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Effects of insulin on human pancreatic cancer progression modeled in vitro.

Chan MT, Lim GE, Skovsø S, Yang YH, Albrecht T, Alejandro EU, Hoesli CA, Piret JM, Warnock GL, Johnson JD - BMC Cancer (2014)

Bottom Line: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types.High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro.Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada. James.D.Johnson@ubc.ca.

ABSTRACT

Background: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined.

Methods: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model.

Results: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.

Conclusions: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

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RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked and expressed as the fold change in the percent of PI and Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved and treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 and 1 mM wortmannin (wort.) for 24 hours and 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test.
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Fig5: RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked and expressed as the fold change in the percent of PI and Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved and treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 and 1 mM wortmannin (wort.) for 24 hours and 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test.

Mentions: Next, we assessed the requirement for RAF1/ERK versus PI3K/AKT signalling on the viability of PANC1 cells. Inhibition of RAF1 significantly increased cell death (Figure 5A-C) and reduced cell viability (Figure 5D,E) in PANC1 cells. A more modest delayed effect on cell viability and cell death was also observed after MEK1/2 inhibition by U0126 (Figure 5A,D,E), similar to the findings in the HPDE cells. AKT inhibition was much less effective at inducing PANC1 cell death as assessed by cell counting, PI incorporation, and cleaved caspase 3 levels (Figure 5A-E). These observations indicate that the RAF1/ERK pathway, and not the PI3K/AKT pathway, may play a more important role in the maintenance of PANC1 cell survival under these basal conditions.Figure 4


Effects of insulin on human pancreatic cancer progression modeled in vitro.

Chan MT, Lim GE, Skovsø S, Yang YH, Albrecht T, Alejandro EU, Hoesli CA, Piret JM, Warnock GL, Johnson JD - BMC Cancer (2014)

RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked and expressed as the fold change in the percent of PI and Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved and treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 and 1 mM wortmannin (wort.) for 24 hours and 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test.
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Fig5: RAF1/ERK signalling is preferentially required for PANC1 cell survival in the absence of exogenous insulin. (A) Effects of different small molecule inhibitors on propidium iodide (PI) incorporation (PI) in PANC1 cells were tracked and expressed as the fold change in the percent of PI and Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t =0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =3) Data points that have been shaded solid black represent statistical significance when compared to non-treated conditions at that time point. # Indicates statistical significance in cells treated with Akti1/2 when compared to control at that time point. (B) Average number of PI positive cells over time of each treated group in Figure 5A is shown as a histogram expressed in arbitrary units (AU). GW5074 exhibited statistical significance, where as other treatments did not yield significance. U0126 p = 0.38, GW5074 *p = 0.0005, Akti-1/2 p = 0.395, Wort. p = 0.292 (n = 3). (C) The effect of 24 hours treatment with inhibitors on cleaved caspase 3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates (n =3). (D-E) PANC1 cells were serum starved and treated with either DMSO, 10 μM GW5074, 10 μM U0126, 200 nM Akti-1/2 and 1 mM wortmannin (wort.) for 24 hours and 120 hours (n =4-5). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control. (C-E) One-way ANOVA analysis with Bonferroni post-test was performed. *Represents statistical significance of p < 0.05 where treated groups are compared to control (-) in the post-hoc test.
Mentions: Next, we assessed the requirement for RAF1/ERK versus PI3K/AKT signalling on the viability of PANC1 cells. Inhibition of RAF1 significantly increased cell death (Figure 5A-C) and reduced cell viability (Figure 5D,E) in PANC1 cells. A more modest delayed effect on cell viability and cell death was also observed after MEK1/2 inhibition by U0126 (Figure 5A,D,E), similar to the findings in the HPDE cells. AKT inhibition was much less effective at inducing PANC1 cell death as assessed by cell counting, PI incorporation, and cleaved caspase 3 levels (Figure 5A-E). These observations indicate that the RAF1/ERK pathway, and not the PI3K/AKT pathway, may play a more important role in the maintenance of PANC1 cell survival under these basal conditions.Figure 4

Bottom Line: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types.High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro.Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada. James.D.Johnson@ubc.ca.

ABSTRACT

Background: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined.

Methods: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model.

Results: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.

Conclusions: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Show MeSH
Related in: MedlinePlus