The Hippo transducer TAZ promotes cell proliferation and tumor formation of glioblastoma cells through EGFR pathway

Abstract

TAZ, a WW-domain-containing transcriptional co-activator, is important for development of various tissues in mammals. Recently, TAZ has been found to be overexpressed in some types of human cancers. However, the role of TAZ in glioblastoma remains unclear. In this study, we found that TAZ was overexpressed in prognostically poor glioblastoma patients. Through knocking down or overexpressing TAZ in U87 and LN229 cells, the expression level of TAZ was found to be positively related to cell proliferation in vitro and tumor formation in vivo. Further investigation indicated that TAZ could significantly promote the acceleration of cell cycle. Moreover, the western blot for p-EGFR, p-AKT, p-ERK1/2, p21, cyclin E and CDK2 proteins, target genes of the EGFR pathway, indicated that TAZ significantly activated EGFR/AKT/ERK signaling. Additionally, the blockage of EGFR pathway resulted in a significantly inhibition of cell proliferation induced by TAZ. Taken together, these results demonstrate that TAZ can promote proliferation and tumor formation in glioblastoma cells by potentiating the EGFR/AKT/ERK pathway, and provide the evidence for promising target for the treatment of glioblastoma.

Keywords: EGFR; TAZ; cell cycle; cell proliferation; glioblastoma.

Figure 1. High TAZ expression is a prognostic indicator of poor survival in glioblastoma patients

(A) Kaplan-Meier analysis of progression-free survival for the TCGA database with the log rank test P value was indicated. Cutoff:400-1094.1: raw p: 4.4e-5 (bonf: 0.021) (B) Kaplan-Meier analysis of progression-free survival for the Frence database with the log rank test P value indicated. Cutoff: 151-1028.0: raw p: 1.4e-11 (bonf: 3.6e-09) (C) Box plot of TAZ expression levels from non-tumor, GBM and recurrent GBM patients was shown. (D) Box plot of TAZ expression levels in the normal, stage 1 to 3 and GBM tumors. (E) Box plot of TAZ expression levels in the stage 2 and 4 tumors. (F) Western blot assay of TAZ expression in GBM cell lines and different tissues was performed. All data are shown as the mean ± SD, *p < 0.05, **p < 0.01. All p values are based on analysis control versus treatment.

Figure 2. TAZ promotes GBM cell growth and proliferation

(A) Western blot assay was used to characterize the expression of TAZ in TAZ-knockdown U87 cells and TAZ-overexpressing U87 cells. (B) The effect of TAZ on the proliferation of U87 cells. (C) The effect of TAZ on the viability of U87 cells. (D) Western blot assay was used to characterize the expression of TAZ in TAZ-knockdown LN229 cells and TAZ-overexpressing LN229 cells. (E) The effect of TAZ on the proliferation of LN229 cells. (F) The effect of TAZ on the viability of LN229 cells. (G, H) Image and quantification of U87 and LN229 cells positive for BrdU staining. All data are shown as the mean ± SD, *p < 0.05, **p < 0.01. All p values are based on analysis control versus treatment.

Figure 3. TAZ promotes colony formation and tumor formation of U87 cells in immunodeficient mice

(A, B) The effects of TAZ on the colony formation in TAZ-knockdown U87 cells and TAZ-overexpressing U87 cells. (C) The tumor growth curve of TAZ-knockdown U87 cells injected into immunodeficient mice. (D) The tumor growth curve of TAZ-overexpressing U87 cells injected into immunodeficient mice. (E) Immunohistochemical staining for Ki67 in tumor tissues, shTAZ: shRNA for TAZ; shCtr: shRNA for control. Values are shown as the mean ± SD. *p < 0.05, **p < 0.01. All p values are based on analysis control versus treatment.

Figure 4. TAZ accelerates the cell cycle in GBM cells

(A) The cell cycle of TAZ-modulated U87 cells was analyzed by flow cytometry. (B) The effect of TAZ on the cell cycle of U87 cells. (C) The cell cycle of TAZ-modulated LN229 cells was analyzed by flow cytometry. (D) The effect of TAZ on the cell cycle of LN229 cells. (E, G) Western blot analysis of cyclin E2, CDK2 and p21 expression in TAZ-modulated cells; representative blots are shown. (F, H) Quantitative analysis of cyclins and CDKs expression in TAZ-modulated cells; GAPDH was used as a loading control; student's t-test was carried out. All data are shown as the mean ± SD, *p < 0.05, **p < 0.01.

Figure 5. TAZ enhances the activity of the EGFR/AKT/ERK pathway

(A) The expression of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 in TAZ-modulated U87 cells was measured by western blot assay. (B) The quantitative analysis of p-EGFR, p-AKT, p-ERK1/2 relative expression in TAZ-modulated U87 cells. (C) The expression of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 in TAZ-modulated LN229 cells was measured by western blot assay. (D) The quantitative analysis of p-EGFR, p-AKT, p-ERK1/2 relative expressions in TAZ-modulated LN229 cells. (E) The expression of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 in tumor xenografts was measured by western blot. (F) The quantitative analysis of p-EGFR, p-AKT, p-ERK1/2 relative expressions in tumor xenografts. All data are shown as the mean ± SD, *p < 0.05, **p < 0.01.

Figure 6. Erlotinib treatment attenuates the increasing proliferation of GBM cells induced by TAZ

TAZ-modulated U87 and LN229 cells were treated with the EGFR inhibitor erlotinib (0.1 μM), and the expression of p-EGFR, p-AKT, and p-ERK1/2 was measured by western blot. (A, B) The representative blot and quantitative analysis of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 expression in U87 cells were shown. (C) The effect of erlotinib on the viability of TAZ-modulated U87 cells was measured by the MTT assay. (D, E) The representative blot and quantitative analysis of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 expression in LN229 cells were shown. (F) The effect of erlotinib on the viability of TAZ-modulated LN229 cells was measured by the MTT assay. Values are shown as the mean ± SD. *p < 0.05, **p < 0.01. All p values are based on analysis control versus treatment.