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. 2009 Oct;100(10):1834-41.
doi: 10.1111/j.1349-7006.2009.01260.x. Epub 2009 Jul 24.

Involvement of Pin1 induction in epithelial-mesenchymal transition of tamoxifen-resistant breast cancer cells

Mi Ra Kim  1 , Hoo-Kyun Choi, Kyoung Bin Cho, Hyung Sik Kim, Keon Wook Kang
Affiliations

Involvement of Pin1 induction in epithelial-mesenchymal transition of tamoxifen-resistant breast cancer cells

Mi Ra Kim et al. Cancer Sci. 2009 Oct.

Abstract

Acquisition of resistance to tamoxifen is a critical therapeutic problem in breast cancer patients. Epithelial-mesenchymal transition (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, is associated with invasion and motility of cancer cells. Here, we found that tamoxifen-resistant (TAMR)-MCF-7 cells had undergone EMT, as evidenced by mesenchymal-like cell shape, downregulation of basal E-cadherin expression, and overexpression of N-cadherin and vimentin, as well as increased Snail transcriptional activity and protein expression. Given the roles of glycogen synthase kinase (GSK)-3beta and nuclear factor (NF)-kappaB in Snail-mediated E-cadherin deregulation during EMT, we examined the role of these signaling pathways in the EMT of TAMR-MCF-7 cells. Both Ser9-phosphorylated GSK-3beta (inactive form) and NF-kappaB reporter activity were increased in TAMR-MCF-7 cells, as was activation of the phosphatase and tensin homolog depleted on chromosome ten (PTEN)-phosphoinositide 3 (PI3)-kinase-Akt pathway. Pin1, a peptidyl-prolyl isomerase, was overexpressed in TAMR-MCF-7 cells, and Snail transcription and the expression of EMT markers could be decreased by Pin1 siRNA treatment. These results imply that Pin1 overexpression in TAMR-MCF-7 cells is involved in the EMT process via PTEN-PI3-kinase-Akt-GSK-3beta and/or GSK-3beta-NF-kappaB-dependent Snail activation, and suggest the potential involvement of Pin1 in EMT during breast cancer development.

