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Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.

Aguirre Ghiso JA, Kovalski K, Ossowski L - J. Cell Biol. (1999)

Bottom Line: We found that uPA/uPAR proteins were physically associated with alpha5beta1, and that in cells with low uPAR the frequency of this association was significantly reduced, leading to a reduced avidity of alpha5beta1 and a lower adhesion of cells to the fibronectin (FN).Disruption of uPAR-alpha5beta1 complexes in uPAR-rich cells with antibodies or a peptide that disrupts uPAR-beta1 interactions, reduced the FN-dependent ERK1/2 activation.In support of this conclusion we found that treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR/beta1 signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR.

View Article: PubMed Central - PubMed

Affiliation: Rochelle Belfer Chemotherapy Foundation Laboratory, Division of Medical Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Mechanisms that regulate the transition of metastases from clinically undetectable and dormant to progressively growing are the least understood aspects of cancer biology. Here, we show that a large ( approximately 70%) reduction in the urokinase plasminogen activator receptor (uPAR) level in human carcinoma HEp3 cells, while not affecting their in vitro growth, induced a protracted state of tumor dormancy in vivo, with G(0)/G(1) arrest. We have now identified the mechanism responsible for the induction of dormancy. We found that uPA/uPAR proteins were physically associated with alpha5beta1, and that in cells with low uPAR the frequency of this association was significantly reduced, leading to a reduced avidity of alpha5beta1 and a lower adhesion of cells to the fibronectin (FN). Adhesion to FN resulted in a robust and persistent ERK1/2 activation and serum-independent growth stimulation of only uPAR-rich cells. Compared with uPAR-rich tumorigenic cells, the basal level of active extracellular regulated kinase (ERK) was four to sixfold reduced in uPAR-poor dormant cells and its stimulation by single chain uPA (scuPA) was weak and showed slow kinetics. The high basal level of active ERK in uPAR-rich cells could be strongly and rapidly stimulated by scuPA. Disruption of uPAR-alpha5beta1 complexes in uPAR-rich cells with antibodies or a peptide that disrupts uPAR-beta1 interactions, reduced the FN-dependent ERK1/2 activation. These results indicate that dormancy of low uPAR cells may be the consequence of insufficient uPA/uPAR/alpha5beta1 complexes, which cannot induce ERK1/2 activity above a threshold needed to sustain tumor growth in vivo. In support of this conclusion we found that treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR/beta1 signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR.

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Effect of β1-integrin–uPAR complex disruption and suPAR treatment on ERK activation. (A) Effect of soluble uPAR on ERK activation. LK25 (top panels) or AS24 (bottom panels) monolayers were serum-starved for 24 h and incubated with suPAR for 5 min in the presence of 200 KIU/ml of aprotinin. The cells were lysed and the proteins were analyzed for phospho-ERK and ERK levels (experiment was repeated three times). (B) Effect of anti–uPAR antibodies on ERK activation. T-HEp3 cells were incubated in suspension with medium alone (lanes 1 and 2), 7 μg/ml of isotype-matched IgG (lane 3), or with 7 μg/ml of anti–uPAR antibody (R2, lane 4) at 37°C for 35 min and inoculated into plates coated with 4 μg/ml PL (lane 1) or FN (lanes 2–4), allowed to adhere for 10 min, lysed, and analyzed for phospho-ERK (upper panel) and ERK (lower panel) levels. The numbers on top of each lane show the ratio of phospho-ERK/ERK determined by densitometry. (Experiment repeated twice). (C) Effect of peptide 25 (P25) on ERK phosphorylation. Subconfluent monolayers of T-HEp3 cells were treated with peptide 25 or its scrambled version (SP25) for 10 min, the cells were lysed, and phospho-ERK and ERK levels were determined. (D) Effect of anti–β1 antibody and peptide 25 on adhesion-activated ERK. Suspensions of T-HEp3 cells were incubated with or without anti–β1 antibodies (AIIB2) or isotype-matched IgG for 30 min, the cells plated on plastic plates coated with 4 μg/ml of PL or a combination of PL + 4 μg/ml FN and after 15 min tested for phospho-ERK and ERK levels. In addition, T-HEp3 cells were plated on FN in the presence or absence of 5 μM peptide 25 and tested as above.
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Figure 7: Effect of β1-integrin–uPAR complex disruption and suPAR treatment on ERK activation. (A) Effect of soluble uPAR on ERK activation. LK25 (top panels) or AS24 (bottom panels) monolayers were serum-starved for 24 h and incubated with suPAR for 5 min in the presence of 200 KIU/ml of aprotinin. The cells were lysed and the proteins were analyzed for phospho-ERK and ERK levels (experiment was repeated three times). (B) Effect of anti–uPAR antibodies on ERK activation. T-HEp3 cells were incubated in suspension with medium alone (lanes 1 and 2), 7 μg/ml of isotype-matched IgG (lane 3), or with 7 μg/ml of anti–uPAR antibody (R2, lane 4) at 37°C for 35 min and inoculated into plates coated with 4 μg/ml PL (lane 1) or FN (lanes 2–4), allowed to adhere for 10 min, lysed, and analyzed for phospho-ERK (upper panel) and ERK (lower panel) levels. The numbers on top of each lane show the ratio of phospho-ERK/ERK determined by densitometry. (Experiment repeated twice). (C) Effect of peptide 25 (P25) on ERK phosphorylation. Subconfluent monolayers of T-HEp3 cells were treated with peptide 25 or its scrambled version (SP25) for 10 min, the cells were lysed, and phospho-ERK and ERK levels were determined. (D) Effect of anti–β1 antibody and peptide 25 on adhesion-activated ERK. Suspensions of T-HEp3 cells were incubated with or without anti–β1 antibodies (AIIB2) or isotype-matched IgG for 30 min, the cells plated on plastic plates coated with 4 μg/ml of PL or a combination of PL + 4 μg/ml FN and after 15 min tested for phospho-ERK and ERK levels. In addition, T-HEp3 cells were plated on FN in the presence or absence of 5 μM peptide 25 and tested as above.

