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The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

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Invasion response of normal epidermal and SCC-derived cell lines. (a) Quantitative analysis of invasion of primary  keratinocytes (HEK), immortalized keratinocytes (TFK104 and  HaCaT), and various SCC-derived cell lines in response to 10 ng/ml  EGF. Invasion assay results were quantitated as described elsewhere (Hennigan et al., 1994; Lamb et al., 1997a) with a Bio-Rad  program (Comos) and represent the average from at least three  independent experiments. (b) A431 invasion in response to EGF.  Shown are confocal images of propidium iodide–stained cell nuclei at the bottom of the filter (0 μm), top of the filter (10 μm),  and at various heights as indicated through the Matrigel in the  absence (top) or presence (bottom) of 10 ng/ml EGF.
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Figure 1: Invasion response of normal epidermal and SCC-derived cell lines. (a) Quantitative analysis of invasion of primary keratinocytes (HEK), immortalized keratinocytes (TFK104 and HaCaT), and various SCC-derived cell lines in response to 10 ng/ml EGF. Invasion assay results were quantitated as described elsewhere (Hennigan et al., 1994; Lamb et al., 1997a) with a Bio-Rad program (Comos) and represent the average from at least three independent experiments. (b) A431 invasion in response to EGF. Shown are confocal images of propidium iodide–stained cell nuclei at the bottom of the filter (0 μm), top of the filter (10 μm), and at various heights as indicated through the Matrigel in the absence (top) or presence (bottom) of 10 ng/ml EGF.

Mentions: SCC-derived cell lines express elevated levels of EGFR compared with normal human keratinocytes in culture (Hendler and Ozanne, 1984; Stanton et al., 1994). We used a quantitative in vitro invasion assay (Hennigan et al., 1994; Lamb et al., 1997a) to test whether EGFR overexpression correlates with increased invasion potential. For our studies we used a number of well characterized SCC-derived cell lines (Edington et al., 1995; Malliri et al., 1996) for which the levels of EGFR expression are known (Stanton et al., 1994). We also included normal HEKs, HPV16 E6 and E7 immortalized HEKs (TFK104), and benign immortal keratinocytes (HaCaT) to determine whether invasiveness is an exclusive property of malignant epithelial cells. Unlike the SCC-derived cell lines, none of the benign keratinocyte cell lines overexpress EGFR (Stanton et al., 1994). Further, none of the benign epidermal cells invaded in this assay, even in the presence of EGF (Fig. 1 a). In contrast, whereas none of the SCC-derived cell lines tested invaded spontaneously, they all invaded after EGF treatment (Fig. 1 a). A431 cells are an example of the SCC-derived cell lines shown in Fig. 1 a. Without EGF no A431 cells migrated to the top of the filter; on addition of EGF cells migrated to a distance of 50 μm from the bottom of the filter (Fig. 1 b). Invasion of A431 cells was dependent upon the concentration of EGF added to the top of the Matrigel and was maximal for 10 ng/ml (data not shown). Invasion was abrogated both by anti-EGFR antibodies demonstrated to inhibit EGFR signaling (Modjtahedi et al., 1993) and by CGP52411 (a gift from Dr. N. Lydon, Ciba Geigy Pharmaceuticals, Basel, Switzerland) that inhibits EGFR tyrosine kinase activity (Buchdunger et al., 1994; data not shown). We conclude that invasion of SCC-derived cells lines correlates with EGFR overexpression and is dependent on EGFR signaling.


The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW - J. Cell Biol. (1998)

Invasion response of normal epidermal and SCC-derived cell lines. (a) Quantitative analysis of invasion of primary  keratinocytes (HEK), immortalized keratinocytes (TFK104 and  HaCaT), and various SCC-derived cell lines in response to 10 ng/ml  EGF. Invasion assay results were quantitated as described elsewhere (Hennigan et al., 1994; Lamb et al., 1997a) with a Bio-Rad  program (Comos) and represent the average from at least three  independent experiments. (b) A431 invasion in response to EGF.  Shown are confocal images of propidium iodide–stained cell nuclei at the bottom of the filter (0 μm), top of the filter (10 μm),  and at various heights as indicated through the Matrigel in the  absence (top) or presence (bottom) of 10 ng/ml EGF.
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Related In: Results  -  Collection

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Figure 1: Invasion response of normal epidermal and SCC-derived cell lines. (a) Quantitative analysis of invasion of primary keratinocytes (HEK), immortalized keratinocytes (TFK104 and HaCaT), and various SCC-derived cell lines in response to 10 ng/ml EGF. Invasion assay results were quantitated as described elsewhere (Hennigan et al., 1994; Lamb et al., 1997a) with a Bio-Rad program (Comos) and represent the average from at least three independent experiments. (b) A431 invasion in response to EGF. Shown are confocal images of propidium iodide–stained cell nuclei at the bottom of the filter (0 μm), top of the filter (10 μm), and at various heights as indicated through the Matrigel in the absence (top) or presence (bottom) of 10 ng/ml EGF.
Mentions: SCC-derived cell lines express elevated levels of EGFR compared with normal human keratinocytes in culture (Hendler and Ozanne, 1984; Stanton et al., 1994). We used a quantitative in vitro invasion assay (Hennigan et al., 1994; Lamb et al., 1997a) to test whether EGFR overexpression correlates with increased invasion potential. For our studies we used a number of well characterized SCC-derived cell lines (Edington et al., 1995; Malliri et al., 1996) for which the levels of EGFR expression are known (Stanton et al., 1994). We also included normal HEKs, HPV16 E6 and E7 immortalized HEKs (TFK104), and benign immortal keratinocytes (HaCaT) to determine whether invasiveness is an exclusive property of malignant epithelial cells. Unlike the SCC-derived cell lines, none of the benign keratinocyte cell lines overexpress EGFR (Stanton et al., 1994). Further, none of the benign epidermal cells invaded in this assay, even in the presence of EGF (Fig. 1 a). In contrast, whereas none of the SCC-derived cell lines tested invaded spontaneously, they all invaded after EGF treatment (Fig. 1 a). A431 cells are an example of the SCC-derived cell lines shown in Fig. 1 a. Without EGF no A431 cells migrated to the top of the filter; on addition of EGF cells migrated to a distance of 50 μm from the bottom of the filter (Fig. 1 b). Invasion of A431 cells was dependent upon the concentration of EGF added to the top of the Matrigel and was maximal for 10 ng/ml (data not shown). Invasion was abrogated both by anti-EGFR antibodies demonstrated to inhibit EGFR signaling (Modjtahedi et al., 1993) and by CGP52411 (a gift from Dr. N. Lydon, Ciba Geigy Pharmaceuticals, Basel, Switzerland) that inhibits EGFR tyrosine kinase activity (Buchdunger et al., 1994; data not shown). We conclude that invasion of SCC-derived cells lines correlates with EGFR overexpression and is dependent on EGFR signaling.

Bottom Line: Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression.However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells.Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom.

ABSTRACT
Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Show MeSH
Related in: MedlinePlus