Suppression of Esophageal Squamous Cell Carcinoma Development by Mechanosensitive Protein Piezo1 Downregulation

Abstract

Esophageal squamous cell carcinoma (ESCC) is a malignant epithelial cancer of the esophageal epithelium. Piezo-type mechanosensitive ion channel component 1 (Piezo1), an essential mechanosensitive protein, plays an important role in maintaining cell biological functions under the stimulation of physiological force. Immunohistochemical and bioinformatic analyses of ESCC tissue samples indicate that Piezo1 expression is higher in ESCC tissues than in paracancerous tissues. shRNA-mediated Piezo1 downregulation in the ESCC lines EC9706 and EC109 showed that proliferation, migration, and invasion were suppressed by Piezo1 knockdown. Piezo1 downregulation suppresses ESCC migration and invasion in both cells and tissues via the epithelial-mesenchymal transition pathway. Moreover, G0/G1 to S-phase cell cycle progression was inhibited, and cell apoptosis was induced by Piezo1 downregulation. Furthermore, we observed an interaction between Piezo1 and p53 using immunoprecipitation. The protein levels of p53, downstream factor Bax, apoptosis executioner cleaved-caspase3, and caspase3 were significantly upregulated by the downregulation of Piezo1. The inhibited growth rate and upregulated expression of these related factors were validated using tumor-bearing mice. Therefore, Piezo1 downregulation induces ESCC apoptosis via a Piezo1-p53-Bax-Caspase 3 axis. In conclusion, Piezo1 downregulation suppresses ESCC development, and mechanosensitive protein Piezo1 can be considered a new target for ESCC therapy.

Figure 1

Piezo1 expression in tissues. (a) Immunohistochemical images of Piezo1 expression in paracancerous tissues (above) and ESCC tissues (below). (b) Relative expression level of Piezo1 in the cancer genome atlas (TCGA) samples.

Figure 2

Construction of Piezo1-downregulated cell lines. (a) Piezo1 mRNA expression in different ESCC cell lines. (b) Protein expression of Piezo1 in transfected EC9706 and EC109 cell lines. (c and d) Relative mRNA expression of Piezo1 in transfected EC9706 and EC109 cell lines. * p < 0.05 vs control and *** p < 0.001 vs control.

Figure 3

FI of intracellular Ca²⁺ and intracellular Ca²⁺ levels increased by Yoda1. (a) FI_before in EC109^shRNA-piezo1 and EC109^{shRNA-control} cells. ** p < 0.001 vs control. (b) FI_before in EC9706^shRNA-piezo1 and EC9706^{shRNA-control} cells. *** p < 0.001 vs control. (c) ΔFI in EC109^shRNA-piezo1 and EC109^{shRNA-control} cells. *** p < 0.05 vs control. (d) ΔFI in EC9706^shRNA-piezo1 and EC9706^{shRNA-control} cells. * p < 0.05 vs control.

Figure 4

Migration and invasion of transfected cells. (a) Migration of EC109^shRNA-piezo1 and EC109^{shRNA-control} cells. (b) Quantitative analysis of panel A, *** p < 0.001 vs control. (c) Invasion of EC109^shRNA-piezo1 and EC109^{shRNA-control} cells, ** p < 0.01 vs control. (d and e) Invasion and migration of EC9706^shRNA-piezo1 and EC9706^{shRNA-control} cells, ** p < 0.01 vs control. (f) Protein expression of E-cadherin and N-cadherin in transfected EC109 and EC9706 cells.

Figure 5

Piezo1 downregulation affects ESCC cell proliferation, cell cycle progression, and apoptosis. Proliferation was suppressed by Piezo1 downregulation in (a) EC109 cells and (b) EC9706 cells, *** p < 0.001 vs control. (c) Cell cycle distribution of transfected EC109 cells. (d) Analysis of cell cycle distribution in panel C, * p < 0.05 vs control. (e) Cell cycle distribution of transfected EC109 cells. (f) Analysis of cell cycle distribution in panel E, * p < 0.05 vs control. Apoptosis of transfected (g) EC109 cells and (h) EC109 cells. (i) Protein expressions of cleaved-caspase3 and caspase3 in transfected EC109 and EC9706 cells, respectively.

Figure 6

Interaction between p53 and Piezo1, and the protein expression of p53 and Bax. (a) Co-IP between Piezo1 and p53 in EC109 and EC9706 cells. Cell lysates underwent IP using control IgG or the indicated antibody, and the precipitated protein was detected using immunoblotting analysis with the indicated antibody. Cell extracts were used as a positive control (input). (b) Relative mRNA levels of p53 and Bax in transfected EC109 and EC9706 cells, respectively. ** p < 0.01 vs control and * p < 0.05 vs control. (c) Protein expressions of p53 and Bax in transfected EC109 and EC9706 cells, respectively. (d) Relative mRNA expression levels of Piezo1 and p53 in EC109 and (e) EC9706 cell lines after the addition of the p53 inhibitor.

Figure 7

Tumor growth in tumor-bearing mice. (a) Images of tumors harvested from mice^shRNA-piezo1 and mice^{shRNA-control}. (b) Final weights of tumors in mice^shRNA-piezo and mice^{shRNA-control}. **p < 0.01 vs control. (c) Tumor volumes measured throughout the experiment. * p < 0.05 vs control. (d) Protein expression of Bax, cleaved-caspase3, caspase3, p53, E-cadherin, and N-cadherin in tumor tissues.

Figure 8

Schema of the Piezo1-p53-Bax-Caspase 3 axis.