Trail resistance induces epithelial-mesenchymal transition and enhances invasiveness by suppressing PTEN via miR-221 in breast cancer

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis of cancer cells and is verified effective to various cancers. However, a variety of breast cancer cell lines are resistant to TRAIL and the mechanisms of resistance are largely unknown. In our present experiment, we successfully utilized breast cancer cell line MDA-MB-231 to establish TRAIL-resistant cell line. We found resistance to TRAIL could induce epithelial-mesenchymal transition (EMT) and enhance invasiveness. We further demonstrated PTEN was down-regulated in TRAIL-resistant cells. Silencing miR-221, PTEN expression was up-regulated, the process of EMT could be reversed, and the ability of migration and invasion were correspondingly weakened. We also demonstrated knockdown of miR-221 could reverse resistance to TRAIL partially by targeting PTEN. Our findings suggest that resistance to TRAIL could induce EMT and enhance invasiveness by suppressing PTEN via miR-221. Re-expression of miR-221 or targeting PTEN might serve as potential therapeutic approaches for the treatment of Trail-resistant breast cancer.

Figure 1. TRAIL-resistant cells (231T) were resistant to TRAIL and had different morphology from MDA-MB-231 cells (231).

A. Sensitization of 231 cells and 231T cells to TRAIL at gradient concentrations was measured by MTT assay. Points represented the average of three independent experiments. Bars stood for SD; *P<0.05; **P<0.01; B and C. Resistance to TRAIL induced morphological change. B: 231 cells. C: 231T cells. Cells were observed under a light microscope.

Figure 2. Resistance to TRAIL induced EMT.

A.The mesenchymal markers, including N-cad, fibronectin, vimentin, and Snail were examined by western blot analysis. β-actin was used to confirm equal loading of samples. B. The mRNA expressions of transcriptional factors including Snail, Twist1, Zeb2, Foxq1 and FoxC2. *P<0.05; **P<0.01.

Figure 3. Resistance to TRAIL enhanced cell invasiveness.

A. Migration assay of 231 cells and 231T cells. C. Invasion assay of 231 and 231T cells. Cells that passed through the membrane were counted in 10 representative fields. Diagrams for migration (B) and invasion (D) were shown, respectively. Data was shown as mean ± SD. *P<0.05; **P<0.01.

Figure 4. Down-regulation of PTEN resulted in resistance to TRAIL of 231 cells, EMT and enhancement of invasiveness.

A. PTEN was down-regulated in 231T cells compared with 231. B. MDA-MB-231 cells transfected with siPTEN and its corresponding NC were named as 231-siPTEN and 231-n respectively. Tolerance of 231-n cells and 231-siPTEN cells to different concentrations of TRAIL was examined by MTT assay. Points represented the average of three independent experiments. Bars stood for SD; C. Migration and invasion assay of 231-n cells and 231-siPTEN cells. Count the cells in 10 representative fields under a light microscope. D. N-cadherin, fibronectin, vimentin and Snail of 231-n and 231-siPTEN cells were detected by western blot assay. Also β-actin was used as control. E. Summary graphs for migration and invasion, respectively. Data were shown as mean ± SD. *P<0.05; **P<0.01.

Figure 5. MiR-21 was over-expressed in 231T cells but not the key regulator in TRAIL-resistance.

A. Several miRNAs were detected in 231 and 231T cells. miR-21 and miR-221 were statistically up-regulated in 231T cells. B. miR-21 was silenced by inhibitor in 231T cells. C. Silence of miR-21 didn't reverse the resistance of 231T cells measured by MTT assay. D. After silencing miR-21, expression of PTEN didn't change. *P<0.05; **P<0.01.

Figure 6. PTEN was the target gene of miR-221.

A. miR-221 was predicted to regulate PTEN. B. After silencing miR-221, the mRNA level of PTEN was significantly up-regulated detected by real-time PCR. C. The corresponding change of PTEN in 231T-n and 231T-si221 cells was detected by western blot assay. *P<0.05; **P<0.01.

Figure 7. MiR-221 knockdown could sensitize 231T cells to TRAIL, reverse EMT and reduce cell mobility.

A. Cultured 231T-n and 231T-si221 cells with different concentrations of TRAIL in 96-well plates. 48 hours later examined their sensitivity to TRAIL by MTT assay. Points represented the average of three independent experiments. Bars stood for SD; B. The mesenchymal markers, N-cadherin, fibronectin, vimentin and Snail expressed in 231T-n and 231T-si221 cells were detected by western blot assay. β-actin was used as control; C. Migration and invasion assay of 231T-n cells and 231T-si221 cells. Staining the migrated cells with hematoxylin–eosin and count them in 10 representative fields under a light microscope. D. Summary graphs for migration and invasion were also shown, respectively. Data was presented as mean ± SD. *P<0.05; **P<0.01.