Mentions: We next examined whether the low level of ERK activation in uPAR-deficient cells can be corrected by exogenously added, soluble uPAR (suPAR). Such an effect would suggest that β1 integrin or other extracellular domains of transmembrane proteins may serve as uPAR adapter molecules mediating its effect on ERK activation. LK25 or AS24 cells were incubated for 5 min with increasing concentrations of suPAR and tested for ERK phosphorylation. While not affecting ERK in LK25 cells, suPAR induced a dose-dependent phosphorylation of ERK in AS24 cells, with maximal stimulation occurring at 5 ng/ml (∼0.5 nM) (Fig. 7 A; T-HEp3 cells behaved like LK25 and D-HEp3 like AS24, results not shown). The addition of suPAR did not fully restore the level of ERK activation found in uPAR-rich cells, suggesting that GPI-anchored uPAR may be more effective in integrin activation. Taken together, these results strongly suggest that a full complement of uPA/uPAR, through an interaction with α5β1 integrin, may be responsible for the high level of ERK activation in LK25 and T-HEp3 cells.


Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.

Aguirre Ghiso JA, Kovalski K, Ossowski L - J. Cell Biol. (1999)

Effect of β1-integrin–uPAR complex disruption and suPAR treatment on ERK activation. (A) Effect of soluble uPAR on ERK activation. LK25 (top panels) or AS24 (bottom panels) monolayers were serum-starved for 24 h and incubated with suPAR for 5 min in the presence of 200 KIU/ml of aprotinin. The cells were lysed and the proteins were analyzed for phospho-ERK and ERK levels (experiment was repeated three times). (B) Effect of anti–uPAR antibodies on ERK activation. T-HEp3 cells were incubated in suspension with medium alone (lanes 1 and 2), 7 μg/ml of isotype-matched IgG (lane 3), or with 7 μg/ml of anti–uPAR antibody (R2, lane 4) at 37°C for 35 min and inoculated into plates coated with 4 μg/ml PL (lane 1) or FN (lanes 2–4), allowed to adhere for 10 min, lysed, and analyzed for phospho-ERK (upper panel) and ERK (lower panel) levels. The numbers on top of each lane show the ratio of phospho-ERK/ERK determined by densitometry. (Experiment repeated twice). (C) Effect of peptide 25 (P25) on ERK phosphorylation. Subconfluent monolayers of T-HEp3 cells were treated with peptide 25 or its scrambled version (SP25) for 10 min, the cells were lysed, and phospho-ERK and ERK levels were determined. (D) Effect of anti–β1 antibody and peptide 25 on adhesion-activated ERK. Suspensions of T-HEp3 cells were incubated with or without anti–β1 antibodies (AIIB2) or isotype-matched IgG for 30 min, the cells plated on plastic plates coated with 4 μg/ml of PL or a combination of PL + 4 μg/ml FN and after 15 min tested for phospho-ERK and ERK levels. In addition, T-HEp3 cells were plated on FN in the presence or absence of 5 μM peptide 25 and tested as above.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164973&req=5

