Loss of miR-200b promotes invasion via activating the Kindlin-2/integrin β1/AKT pathway in esophageal squamous cell carcinoma: An E-cadherin-independent mechanism

Abstract

Our previous studies have shown that loss of miR-200b enhances the invasiveness of esophageal squamous cell carcinoma (ESCC) cells. However, whether the miR-200-ZEB1/2-E-cadherin regulatory cascade, a master regulator of epithelial-to-mesenchymal transition (EMT), is involved in the regulation of ESCC invasion remains elusive. Here, we show that miR-200b represses ESCC cell invasion in vivo without altering the expression of E-cadherin and vimentin, two surrogate markers of EMT. However, an inverse correlation was observed between the expression levels of miR-200b and ZEB1/2 in both ESCC cell lines (n = 7, P < 0.05) and ESCC tumor samples (n = 88, P < 0.05). Methylation of E-cadherin gene was found to block the regulation of E-cadherin by the miR-200b-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b in ESCC. We revealed that miR-200b suppresses the integrin β1-AKT pathway via targeting Kindlin-2 to mitigate ESCC cell invasiveness. In two independent cohorts of ESCC samples (n = 20 and n = 53, respectively), Kindlin-2 expression positively correlated with the activation status of both the integrin signaling pathway and the PI3K-AKT signaling pathway (both P < 0.01). These data highlight that suppression of the Kindlin-2-integrin β1-AKT regulatory axis is an alternative mechanism underlying the tumor suppressor function of miR-200b in ESCC.

Keywords: E-cadherin; esophageal squamous cell carcinoma; invasion; miR-200; prognosis.

Figure 1. miR-200b suppresses ESCC tumor invasion in vivo

A. Left panel: representative mice showing the impact of miR-200b on ESCC tumor invasion in vivo. Note that the invasion sites on the thigh were magnified, and the arrows indicate the invaded tumor nodules. Right panel: the local invasion areas of tumors formed by EC109-NC and EC109–200b cells were compared. Data are presented as mean ± SD. Student's t test was used in the statistical analysis. B. The proportions of mice that developed invasive tumors in the thigh region were compared. n = 10 for NC transfected group, and n = 9 for miR-200b transfected group. C. Representative H&E staining showing the invaded tumor nodules within the muscle of the thigh area. Upper panel scale bars: 100 μm; lower panel scale bars: 50 μm. D–E. Immunostaining of E-cadherin and vimentin in tumor xenografts dissected from the mice. Upper panel scale bars: 100 μm; lower panel scale bars: 50 μm.

Figure 2. The miR-200b-ZEB1/2 axis in ESCC cell lines and patient tumors

A–B. The expression of miR-200b and ZEB1/2 were determined using real-time PCR in a panel of cell lines. SHEE, an immortalized cell line was used as a normal control. Data are presented as mean ± SD. **P < 0.01, Student's t test. C. In EC109 cells, the influence of the enforced expression of miR-200b on the expression of ZEB1/2 mRNA was determined using real-time PCR. D. The expression of ZEB1 and ZEB2 mRNA were compared between ESCC tumors expressing low or high levels of miR-200b (Median miR-200b expression level was used in the stratification). Mann Whitney U test was used in the statistical analysis. E. The association of ZEB1 and ZEB2 expression with the overall survival of ESCC patients was analyzed using Kaplan-Meier survival analysis, and log-rank test was used in the statistical analysis.

Figure 3. DNA methylation of E-cadherin gene blocks the control of E-cadherin expression by the miR-200b-ZEB1/2 axis

A. RT-PCR and Western blot assays were performed to determine the impact of miR-200b mimic transfection on the expression of E-cadherin and vimentin in ESCC cell lines (EC109, KYSE510 and EC9706) and an immortalized esophageal cell line NE2. B. RT-PCR and Western blot assays were performed to determine the impact of 5-aza-dC treatment (72 h) on the expression of E-cadherin and vimentin in EC109 and EC9706 cells. GAPDH and Actin were used as loading controls for RT-PCR and Western blot, respectively. C. Western blot was performed to determine the impact of 5-aza-dC treatment and/or miR-200b mimic transfection on the expression of E-cadherin. Actin was used as a loading control.

Figure 4. AKT mediates the biological effect of miR-200b in suppressing ESCC cell invasiveness

A. The effects of miR-200b or miR-200b inhibitor (i200b) transfection on the expression of phospho-AKT^Ser473 (pAKT^S473) was determined by Western blot. Actin was used as a loading control. B. Left panel: the inhibitor effect of the PI3K inhibitor LY294002 on the expression of pAKT^S473 was determined by Western blot. Actin was used as a loading control. Right panel: The impact of LY294002 on cell morphology. Dashed lines were used to note the morphological changes. C. The impact of LY294002 on cell invasiveness was detected using transwell invasiveness assays. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, Student's t test. D. LY294002 (LY) suppresses the biological effects of miR-200b inhibitor (i200b) in KYSE150 cells. Data are presented as mean ± SD. **P < 0.01, Student's t test. E. A constitutively active form of AKT, myristoylated AKT (myr-AKT), restored cell invasiveness that was suppressed by miR-200b mimic transfection. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, Student's t test.

Figure 5. miR-200b inhibits the activation of AKT via suppressing the Kindin-2- integrin β1 axis

A. In EC109 cells, Western blot was performed to determine the expression of Kindlin-2 and pAKT^S473 after transfection. Actin was used as a loading control. B–C. In EC109 cells, flow cytometry was performed to assess the effects of enforced expression of miR-200b and Kindlin-2 knockdown on the expression of active/total integrin β1. Data are presented as mean ± SD. *P < 0.05, Student's t test. D. Cell adhesion assay was performed to determine the impact of enforced expression or inhibition of miR-200b on cell adhesion on fibronectin (FN). Data are presented as mean ± SD. *P < 0.05, Student's t test.

Figure 6. Correlation between Kindlin-2 and the integrin signaling pathway and the PI3K-AKT signaling pathway in ESCC tumors

A–B. GSEA (Gene Set Enrichment Analysis) was performed to analyze the correlation between Kindlin-2 and the integrin signaling pathway or the PI3K-AKT signaling pathway in two independent cohorts of ESCC patients (NCBI/GEO/GSE20991, n = 20; and NCBI/GEO/GSE23400, n = 53). C. A schematic model showing the possible mechanisms underlying the invasiveness-suppressing role of miR-200b in ESCC. Specifically, E-cadherin gene methylation can block the control of E-cadherin expression by the miR-200-ZEB1/2 axis, indicating that an E-cadherin-independent mechanism can mediate the biological function of miR-200b. Our findings show that miR-200b can suppress the Kindlin-2-integrin β1-AKT pathway to mitigate ESCC cell invasiveness. Note that arrows indicate stimulation effects, while blunt arrows indicate inhibitory effects.