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1.
Theranostics ; 10(21): 9443-9457, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32863938

RESUMO

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC. Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo. Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo. Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays. Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites. Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Via de Sinalização Hippo , Humanos , Imuno-Histoquímica/métodos , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Ubiquitinação/genética
2.
J Cell Mol Med ; 24(20): 11912-11921, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32896069

RESUMO

Oesophageal cancer is one of the most lethal malignancies worldwide, whereas the 5-year survival is less than 20%. Although the detailed carcinogenic mechanisms are not totally clear, recent genomic sequencing data showed dysregulation of Hippo signalling could be a critical factor for oesophageal squamous cell carcinoma (ESCC) progression. Therefore, understanding of the molecular mechanisms that control Hippo signalling activity is of great importance to improve ESCC diagnostics and therapeutics. Our current study revealed RACO-1 as an inhibitory protein for YAP/TEAD axis. Depletion of RACO-1 increases the protein level of YAP and expression of YAP/TEAD target gene. Besides, RACO-1 silencing could promote ESCC cell invasion and migration, which effect could be rescued by YAP depletion in ESCC cells. Immunoprecipitation showed that RACO-1 associated with YAP and promote ubiquitination and degradation of YAP at k48 poly-ubiquitination site. Our research discovered a new regulator of Hippo signalling via modulating YAP stability. RACO-1 could be a promising factor, which serves cancer diagnostics and therapeutics in ESCC patients.


Assuntos
Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Via de Sinalização Hippo , Humanos , Modelos Biológicos , Invasividade Neoplásica , Estabilidade Proteica , Proteólise , Ubiquitinação
3.
Neoplasia ; 22(2): 76-85, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31884247

RESUMO

Esophageal cancer is one of the leading malignancies worldwide, while around sixty percent of newly diagnosed cases are in China. In recent years, genome-wide sequencing studies and cancer biology studies show that Hippo signaling functions a critical role in esophageal squamous cell carcinoma (ESCC) progression, which could be a promising therapeutic targets in ESCC treatment. However, the detailed mechanisms of Hippo signaling dys-regulation in ESCC remain not clear. Here we identify SHARPIN protein as an endogenous inhibitor for YAP protein. SHARPIN depletion significantly decreases cell migration and invasion capacity in ESCC, which effects could be rescued by further YAP depletion. Depletion SHARPIN increases YAP protein level and YAP/TEAD target genes, such as CTGF and CYR61 in ESCC. Immuno-precipitation assay shows that SHARPIN associates with YAP, promoting YAP degradation possibly via inducing YAP K48-dependent poly-ubiquitination. Our study reveals a novel post-translational mechanism in modulating Hippo signaling in ESCC. Overexpression or activation of SHARPIN could be a promising strategy to target Hippo signaling for ESCC patients.


Assuntos
Proteínas de Ciclo Celular/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ubiquitinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Via de Sinalização Hippo , Humanos , Masculino , Invasividade Neoplásica/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Ubiquitina/genética , Ubiquitinas/antagonistas & inibidores
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