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Figures

Figure 1
Figure 1
Snail‐mediated epithelial–mesenchymal transition (EMT) induction in tamoxifen‐resistant (TAMR)‐MCF‐7 cells. (a) Cell morphology of MCF‐7 and TAMR‐MCF‐7 cells. (b) Immunoblot analysis of E‐cadherin, N‐cadherin, and vimentin. Equal loading of proteins was verified by actin immunoblot. (c) Immunoblot analysis of Snail, Slug, and Twist. Representative immunoblots show the protein levels of Snail, Slug, and Twist in both MCF‐7 and TAMR‐MCF‐7 cells deprived of serum for 18 h.
Figure 2
Figure 2
Role of glycogen synthase kinase (GSK)‐3β inactivation in Snail upregulation. (a) Basal reporter activity of Snail promoters (Snail‐Luc) in MCF‐7 and tamoxifen‐resistant (TAMR)‐MCF‐7 cells. Each cell type was transiently transfected with –1.5kb or –2.5kb Snail‐Luc plasmid. Dual luciferase reporter assays were carried out on the lysed cells cotransfected with Snail‐Luc plasmids (firefly luciferase) and phRL‐SV (hRenilla luciferase) (a ratio of 100:1) 18 h after transfection. Reporter gene activation was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent means ± SD with four different samples (significant vs MCF‐7 cells, **P < 0.01, ## P < 0.01). (b) Immunoblot analysis of GSK‐3β and phosphorylated (p)‐GSK‐3β. Representative immunoblots show each protein in both MCF‐7 and TAMR‐MCF‐7 cells serum‐deprived for 18 h. (c) Inhibition of Snail transactivation by GSK‐3β overexpression vector. Left upper panel: Levels of GSK‐3β and Snail were determined by immunoblotting in TAMR‐MCF‐7 cells transfected with GSK‐3β overexpression vector (1 µg/well) or mock vector PCMV5 (1 µg/well). Right upper panel: Snail mRNA expression was determined by RT‐PCR analysis in TAMR‐MCF‐7 cells transfected with GSK‐3β overexpression vector or PCMV5 (1 µg/well). Lower panel: MCF‐7 and TAMR‐MCF‐7 cells were cotransfected with Snail –2.5kb reporter plasmid in combination with GSK‐3β overexpression vector or PCMV5 (1 µg/well). Data represents means ± SD with four different samples (significant vs the control, **P < 0.01; significant vs PCMV5‐transfected group, ## P < 0.01; control level = 1). (d) Effect of MG‐132, a proteasome inhibitor, on the expression of Snail. GSK‐3β overexpression vector‐ or PCMV5 (1 µg/well)‐transfected TAMR‐MCF‐7 cells were incubated with or without MG‐132 (1 µM) for 24 h and total cell lysates were subjected to immunoblotting.
Figure 3
Figure 3
Role of phosphatase and tensin homolog depleted on chromosome ten (PTEN)–PI3‐kinase–Akt in Snail upregulation of tamoxifen‐resistant (TAMR)‐MCF‐7 cells. Immunoblot analyses of (a) phosphorylated (p)‐Akt and (b) PTEN. Equal loading of proteins was verified by actin immunoblot. (c) Inhibition of Snail transactivation by PTEN overexpression vector. Left panel: Reporter gene assay. MCF‐7 and TAMR‐MCF‐7 cells were cotransfected with –2.5 kb Snail‐Luc reporter plasmid in combination with PTEN overexpression vector (0.1 µg/well) or mock vector PCMV5 (0.1 µg/well). Data represents means ± SD with four different samples (significant vs the MCF‐7 cells, **P < 0.01; significant vs PCMV5‐transfected TAMR‐MCF‐7 cells, ## P < 0.01). Right panel: Immunoblotting of Snail in TAMR‐MCF‐7 cells transfected with PCMV5 or PTEN overexpression vector. (d) Inhibition of Snail transactivation by myc‐p85 overexpression vector. Left panel: Reporter gene assay. MCF‐7 and TAMR‐MCF‐7 cells were cotransfected with –2.5 kb Snail‐Luc reporter plasmid in combination with myc‐p85 overexpression vector (0.1 µg/well) or control mock vector PCMV5 (0.1 µg/well). Data represent means ± SD with four different samples (significant vs the MCF‐7 cells, **P < 0.01; significant vs PCMV5‐transfected TAMR‐MCF‐7 cells, ## P < 0.01). Right panel: Immunoblotting of Snail in TAMR‐MCF‐7 cells transfected with PCMV5 or myc‐p85 overexpression vector.
Figure 4
Figure 4
