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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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Analysis of uPA/uPAR–dependent signaling to ERK. (A) Effect of scuPA on phospho-ERK. Subconfluent cultures of LK25 or AS24 in 6-well plates were serum-starved for 24 h, acid-stripped (to remove uPAR-bound uPA), and incubated for 5 min with the indicated doses of scuPA and 200 KIU/ml of aprotinin (aprotinin was also used in B and C). The levels of phospho-ERK1/2 and ERK (lower panels) were determined by Western blot (see Materials and Methods); the exposure time in upper left panel is 1 s and in the upper right panel is 10 s (the experiment was repeated three times); the graphs show the Phospho-ERK/ERK ratio (R) obtained by densitometric scanning. (B) Effect of MEK-1 inhibitor on scuPA-induced ERK activation. LK25 or AS24 serum-starved cells were pretreated for 15 min with 10 μM of MEK-1 inhibitor PD98059, or medium alone. The monolayers were acid-stripped, incubated with 10 nM scuPA or medium alone, for 5 min, with or without 10 μM PD98059, lysed, and analyzed for phospho-ERK (upper panels) or ERK levels (lower panels) (experiment was repeated twice). (C) Time dependence of ERK activation by scuPA. LK25 or AS24 cell monolayers were acid-stripped and incubated for the indicated times with 10 nM scuPA. Phospho-ERK (upper panels) and ERK (lower panels) levels were determined (experiment was repeated twice).
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Figure 3: Analysis of uPA/uPAR–dependent signaling to ERK. (A) Effect of scuPA on phospho-ERK. Subconfluent cultures of LK25 or AS24 in 6-well plates were serum-starved for 24 h, acid-stripped (to remove uPAR-bound uPA), and incubated for 5 min with the indicated doses of scuPA and 200 KIU/ml of aprotinin (aprotinin was also used in B and C). The levels of phospho-ERK1/2 and ERK (lower panels) were determined by Western blot (see Materials and Methods); the exposure time in upper left panel is 1 s and in the upper right panel is 10 s (the experiment was repeated three times); the graphs show the Phospho-ERK/ERK ratio (R) obtained by densitometric scanning. (B) Effect of MEK-1 inhibitor on scuPA-induced ERK activation. LK25 or AS24 serum-starved cells were pretreated for 15 min with 10 μM of MEK-1 inhibitor PD98059, or medium alone. The monolayers were acid-stripped, incubated with 10 nM scuPA or medium alone, for 5 min, with or without 10 μM PD98059, lysed, and analyzed for phospho-ERK (upper panels) or ERK levels (lower panels) (experiment was repeated twice). (C) Time dependence of ERK activation by scuPA. LK25 or AS24 cell monolayers were acid-stripped and incubated for the indicated times with 10 nM scuPA. Phospho-ERK (upper panels) and ERK (lower panels) levels were determined (experiment was repeated twice).

Mentions: Since in tumorigenic HEp3 cells 80–90% of uPAR is occupied by endogenously produced uPA (Ossowski 1992), and since binding of uPA, scuPA, or amino terminal fragment of uPA to uPAR is known to initiate signal transduction (Busso et al. 1994; Nguyen et al. 1998), we wished to test whether removal of the endogenous ligand interrupts the signal leading to ERK activation and whether its addition restores it. LK25 and AS24 cells stripped of endogenous uPA were incubated for 5 min with increasing concentrations of scuPA and tested for ERK phosphorylation. To avoid scuPA activation and protease-dependent effects, the reaction was carried out in the presence of 200 KIU/ml aprotinin. In LK25 cells, scuPA binding induced a strong, dose-dependent increase in phospho-ERK levels, with a maximal effect at ∼10 nM (Fig. 3 A, left). Neither the basal nor the stimulated levels of phospho-ERK were detectable in AS24 cells at the same time of exposure (data not shown). However, after a 10 times longer exposure of the blot (10 s versus 1 s used in Fig. 3 A, left) both basal and scuPA-induced active ERK became detectable. Densitometric analysis revealed that 1 nM concentration of scuPA (approximately its Kd value) caused a maximal (approximately fourfold) stimulation of active ERK in LK25 cells, whereas causing no modulation in AS24 cells. The highest concentration of scuPA (80 nM) added to AS24 cells produced a large stimulation over the basal level (Fig. 3 A, right), but it still represented only <4% of active ERK in LK25 cells (Fig. 3 A, left). This result suggests that it is not the fold increase, but the absolute level of active ERK that needs to be high enough to surpass the required threshold for proliferation in vivo. Similar results were observed when T-HEp3 was compared with D-HEp3 cells (data not shown). Cells stripped of endogenous uPA had a very low level of active ERK that could be restored by incubation with exogenous scuPA, but not in the presence of the MEK-1 inhibitor PD98059 (Fig. 3 B), indicating that as shown before (Nguyen et al. 1998) the uPA-induced signal was propagated through the MEK1-ERK pathway. Also, time dependence of ERK activation by scuPA was different: whereas 10 nM scuPA induced a large increase in the level of phospho-ERK in LK25 cells, which at 10 min reached a plateau sustainable for at least 60 min, AS24 cells showed a less pronounced and much delayed activation of ERK by scuPA, peaking at 30 min and decreasing at 60 min (Fig. 3 C), suggesting that the ∼80% lower uPAR level in AS24 cells is incapable of evoking an optimal signal through this pathway.


