., June 1, 2005; 65(11): 4698 - 4706.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/46/48342    most recent
M407953200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Y.
Right arrow Articles by Kapila, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Y.
Right arrow Articles by Kapila, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement