RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transformação Celular Neoplásica , Células-Tronco Neoplásicas , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Proteínas de Sinalização YAP , Humanos , Feminino , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/metabolismo , Animais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismo , Linhagem Celular Tumoral , Camundongos , Efeito Warburg em Oncologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proliferação de Células/genética , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologiaRESUMO
BACKGROUND: In advanced and recurrent endometrial carcinoma (EC), the current state of immuno- or targeted therapy remains in the clinical research phase. Our study aimed to explore the role of the ESCRT machinery in maintaining cell membrane integrity and reversing pyroptotic cell death. METHODS: Immunohistochemistry, western blotting, and co-immunoprecipitation were performed to determine the expression and relationship between GSDMD, CHMP4B, and VPS4A. We employed techniques such as FITC Annexin V/propidium iodide staining, Ca2+ fluorescence intensity, IL-1ß enzyme-linked immunosorbent assay, and lactate dehydrogenase release assay to detect pyroptosis in endometrial cancer cells. Plasma membrane perforations and CHMP4B/VPS4A puncta were observed through electron and fluorescence confocal microscopy. RESULTS: We showed that GSDMD, CHMP4B, and VPS4A were differentially expressed in the pyroptotic EC xenograft mouse model group, as well as high, moderate, and mild expression in EC cells treated with LPS and nigericin compared to endometrial epithelial cells. Co-IP confirmed the interaction between GSDMD, CHMP4B, and VPS4A. We found that GSDMD knockdown reduced PI-positive cells, Ca2+ efflux, IL-1ß, and LDH release, while CHMP4B and VPS4A depletion enhanced these indicators in HEC1A and AN3CA cells. Electron microscopy showed membrane perforations correspondingly decreased with inactivated GSDMD and increased or decreased after CHMP4B and VPS4A depletion or overexpression in EC cells. Fluorescence confocal microscopy detected CHMP4B protein puncta associated with VPS4A at the injured plasma membrane in GSDMDNT cells. CONCLUSIONS: We preliminary evidenced that CHMP4B and VPS4A reverses GSDMD-mediated pyroptosis by facilitating cell membrane remodeling in endometrial carcinoma. Targeting CHMP4B related proteins may promote pyroptosis in endometrial tumors.