TGF-β1 stimulates migration of type II endometrial cancer cells by down-regulating PTEN via activation of SMAD and ERK1/2 signaling pathways

Abstract

PTEN acts as a tumor suppressor primarily by antagonizing the PI3K/AKT signaling pathway. PTEN is frequently mutated in human cancers; however, in type II endometrial cancers its mutation rate is very low. Overexpression of TGF-β1 and its receptors has been reported to correlate with metastasis of human cancers and reduced survival rates. Although TGF-β1 has been shown to regulate PTEN expression through various mechanisms, it is not yet known if the same is true in type II endometrial cancer. In the present study, we show that treatment with TGF-β1 stimulates the migration of two type II endometrial cancer cell lines, KLE and HEC-50. In addition, TGF-β1 treatment down-regulates both mRNA and protein levels of PTEN. Overexpression of PTEN or inhibition of PI3K abolishes TGF-β1-stimulated cell migration. TGF-β1 induces SMAD2/3 phosphorylation and knockdown of common SMAD4 inhibits the suppressive effects of TGF-β1 on PTEN mRNA and protein. Interestingly, TGF-β1 induces ERK1/2 phosphorylation and pre-treatment with a MEK inhibitor attenuates the suppression of PTEN protein, but not mRNA, by TGF-β1. This study provides important insights into the molecular mechanisms mediating TGF-β1-induced down-regulation of PTEN and demonstrates an important role of PTEN in the regulation of type II endometrial cancer cell migration.

Keywords: PTEN; TGF-β1; migration; type II endometrial cancer.

Figure 1. TGF-β1 stimulates type II endometrial cancer cell migration

(A) KLE and HEC-50 cells were treated without (Ctrl) or with 10 ng/mL TGF-β1 for 24 h and then seeded into transwell inserts for the 24-hour migration assay. Upper panels show representative photomicrographs of migrating cells, while lower panels show summarized quantitative results. (B) KLE and HEC-50 cells were pre-treated with vehicle (DMSO) or SB431542 (10 μM) for 1 h and then treated with 10 ng/mL TGF-β1 for 24 h. After treatment, the levels of cell migration were examined by the transwell migration assay (24 h). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 2. TGF-β1 down-regulates PTEN expression in type II endometrial cancer cells

(A) KLE and HEC-50 cells were treated without (Ctrl) or with 10 ng/mL TGF-β1 for different periods of time and PTEN mRNA levels were examined by RT-qPCR (normalized to GAPDH mRNA levels). (B) KLE and HEC-50 cells were pre-treated with vehicle (DMSO) or SB431542 (10 μM) for 1 h and then treated with 10 ng/mL TGF-β1 (T) for 24 h. PTEN protein levels were examined by Western blot (normalized to α-tubulin protein levels). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 3. Overexpression of PTEN abolishes TGF-β1-stimulated cell migration

(A) KLE and HEC-50 cells were transfected for 48 h with 1 μg control vector (Vec; pcDNA-GFP) or vector encoding PTEN (pcDNA-PTEN-GFP) and PTEN-GFP protein levels were examined by Western blot. (B) KLE and HEC-50 cells were transfected with 1 μg vector control or PTEN for 48 h and then treated without (Ctrl) or with 10 ng/mL TGF-β1 for 24 h. After treatment, the levels of cell migration were examined by the transwell migration assay (24 h). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 4. Inhibition of AKT signaling attenuates TGF-β1-stimulated cell migration

(A) KLE and HEC-50 cells were transfected with 1 μg vector control (Vec; pcDNA-GFP) or vector encoding PTEN (pcDNA-PTEN-GFP) for 48 h and then treated without (C) or with 10 ng/mL TGF-β1 (T) or 10% FBS for a further 24 h. Western blot was used to confirm PTEN-GFP overexpression and to examine the levels of phosphorylated AKT (p-AKT) in relation to its total levels from the same membrane (AKT). (B) KLE and HEC-50 cells were pre-treated with vehicle (DMSO), LY294002 (10 μM) or Wortmannin (1 μM) for 1 h and then treated without (Ctrl) or with 10 ng/mL TGF-β1 for 24 h. After treatment, the levels of cell migration were examined by the transwell migration assay (24 h). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 5. Activation of SMAD signaling is required for the down-regulation of PTEN by TGF-β1

(A) KLE and HEC-50 cells were treated without (C) or with 10 ng/mL of TGF-β1 (T) for 30 and 60 minutes. Western blot was used to examine the levels of phosphorylated SMAD2 (p-SMAD2) or SMAD3 (p-SMAD3) in relation to their total levels from the same membrane (SMAD2 and SMAD3, respectively). (B) KLE and HEC-50 cells were transfected for 48 h with 20 nM control siRNA (si-Ctrl) or SMAD4 siRNA (si-SMAD4) and then treated without (Ctrl) or with 10 ng/mL of TGF-β1 for 6 or 24 h. PTEN mRNA levels were examined by RT-qPCR (normalized to GAPDH mRNA levels). (C) KLE and HEC-50 cells were transfected for 48 h with 20 nM control or SMAD4 siRNA, treated for a further 24 h with or without 10 ng/mL of TGF-β1, and PTEN protein levels were examined by Western blot (normalized to α-tubulin protein levels). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 6. Activation of ERK1/2 signaling is required for the down-regulation of PTEN by TGF-β1

(A) KLE and HEC-50 cells were pre-treated for 1 h with vehicle (DMSO), SB431542 (10 μM) or U0126 (10 μM) and then treated without (C) or with 10 ng/mL TGF-β1 (T) for 10 min. Western blot was used to examine the levels of phosphorylated ERK1/2 (p-ERK1/2) in relation to its total levels from the same membrane (ERK1/2). Summarized quantitative results are displayed numerically as the mean fold change with superscripted letters indicating statistical significance as described below. (B) KLE and HEC-50 cells were pre-treated for 1 h with or without U0126 (10 μM) and then treated without (Ctrl) or with 10 ng/mL TGF-β1 for 6 or 24 h. PTEN mRNA levels were examined by RT-qPCR (normalized to GAPDH mRNA levels). (C) KLE and HEC-50 cells were pre-treated for 1 h with U0126 (10 μM), treated for a further 24 h with 10 ng/mL TGF-β1, and PTEN protein levels were examined by Western blot (normalized to α-tubulin protein levels). Results are expressed as the mean ± SEM of at least three independent experiments and values without common letters are significantly different (P < 0.05).

Figure 7. Proposed model for the actions of TGF-β1 on PTEN, PI3K-AKT signaling and cell migration in type II endometrial cancer cells

TGF-β1 binds to a complex of type I and II receptors leading to the phosphorylation/activation of receptor-regulated SMAD2/3 which bind to common SMAD4 and translocate into the nucleus to decrease the transcription of PTEN. In parallel, the ligand-receptor complex subsequently activates MEK leading to the phosphorylation/activation of ERK1/2 which acts post-transcriptionally to suppress PTEN protein. The down-regulation of PTEN enhances PI3K-AKT signaling which is an essential mediator of TGF-β1-induced type II endometrial cancer cell migration.