Figure 7: Effect of β1-integrin–uPAR complex disruption and suPAR treatment on ERK activation. (A) Effect of soluble uPAR on ERK activation. LK25 (top panels) or AS24 (bottom panels) monolayers were serum-starved for 24 h and incubated with suPAR for 5 min in the presence of 200 KIU/ml of aprotinin. The cells were lysed and the proteins were analyzed for phospho-ERK and ERK levels (experiment was repeated three times). (B) Effect of anti–uPAR antibodies on ERK activation. T-HEp3 cells were incubated in suspension with medium alone (lanes 1 and 2), 7 μg/ml of isotype-matched IgG (lane 3), or with 7 μg/ml of anti–uPAR antibody (R2, lane 4) at 37°C for 35 min and inoculated into plates coated with 4 μg/ml PL (lane 1) or FN (lanes 2–4), allowed to adhere for 10 min, lysed, and analyzed for phospho-ERK (upper panel) and ERK (lower panel) levels. The numbers on top of each lane show the ratio of phospho-ERK/ERK determined by densitometry. (Experiment repeated twice). (C) Effect of peptide 25 (P25) on ERK phosphorylation. Subconfluent monolayers of T-HEp3 cells were treated with peptide 25 or its scrambled version (SP25) for 10 min, the cells were lysed, and phospho-ERK and ERK levels were determined. (D) Effect of anti–β1 antibody and peptide 25 on adhesion-activated ERK. Suspensions of T-HEp3 cells were incubated with or without anti–β1 antibodies (AIIB2) or isotype-matched IgG for 30 min, the cells plated on plastic plates coated with 4 μg/ml of PL or a combination of PL + 4 μg/ml FN and after 15 min tested for phospho-ERK and ERK levels. In addition, T-HEp3 cells were plated on FN in the presence or absence of 5 μM peptide 25 and tested as above.
Mentions: We next examined whether the low level of ERK activation in uPAR-deficient cells can be corrected by exogenously added, soluble uPAR (suPAR). Such an effect would suggest that β1 integrin or other extracellular domains of transmembrane proteins may serve as uPAR adapter molecules mediating its effect on ERK activation. LK25 or AS24 cells were incubated for 5 min with increasing concentrations of suPAR and tested for ERK phosphorylation. While not affecting ERK in LK25 cells, suPAR induced a dose-dependent phosphorylation of ERK in AS24 cells, with maximal stimulation occurring at 5 ng/ml (∼0.5 nM) (Fig. 7 A; T-HEp3 cells behaved like LK25 and D-HEp3 like AS24, results not shown). The addition of suPAR did not fully restore the level of ERK activation found in uPAR-rich cells, suggesting that GPI-anchored uPAR may be more effective in integrin activation. Taken together, these results strongly suggest that a full complement of uPA/uPAR, through an interaction with α5β1 integrin, may be responsible for the high level of ERK activation in LK25 and T-HEp3 cells.

Bottom Line: We found that uPA/uPAR proteins were physically associated with alpha5beta1, and that in cells with low uPAR the frequency of this association was significantly reduced, leading to a reduced avidity of alpha5beta1 and a lower adhesion of cells to the fibronectin (FN).Disruption of uPAR-alpha5beta1 complexes in uPAR-rich cells with antibodies or a peptide that disrupts uPAR-beta1 interactions, reduced the FN-dependent ERK1/2 activation.In support of this conclusion we found that treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR/beta1 signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR.

View Article: PubMed Central - PubMed

Affiliation: Rochelle Belfer Chemotherapy Foundation Laboratory, Division of Medical Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Mechanisms that regulate the transition of metastases from clinically undetectable and dormant to progressively growing are the least understood aspects of cancer biology. Here, we show that a large ( approximately 70%) reduction in the urokinase plasminogen activator receptor (uPAR) level in human carcinoma HEp3 cells, while not affecting their in vitro growth, induced a protracted state of tumor dormancy in vivo, with G(0)/G(1) arrest. We have now identified the mechanism responsible for the induction of dormancy. We found that uPA/uPAR proteins were physically associated with alpha5beta1, and that in cells with low uPAR the frequency of this association was significantly reduced, leading to a reduced avidity of alpha5beta1 and a lower adhesion of cells to the fibronectin (FN). Adhesion to FN resulted in a robust and persistent ERK1/2 activation and serum-independent growth stimulation of only uPAR-rich cells. Compared with uPAR-rich tumorigenic cells, the basal level of active extracellular regulated kinase (ERK) was four to sixfold reduced in uPAR-poor dormant cells and its stimulation by single chain uPA (scuPA) was weak and showed slow kinetics. The high basal level of active ERK in uPAR-rich cells could be strongly and rapidly stimulated by scuPA. Disruption of uPAR-alpha5beta1 complexes in uPAR-rich cells with antibodies or a peptide that disrupts uPAR-beta1 interactions, reduced the FN-dependent ERK1/2 activation. These results indicate that dormancy of low uPAR cells may be the consequence of insufficient uPA/uPAR/alpha5beta1 complexes, which cannot induce ERK1/2 activity above a threshold needed to sustain tumor growth in vivo. In support of this conclusion we found that treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR/beta1 signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR.

Show MeSH
Related in: MedlinePlus