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Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells.

Wey JS, Gray MJ, Fan F, Belcheva A, McCarty MF, Stoeltzing O, Somcio R, Liu W, Evans DB, Klagsbrun M, Gallick GE, Ellis LM - Br. J. Cancer (2005)

Bottom Line: Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU.In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine.Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, Unit 444, The University of Texas, MD Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402, USA.

ABSTRACT
Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

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Effect of conditioned medium from NRP-1-transfected FG cells on signalling in mock-transfected FG cells. Mock-transfected (Control) or NRP-1-transfected FG cells were grown for 48 h in medium containing 1% FBS. Conditioned medium was collected and applied to mock-transfected FG cells. Mock-transfected control cells were left untreated. After incubation for 5 min (ERK) or 15 min (JNK), cells were lysed, and Western blot analysis for phosphorylated ERK1/2 or JNK was performed. Treatment with conditioned medium from NRP-1-transfected cells (NRP-1 C#1 and C#9) led to moderate upregulation in ERK1/2 phosphorylation and at least three-fold upregulation of JNK phosphorylation relative to cells treated with conditioned medium from mock-transfectants. Control-treated cells had relatively unchanged signalling compared to untreated cells (data not shown).
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fig3: Effect of conditioned medium from NRP-1-transfected FG cells on signalling in mock-transfected FG cells. Mock-transfected (Control) or NRP-1-transfected FG cells were grown for 48 h in medium containing 1% FBS. Conditioned medium was collected and applied to mock-transfected FG cells. Mock-transfected control cells were left untreated. After incubation for 5 min (ERK) or 15 min (JNK), cells were lysed, and Western blot analysis for phosphorylated ERK1/2 or JNK was performed. Treatment with conditioned medium from NRP-1-transfected cells (NRP-1 C#1 and C#9) led to moderate upregulation in ERK1/2 phosphorylation and at least three-fold upregulation of JNK phosphorylation relative to cells treated with conditioned medium from mock-transfectants. Control-treated cells had relatively unchanged signalling compared to untreated cells (data not shown).

Mentions: Since NRP-1 overexpression resulted in such a consistent increase in ERK1/2 and JNK signalling, we sought to determine whether this was caused by induction of an autocrine pathway. Conditioned medium from mock-transfected or NRP-1-transfected FG cells was collected and applied to mock-transfected FG cells, and the effect on signalling intermediates was assessed. Nearly a two-fold induction of ERK1/2 phosphorylation was consistently detected in cells exposed to conditioned medium from NRP-1-overexpressing cells, and a greater than three-fold increase was seen in JNK phosphorylation, similar to levels seen in NRP-1-overexpressing cells themselves (Figure 3). Low levels of phosphorylated ERK1/2 and JNK were seen in control cells grown in 1% medium (data not shown). These findings suggest that the upregulated MAPK signalling observed in NRP-1-overexpressing cells is due to an autocrine pathway regulated by a secreted factor.


Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells.

Wey JS, Gray MJ, Fan F, Belcheva A, McCarty MF, Stoeltzing O, Somcio R, Liu W, Evans DB, Klagsbrun M, Gallick GE, Ellis LM - Br. J. Cancer (2005)

Effect of conditioned medium from NRP-1-transfected FG cells on signalling in mock-transfected FG cells. Mock-transfected (Control) or NRP-1-transfected FG cells were grown for 48 h in medium containing 1% FBS. Conditioned medium was collected and applied to mock-transfected FG cells. Mock-transfected control cells were left untreated. After incubation for 5 min (ERK) or 15 min (JNK), cells were lysed, and Western blot analysis for phosphorylated ERK1/2 or JNK was performed. Treatment with conditioned medium from NRP-1-transfected cells (NRP-1 C#1 and C#9) led to moderate upregulation in ERK1/2 phosphorylation and at least three-fold upregulation of JNK phosphorylation relative to cells treated with conditioned medium from mock-transfectants. Control-treated cells had relatively unchanged signalling compared to untreated cells (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2361553&req=5

fig3: Effect of conditioned medium from NRP-1-transfected FG cells on signalling in mock-transfected FG cells. Mock-transfected (Control) or NRP-1-transfected FG cells were grown for 48 h in medium containing 1% FBS. Conditioned medium was collected and applied to mock-transfected FG cells. Mock-transfected control cells were left untreated. After incubation for 5 min (ERK) or 15 min (JNK), cells were lysed, and Western blot analysis for phosphorylated ERK1/2 or JNK was performed. Treatment with conditioned medium from NRP-1-transfected cells (NRP-1 C#1 and C#9) led to moderate upregulation in ERK1/2 phosphorylation and at least three-fold upregulation of JNK phosphorylation relative to cells treated with conditioned medium from mock-transfectants. Control-treated cells had relatively unchanged signalling compared to untreated cells (data not shown).
Mentions: Since NRP-1 overexpression resulted in such a consistent increase in ERK1/2 and JNK signalling, we sought to determine whether this was caused by induction of an autocrine pathway. Conditioned medium from mock-transfected or NRP-1-transfected FG cells was collected and applied to mock-transfected FG cells, and the effect on signalling intermediates was assessed. Nearly a two-fold induction of ERK1/2 phosphorylation was consistently detected in cells exposed to conditioned medium from NRP-1-overexpressing cells, and a greater than three-fold increase was seen in JNK phosphorylation, similar to levels seen in NRP-1-overexpressing cells themselves (Figure 3). Low levels of phosphorylated ERK1/2 and JNK were seen in control cells grown in 1% medium (data not shown). These findings suggest that the upregulated MAPK signalling observed in NRP-1-overexpressing cells is due to an autocrine pathway regulated by a secreted factor.

Bottom Line: Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU.In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine.Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical Oncology, Unit 444, The University of Texas, MD Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402, USA.

ABSTRACT
Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

Show MeSH
Related in: MedlinePlus