Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer. Methods: Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined. Results: Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. Conclusion: The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer. © 2013 Kitano et al

Aberrant glycogen synthase kinase 3β is involved in pancreatic cancer cell invasion and resistance to therapy.

BACKGROUND AND PURPOSE: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer. METHODS: Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined. RESULTS: Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. CONCLUSION: The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer

Effects of GSK3β inhibition on the migration and invasion of pancreatic cancer cells.

<p>(A) Upper panels show the time course for PANC-1 cell migration in a wound-healing assay in the presence of DMSO or AR-A014418 (AR). The lower panel shows the relative widths of wounds measured as a percentage of the initial gap at time zero. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418. (B) Migrating cells through uncoated transwell and invading cells through matrigel-coated transwell were scored for PANC-1 and MIA PaCa-2 cells treated with DMSO or AR-A014418 (AR) for 22 hrs. Representative photomicroscopic findings in each assay are shown below the columns. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418.</p

Combined effect of gemcitabine or ionizong radiation and GSK3β inhibitor against cancer cells and xenografts.

<p>(A) The influence of AR-A014418 on the effect of gemcitabine was analyzed using the isobologram <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Tallarida1" target="_blank">[21]</a> by plotting the IC₅₀ of the combination therapy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289.s002" target="_blank">Fig. S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289.s007" target="_blank">Table S4</a>). (B) The combined effect of ionizing radiation and AR-A014418 was tested in PANC-1 and MIA PaCa-2 cells by colony formation assay. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418. (C) The combined effect of gemcitabine and AR-A014418 was tested in PANC-1 xenografts. Athymic mice with PANC-1 xenograft were assigned to four groups for treatment with intraperitoneal injection (twice a week) of DMSO (control; 8 mice), gemcitabine (GEM; 20 mg/kg body weight; 9 mice) and AR-A014418 (AR; 2 mg/kg body weight; 8 mice), alone or in combination (GEM+AR; 9 mice). At the time after treatment for 10 weeks, tumor volume (cm³) was calculated using the formula 0.5×S²×L, where S is the smallest tumor diameter (cm) and L is the largest (cm) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Shakoori2" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Mai1" target="_blank">[12]</a>. The mean tumor volume was compared between the 4 groups. *<i>p</i><0.05, statistically significant difference between data.</p

Changes in expression and phosphorylation of the proteins in cancer cells following GSK3β inhibition.

<p>(A) Immunoblotting analysis compares the expression of Rb, CDK4, CDK6 and cyclin D1, and the phosphorylation of Rb at S780 and S807/811 residues (p-Rb^S780, p-Rb^S807/811) between cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (B) Changes in levels of p-Rb^S780 and p-Rb^S807/811 and expression of Rb and cyclin D1 were examined in MIA PaCa-2 cells at the indicated time points after treatment with 10 µM AR-A014418. (C) Expression of Rb, cyclin D1, GSK3α and GSK3β proteins and levels of Rb phosphorylation (p-Rb^S780) were examined and compared between the same pancreatic cancer cells transfected with non-specific siRNA (NS) or GSK3β-specific siRNA (S) (10 nM each). (A–C) β-actin expression was monitored as a loading control.</p