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)

Analysis of uPA/uPAR–dependent signaling to ERK. (A) Effect of scuPA on phospho-ERK. Subconfluent cultures of LK25 or AS24 in 6-well plates were serum-starved for 24 h, acid-stripped (to remove uPAR-bound uPA), and incubated for 5 min with the indicated doses of scuPA and 200 KIU/ml of aprotinin (aprotinin was also used in B and C). The levels of phospho-ERK1/2 and ERK (lower panels) were determined by Western blot (see Materials and Methods); the exposure time in upper left panel is 1 s and in the upper right panel is 10 s (the experiment was repeated three times); the graphs show the Phospho-ERK/ERK ratio (R) obtained by densitometric scanning. (B) Effect of MEK-1 inhibitor on scuPA-induced ERK activation. LK25 or AS24 serum-starved cells were pretreated for 15 min with 10 μM of MEK-1 inhibitor PD98059, or medium alone. The monolayers were acid-stripped, incubated with 10 nM scuPA or medium alone, for 5 min, with or without 10 μM PD98059, lysed, and analyzed for phospho-ERK (upper panels) or ERK levels (lower panels) (experiment was repeated twice). (C) Time dependence of ERK activation by scuPA. LK25 or AS24 cell monolayers were acid-stripped and incubated for the indicated times with 10 nM scuPA. Phospho-ERK (upper panels) and ERK (lower panels) levels were determined (experiment was repeated twice).
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Related In: Results  -  Collection

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Figure 3: Analysis of uPA/uPAR–dependent signaling to ERK. (A) Effect of scuPA on phospho-ERK. Subconfluent cultures of LK25 or AS24 in 6-well plates were serum-starved for 24 h, acid-stripped (to remove uPAR-bound uPA), and incubated for 5 min with the indicated doses of scuPA and 200 KIU/ml of aprotinin (aprotinin was also used in B and C). The levels of phospho-ERK1/2 and ERK (lower panels) were determined by Western blot (see Materials and Methods); the exposure time in upper left panel is 1 s and in the upper right panel is 10 s (the experiment was repeated three times); the graphs show the Phospho-ERK/ERK ratio (R) obtained by densitometric scanning. (B) Effect of MEK-1 inhibitor on scuPA-induced ERK activation. LK25 or AS24 serum-starved cells were pretreated for 15 min with 10 μM of MEK-1 inhibitor PD98059, or medium alone. The monolayers were acid-stripped, incubated with 10 nM scuPA or medium alone, for 5 min, with or without 10 μM PD98059, lysed, and analyzed for phospho-ERK (upper panels) or ERK levels (lower panels) (experiment was repeated twice). (C) Time dependence of ERK activation by scuPA. LK25 or AS24 cell monolayers were acid-stripped and incubated for the indicated times with 10 nM scuPA. Phospho-ERK (upper panels) and ERK (lower panels) levels were determined (experiment was repeated twice).
Mentions: Since in tumorigenic HEp3 cells 80–90% of uPAR is occupied by endogenously produced uPA (Ossowski 1992), and since binding of uPA, scuPA, or amino terminal fragment of uPA to uPAR is known to initiate signal transduction (Busso et al. 1994; Nguyen et al. 1998), we wished to test whether removal of the endogenous ligand interrupts the signal leading to ERK activation and whether its addition restores it. LK25 and AS24 cells stripped of endogenous uPA were incubated for 5 min with increasing concentrations of scuPA and tested for ERK phosphorylation. To avoid scuPA activation and protease-dependent effects, the reaction was carried out in the presence of 200 KIU/ml aprotinin. In LK25 cells, scuPA binding induced a strong, dose-dependent increase in phospho-ERK levels, with a maximal effect at ∼10 nM (Fig. 3 A, left). Neither the basal nor the stimulated levels of phospho-ERK were detectable in AS24 cells at the same time of exposure (data not shown). However, after a 10 times longer exposure of the blot (10 s versus 1 s used in Fig. 3 A, left) both basal and scuPA-induced active ERK became detectable. Densitometric analysis revealed that 1 nM concentration of scuPA (approximately its Kd value) caused a maximal (approximately fourfold) stimulation of active ERK in LK25 cells, whereas causing no modulation in AS24 cells. The highest concentration of scuPA (80 nM) added to AS24 cells produced a large stimulation over the basal level (Fig. 3 A, right), but it still represented only <4% of active ERK in LK25 cells (Fig. 3 A, left). This result suggests that it is not the fold increase, but the absolute level of active ERK that needs to be high enough to surpass the required threshold for proliferation in vivo. Similar results were observed when T-HEp3 was compared with D-HEp3 cells (data not shown). Cells stripped of endogenous uPA had a very low level of active ERK that could be restored by incubation with exogenous scuPA, but not in the presence of the MEK-1 inhibitor PD98059 (Fig. 3 B), indicating that as shown before (Nguyen et al. 1998) the uPA-induced signal was propagated through the MEK1-ERK pathway. Also, time dependence of ERK activation by scuPA was different: whereas 10 nM scuPA induced a large increase in the level of phospho-ERK in LK25 cells, which at 10 min reached a plateau sustainable for at least 60 min, AS24 cells showed a less pronounced and much delayed activation of ERK by scuPA, peaking at 30 min and decreasing at 60 min (Fig. 3 C), suggesting that the ∼80% lower uPAR level in AS24 cells is incapable of evoking an optimal signal through this pathway.

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