Role of Pin1 overexpression in epithelial–mesenchymal transition of tamoxifen‐resistant (TAMR)‐MCF‐7 cells. (a) Immunoblot analysis of Pin1. A representative immunoblot shows Pin1 protein in both MCF‐7 and TAMR‐MCF‐7 cells serum‐deprived for 18 h. Equal loading of proteins was verified by actin immunoblot. (b) Effects of Pin1 siRNA on the E‐cadherin expression and Snail transcription activity in TAMR‐MCF‐7 cells. Upper panel: Levels of Pin1 and E‐cadherin were determined by immunoblotting in TAMR‐MCF‐7 cells transfected with Pin1 siRNA (60 pmol) or control siRNA. Lower panel: TAMR‐MCF‐7 cells were cotransfected with –2.5 kb Snail‐Luc reporter plasmid in combination with Pin1 siRNA (20 pmol) or control siRNA. Data represent means ± SD with four different samples (significant vs the control, **P < 0.01; significant vs the control siRNA‐transfected group, # P < 0.05). (c) Decrease in the expression levels of phosphorylated (p)‐Akt, p‐glycogen synthase kinase (GSK)‐3β, Snail and increase in phosphatase and tensin homolog depleted on chromosome ten (PTEN) level by Pin1 siRNA introduction. Each protein expression was determined by immunoblotting in TAMR‐MCF‐7 cells transfected with Pin1 siRNA (60 pmol) or control siRNA.
Figure 5
Figure 5
Involvement of nuclear factor (NF)‐κB in Pin1‐mediated epithelial–mesenchymal transition of tamoxifen‐resistant (TAMR)‐MCF‐7 cells. (a) Left panel: NF‐κB reporter activity in TAMR‐MCF‐7 cells. Dual luciferase reporter assays were carried out on the lysed cells co‐transfected with NF‐κB plasmid (firefly luciferase) and phRL‐SV (hRenilla luciferase) (a ratio of 100:1) 18 h after transfection. Reporter gene activation was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent means ± SD with four different samples (significant vs the MCF‐7 cells, **P < 0.01). Right panel: Role of glycogen synthase kinase (GSK)‐3β in the activation of NF‐κB. TAMR‐MCF‐7 cells were cotransfected with NF‐κB plasmid in combination with GSK‐3β overexpression vector or PCMV5 (1 µg/well). Data represent means ± SD with four different samples (significant vs PCMV5‐transfected group, **P < 0.01). (b) Inhibition of NF‐κB transactivation by tosyl‐phenylalanine chloremethyl‐ketone (TPCK), a NF‐κB inhibitor. Left panel: Reporter gene assay. MCF‐7 and TAMR‐MCF‐7 cells were transfected with –2.5kb Snail‐Luc reporter plasmid and treated with TPCK for 24 h. Data represent means ± SD with four different samples (significant vs the control, *P < 0.05, **P < 0.01; control level = 1). Right panel: Immunoblotting of Snail in TAMR‐MCF‐7 cells treated with 20 µM TPCK. (c) Decrease in E‐cadherin level by TPCK treatment. The level of E‐cadherin was determined by immunoblotting in TAMR‐MCF‐7 cells treated with TPCK (0, 20, or 50 µM). (d) Inhibition of NF‐κB transactivation by Pin1 siRNA. TAMR‐MCF‐7 cells were cotransfected with NF‐κB reporter plasmid in combination with Pin1 siRNA (20 pmol) or control siRNA. Data represent means ± SD with four different samples (significant vs the control siRNA‐transfected MCF‐7 cells, **P < 0.01; significant vs the control siRNA‐transfected TAMR‐MCF‐7 cells, ## P < 0.01).
Figure 6
Figure 6
No induction of epithelial–mesenchymal transition (EMT) in Pin1‐overexpressed MCF‐7 cells. (a) Effect of Pin1 overexpression on the EMT phenotypes in MCF‐7 cells. Representative immunoblots show E‐cadherin, N‐cadherin, and vimentin protein levels in both GFP‐MCF‐7 (control) and Pin1‐MCF‐7 cells serum‐deprived for 18 h. (b) Basal reporter activity of Snail promoters (Snail‐Luc) in GFP‐MCF‐7 and Pin1‐MCF‐7 cells. Each cell type was transiently transfected with –2.5 kb Snail‐Luc plasmid. Dual luciferase reporter assays were carried out on the lysed cells cotransfected with Snail‐Luc plasmids (firefly luciferase) and phRL‐SV (hRenilla luciferase) (a ratio of 100:1) 18 h after transfection. Reporter gene activation was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent means ± SD with six different samples. (c) Cell morphology of GFP‐MCF‐7 and Pin1‐MCF‐7 cells. (d) Phosphatase and tensin homolog depleted on chromosome ten (PTEN) expression in GFP‐MCF‐7 and Pin1‐MCF‐7 cells. A representative immunoblot shows PTEN protein in both GFP‐MCF‐7 and Pin1‐MCF‐7 cells serum‐deprived for 18 h.
Figure 7
Figure 7
Possible mechanism of epithelial–mesenchymal transition (EMT) induction in tamoxifen‐resistant (TAMR) breast cancer. Pin1 is overexpressed in TAMR‐MCF‐7 cells. The increased Pin1 activity persistently activates Akt presumably through phosphatase and tensin homolog depleted on chromosome ten (PTEN) downregulation and subsequently inactivates glycogen synthase kinase (GSK)‐3β. GSK‐3β inactivation enhances cellular Snail level via transcriptional activation or inhibition of proteasomal degradation. Direct (p65 accumulation in nucleus) or indirect (GSK‐3β‐mediated) nuclear factor (NF)‐κB activation is also involved in Snail upregulation in TAMR‐MCF‐7 cells. The increased Snail suppresses transcription of the E‐cadherin gene and finally causes EMT in TAMR‐MCF‐7 cells.
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