Capn4 promotes esophageal squamous cell carcinoma metastasis by regulating ZEB1 through the Wnt/β-catenin signaling pathway

Abstract

Background: Capn4 and ZEB1 play important roles in the metastasis of several types of cancer. However, the roles and relationship of Capn4 and ZEB1 in esophageal squamous cell carcinoma (ESCC) remain unclear.

Methods: ESCC tumor tissues and corresponding normal esophageal epithelial tissues were obtained from 86 patients undergoing resection surgery at the Department of General Surgery, First Affiliated Hospital of Chinese PLA General Hospital from 2012 to 2017. Cell migration and invasion were examined via quantitative real-time PCR and Western blot assay.

Results: Our results indicate that both Capn4 and ZEB1 are significantly upregulated in ESCC tissues compared to corresponding adjacent tissues, and a positive correlation between expression and associated malignant characteristics was found. Silencing of Capn4 expression markedly inhibited ESCC invasion and metastasis in vitro and in vivo, and was accompanied by decreased ZEB1 expression. Furthermore, the anti-metastasis role of Capn4 silencing was reversed by ZEB1 overexpression, whereas knockdown of ZEB1 decreased ESCC metastasis driven by the upregulation of Capn4. Mechanistically, Capn4 regulated ZEB1 expression via activation of the Wnt/β-catenin signaling pathway in ESCC cells.

Conclusion: Overall, our results show that enhanced Capn4 expression activates the Wnt/β-catenin signaling pathway, resulting in increased ZEB1 expression and the promotion of ESCC cell metastasis.

Keywords: Capn4; ZEB1; esophageal squamous cell carcinoma (ESCC); metastasis.

Figure 1

Capn4 overexpression is correlated with ZEB1 expression in human esophageal squamous cell carcinoma (ESCC). (a,b) Quantitative real‐time PCR analysis of Capn4 and ZEB1 messenger RNA (mRNA) expression in 86 ESCC and paired non‐tumor tissues (**P < 0.01). (c–e) Determination and quantification of Capn4 and ZEB1 protein levels in ESCC and in paired non‐tumor tissues by Western blotting. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control. (f,g) Scatter plots show a positive correlation between Capn4 and ZEB1 at the mRNA and protein level in 30 ESCC samples (r = 0.416, *P < 0.05 and r = 0.373, *P < 0.05, **P < 0.01).

Figure 2

Stable knockdown of Capn4 reduced ZEB1 expression and inhibited esophageal squamous cell carcinoma (ESCC) invasion and metastasis in vitro and in vivo. (a) Quantitative real‐time PCR (qRT‐PCR) analysis of Capn4 and ZEB1 expression in human esophageal epithelial and ESCC cell lines. (b,c) qRT‐PCR analyses were used to detect Capn4 and ZEB1 messenger RNA (mRNA) expression in ECA109 and TE7 cells stably transfected with the short hairpin negative control (shNC) vector or shCapn4 plasmid (**P < 0.01). (d) Determination (left) and quantification (right) of Capn4 and ZEB1 protein levels in ECA109 and TE7 cells stably transfected with the shNC vector or the shCapn4 plasmid. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control (*P < 0.05). (e,g) Transwell migration assays of ECA109 and TE7 cells with stable Capn4 knockdown (*P < 0.05). (f,h) Transwell invasion assays of ECA109 and TE7 cells transfected with shCapn4 plasmid (*P < 0.05). (i) Statistical analysis of lung metastatic nodules. (n = 6/group; **P < 0.01). (j) Hematoxylin and eosin staining of the paraffin embedded sections of lung metastatic nodules.

Figure 3

Capn4 promotes esophageal squamous cell carcinoma (ESCC) invasion and metastasis by upregulating ZEB1 expression. (a) Determination (left) and quantification (right) of Capn4 and ZEB1 protein levels in the indicated cells by Western blotting. ZEB1 upregulation attenuated the loss of ZEB1 expression in ECA109‐short hairpin Capn4 (shCapn4) cells. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control (*P < 0.05). (b,c) Transwell assays showed that the upregulation of ZEB1 significantly rescued the cell migration and invasion in ECA109‐shCapn4 cells (*P < 0.05). (d) Statistical analysis of lung metastatic nodules. (n = 6/group; **P < 0.01). (e) Western blotting was used to detect Capn4 and ZEB1 expression. (f,g) Transwell assays showed that ZEB1 inhibition reduced Capn4‐enhanced cell migration and invasion (*P < 0.05). (h) Statistical analysis of lung metastatic nodules (n = 6/group; **P < 0.01).

Figure 4

Capn4 regulates ZEB1 expression through the Wnt/β‐catenin pathway in esophageal squamous cell carcinoma (ESCC). (a) The total and nuclear protein β‐catenin levels were assessed by Western blotting in ECA109 cells transfected with short hairpin Capn4 (shCapn4). (b) The relative luciferase activity levels in cells transfected with TOP‐flash and FOP‐flash vectors in ECA109 cells transfected with short hairpin negative control (shNC) and cells transfected with shRNA against Capn4 are shown (*P < 0.05). (c) Determination (left) and quantification (right) of Capn4, ZEB1, and β‐catenin protein levels in the indicated cells by Western blotting. (d,e) Upregulation of β‐catenin reversed cell migration and invasion in ECA109‐shCapn4 cells (*P < 0.05). (f) Western blotting was used to assess the total and nuclear protein β‐catenin levels in KYSE410 cells transfected with p‐Capn4. (g) The relative luciferase activity levels in cells transfected with TOP‐flash and FOP‐flash vectors in ECA109 cells transfected with the Capn4 plasmid (*P < 0.05). (h) Determination (left) and quantification (right) of Capn4, ZEB1, and β‐catenin protein levels in the indicated cells by Western blotting. (i,j) Downregulation of β‐catenin reduced Capn4‐enhanced migration and invasion in KYSE410 cells (*P < 0.05). Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a loading control (*P < 0.05).