YAP/TAZ-TEAD activity promotes the malignant transformation of cervical intraepithelial neoplasia through enhancing the characteristics and Warburg effect of cancer stem cells

Abstract

A number of studies have confirmed that Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ)-transcriptional enhanced associate domain (TEAD) activity is the driver of cancer development. However, the role and mechanism of the YAP/TAZ-TEAD pathway in cervical intraepithelial neoplasia (CIN) remain to be clarified. Therefore, this study was designed to observe the effect of YAP/TAZ-TEAD activity on the development of CIN and provide new ideas for the diagnosis and treatment of CIN. Firstly, cervical tissues were collected from CIN patients in different stages [CIN grade 1 (CIN1) tissue, CIN grade 2/3 (CIN 2/3) and squamous cell carcinoma (SCC)] and healthy volunteers. Next, the expression levels of YAP, TAZ and TEAD in cervical tissues and cells were observed by immunohistochemistry, qRT-PCR and western blot. Besides, Z172 and Z183 cells were transfected with siRNA-YAP/TAZ (si-YAP/TAZ) and YAP/TAZ overexpression vector (YAP-5SA). Also, Z172 cells were co-transfected with YAP-5SA and si-TEAD2/4. Subsequently, the stemness characteristics, glycolysis level and malignant transformation of cells in each group were observed by sphere-formation assay, commercial kit, MTT, Transwell, scratch experiment, xenotransplantation and western blot.The expression of YAP, TAZ and TEAD increased significantly in cervical cancer tissue and cell line at the stage of CIN2/3 and SCC. When YAP/TAZ was knocked down, the stemness characteristics, glycolysis level and malignant transformation of cancer cells were notably inhibited; while activating YAP/TAZ exhibited a completely opposite result. In addition, activating YAP/TAZ and knocking down the TEAD expression at the same time significant weakened the effect of activated YAP/TAZ signal on precancerous cells and reduced inhibitory effect of knocking down TEAD alone. YAP/TAZ-TEAD signal activates the characteristics and Warburg effect of cancer stem cells, thereby promoting the malignant transformation of CIN.

Keywords: Cervical intraepithelial neoplasia (CIN); Characteristics of cancer stem cells; Transcriptional enhanced associate domain (TEAD); Warburg effect; Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ).

Fig. 1

Association between YAP/TAZ and the progression of cervical lesions. A and B qRT-PCR to detect the mRNA expression levels of YAP (A) and TAZ (B) in cervical tissues of patients with different stages of cervical lesions; C and D Western blot to assess the protein expression levels of YAP and TAZ in cervical tissues of patients with different stages of cervical lesions. **P < 0.01, vs normal. YAP Yes-associated protein; TAZ transcriptional co-activator with PDZ-binding motif

Fig. 2

Up-regulation of YAP/TAZ expression in cervical cancer cell lines. A qRT-PCR to measure the mRNA expression levels of YAP and TAZ in different cervical cancer cell lines; B Western blot to test the protein expression levels of YAP and TAZ in different cervical cancer cell lines; C and D qRT-PCR to assess the mRNA expression levels of YAP and TAZ in Z172 (C) and Z183 (D) cells with different treatments. **P < 0.01, vs Z172, Z183 or NC. YAP Yes-associated protein; TAZ transcriptional co-activator with PDZ-binding motif

Fig. 3

YAP/TAZ promotes the stemness of precancerous cells. A and B Sphere-formation assay to observe the spherical formation ability of Z172 and Z183 cells with different treatments (scalar bar = 20 μm). C and D Western blot to check the expression of proteins related to cancer stem cells with different treatments. **P < 0.01, vs NC

Fig. 4

YAP/TAZ promotes glycolysis and malignant characteristics of precancerous cells. A–C The effects of different treatments on glucose consumption, lactic acid production and ECAR of Z172 and Z183 cells were observed by biochemical kits; D MTT assay to assess the effects of different treatments on the viability of Z172 and Z183 cells; E and F transwell to observe the impact of different treatments on the invasion ability of Z172 and Z183 cells; G and H the scratch test to observe the effects of different treatments on the migration ability of Z172 and Z183 cells. **P < 0.01, vs NC. ECAR extracellular acidification rate

Fig. 5

YAP/TAZ encourages the tumorigenesis of precancerous cells in vivo. A Tumorigenesis of rats in each group; B isolated tumours in each group of mice; C the tumor volume of mice in each group was observed every week after treatment; D tumor weight of mice in each group. **P < 0.01, vs si-NC

Fig. 6

TEAD participates in the effect of YAP/TAZ on precancerous cells. A qRT-PCR to detect the expression levels of TEAD1, TEAD2, TEAD3 and TEAD4 in Z172 and Z183 cells, **P < 0.01, vs TEAD1 or TEAD3; B and C qRT-PCR to check the expression levels of TEAD1, TEAD2, TEAD3 and TEAD4 in Z172 (B) and Z183 (C) cells with different treatments, **P < 0.01, vs NC; D Western blot to observe the protein expression levels of YAP, TAZ, TEAD2 and TEAD4 after knocking down TEAD2/ TEAD4, **P < 0.01, vs si-NC; E–F, qRT-PCR to assess the expression levels of TEAD2 and TEAD4 in Z172 (E) and Z183 (F) cells after knocking down TEAD2/TEAD4. YAP Yes-associated protein; TAZ transcriptional co-activator with PDZ-binding motif; TEAD transcriptional enhanced associate domain

Fig. 7

Knocking down TEAD2/4 inhibits the effect of YAP/TAZ on precancerous cells. A Sphere-formation assay to observe the effect of knocking down TEAD2/4 and over-expressing YAP-5SA on the spherical formation ability of cells (scalar bar = 20 μm); B Western blot to check the effect of knocking down TEAD2/4 and over-expressing YAP-5SA on the expression of proteins related to cancer stem cells; C–E effects of knocking down TEAD2/4 and over-expressing YAP-5SA on the glucose consumption, lactic acid production and ECAR in Z172 and Z183 cells; F–H the effect of knocking down TEAD2/4 and over-expressing YAP-5SA on the viability, invasion and migration of Z172 and Z183 cells was observed by MTT (F), Transwell (G) and scratch assay (H). **P < 0.01, vs NC group; ^##P < 0.01, vs si-TEAD2/4 group; ^&&P < 0.01, vs YAP-5SA group. TEAD transcriptional enhanced associate domain; YAP Yes-associated protein; TAZ transcriptional co-activator with PDZ-binding motif

Fig. 8

YAP/TAZ-TEAD activity promotes malignant transformation of precancerous cells. A and B Western blot to check the expression of proteins related to YAP/TAZ-TEAD in cells with different treatments. **P < 0.01, vs NC

Fig. 9

Mechanism diagram. Activation of Warburg effect in precancerous cervical epithelial cells increased the dryness and glycolysis of precancerous cervical epithelial cells, promoted the expression of YAP/TAZ signal transduction TEAD, and thus accelerated the malignant transformation of cervical intraepithelial neoplasia into cervical cancer and promoted the metastasis of cancer cells