Role of Integrin β1 in the progression and chemo-resistance of esophageal squamous cell carcinoma

Abstract

Objective: Integrins have been shown to play an important role in the tumorigenesis of many cancers. In this work, we aimed to explore the expression and clinical value of Integrin α5β1 in esophageal squamous cell carcinoma (ESCC), and the effect of integrin β1 on the development and chemo-resistance of ESCC cells. Methods: The expression profiling of integrins was analyzed in the mRNA expression dataset of ESCC. The expression of Integrin α5β1 in 278 cases of ESCC tissues and 62 cases of paracancerous tissues was detected by immunohistochemistry (IHC). The association between the expression of Integrin α5β1 and the survival of ESCC patients was analyzed by Kaplan-Meier analysis. The effect of Integrin β1 on the proliferation, migration, and invasion of ESCC cells was examined by MTS, Transwell migration, and Transwell invasion assay. The effect of Integrin β1 and L1 cell adhesion molecule (L1CAM) on cisplatin resistance was detected by MTS and the signal pathways involved were analyzed by Western blotting. Results: Integrin β1 and Integrin α5 were significantly up-regulated in ESCC. High expression of Integrin β1 was also related to worse overall survival of ESCC patients and patients with low levels of both Integrin β1 and Integrin α5 showed the shortest survival. Results of IHC revealed that Integrin α5β1 was up-regulated in ESCC and its high expression was associated with poor prognosis and could serve as an independent prognostic factor. siRNA-mediated Integrin β1 silencing or antibody blocking restrained the proliferation, migration, and invasion of ESCC cells. Simultaneous knockdown of Integrin β1 and L1CAM reduced the cisplatin resistance of ESCC cells. Further studies showed that knockdown of Integrin β1 and L1CAM suppressed the activity of Akt signaling with or without cisplatin treatment. Moreover, dual high expression of Integrin β1 and L1CAM was related to worse overall survival of ESCC patients treated with preoperative chemotherapy. Conclusion: Integrin α5β1 was up-regulated in ESCC and could be used as a new prognostic indicator for ESCC patients. In addition, Integrin β1 was involved in the proliferation, invasion, and chemo-resistance of ESCC cells.

Keywords: Biomarker; Chemo-resistance; ESCC; Integrin α5β1; Prognosis.

Figure 1

Expression profiling of integrins in ESCC. (A) The integrins mRNA expression profile of paired cancer and adjacent normal tissues from 179 ESCC patients (data extracted from GSE53625 dataset in GEO database). Mean ± SD. Multiple t-tests. **, P < 0.01, ***, P < 0.001. (B) Kaplan-Meier estimates of the overall survival by Integrin β1 expression in ESCC samples. (C) Kaplan-Meier estimates of the overall survival by Integrin α5 expression in ESCC samples. (D) Kaplan-Meier estimates of the overall survival by Integrin β1 and Integrin α5 expression in ESCC samples. A P value of less than 0.05 was considered statistically significant.

Figure 2

Integrin α5β1 was up-regulated in ESCC and associated with poor prognosis. (A) Representative IHC staining images of Integrin α5β1 in normal esophageal epithelium samples. (B) Representative IHC staining images of Integrin α5β1 in ESCC tissues. (C) Scoring analysis of Integrin α5β1 in normal esophageal tissues and ESCC tissues. (D) Kaplan-Meier estimates of the overall survival (left) and disease-free survival (right) by Integrin α5β1 expression in ESCC samples. A P value of less than 0.05 was considered statistically significant. Bar, 50 μm.

Figure 3

Integrin β1 silencing suppressed the proliferation, migration, and invasion of ESCC cells. (A) The expression of Integrin β1 protein in ESCC cell lines (KYSE30, KYSE150, KYSE180, KYSE450, KYSE510, TE7, TE10, EC109, SHEEC), immortalized esophageal epithelial cell lines (NE2, NE3, NECA6, SHEE) and 293T cell line by qRT-PCR (left) and Western blot (right). (B) KYSE180 (left) and KYSE150 (right) were transfected with negative control siRNA/shRNA (NC) or anti-Integrin β1 siRNA/shRNA. The knockdown of Integrin β1 was evaluated by Western blotting. β-actin was served as a loading control. (C) MTS assay was conducted to measure the proliferation of ESCC cells after Integrin β1 knockdown by siRNA/shRNA or blocked by JB1A, a blocking antibody of Integrin β1. (D) Transwell Migration Assays and Transwell Invasion Assays were performed to measure the migration and invasion of ESCC cells after Integrin β1 knockdown by siRNA/shRNA or blocked by JB1A. The independent sample t-test was used to determine the significance of differences between groups and data was obtained in at least three independent experiments in (C) and (D). Average values are given ± SD. *, P < 0.05; **, P < 0.01.

Figure 4

Integrin β1 and L1CAM synergistically enhanced the cisplatin resistance of ESCC cells by suppressing AKT signaling. (A) Correlation analysis of Integrin β1 and L1CAM expression in esophageal carcinoma. The proteome datasets were obtained from PRIDE database (accession number PXD021701). (B) The cisplatin resistance of ESCC cell lines was detected by MTS. Above: KYSE180 and KYSE150 cells were exposed to cisplatin (4 μM), and cell viability was detected at the indicated time. Below: KYSE180 and KYSE150 cells were exposed to the indicated concentrations of cisplatin for 72 hours, and then the cell viability was detected. (C) The expression of Integrin β1 and L1CAM in KYSE150 was detected by Western blotting. KYSE150 was transfected with integrin β1 and/or L1CAM siRNA. After 36 hours, some cells were used for the cisplatin resistance test, and the remaining cells were cultured for 12 hours to collect cell lysates, followed by Western blot. (D) After Integrin β1 and/or L1CAM silence by siRNA, KYSE150 was exposed to the indicated concentrations of cisplatin for 72 hours, and then the cell viability was detected by MTS. The independent sample t-test was used to determine the significance of differences between groups. (E) The signaling pathways involved in the Integrin β1/L1CAM-mediated cisplatin resistance were detected by Western blotting. After Integrin β1 and/or L1CAM silence by siRNA, KYSE150 was cultured in medium with or without 4 μM for 24 hours, and then the cell lysates were collected for Western blotting. Data were obtained in at least three independent experiments in (C), (D), and (E). Average values are given ± SD. *, P < 0.05. (F-H) The associations between the expression of Integrin β1 or/and L1CAM with the overall survival of ESCC patients treated with preoperative chemotherapy were determined.