Theranostics 2020; 10(21):9443-9457. doi:10.7150/thno.46078

Research Paper

Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin ligase PARK2

Xiaofeng Zhou1,4*, Yajie Li1,2*, Weilong Wang1,2*, Sujie Wang1, Jinghan Hou1,2, Aijia Zhang1,2, Benjie Lv1,2, Can Gao1,2, Ziyi Yan1, Dan Pang1,2, Kui Lu1,2, Nor Hazwani Ahmad3, Lidong Wang7✉, Jian Zhu8,5,6✉, Lichen Zhang4✉, Ting Zhuang4,5,6✉, Xiumin Li1,2✉

1. Xinxiang Key Laboratory for Molecular Therapy of Cancer, Xinxiang Medical University, Xinxiang 453003, Henan Province, P.R. China.
2. Department of Gastroenterology, the Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, Henan Province, P.R. China.
3. Oncological and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Penang, Malaysia.
4. Henan Key Laboratory of immunology and targeted therapy, School of Laboratory Medicine, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang Medical University, Xinxiang, 453003, Henan Province, P.R. China.
5. Xinxiang Key Laboratory of Tumor Migration, Invasion and Precision Medicine, Xinxiang, 453003, Henan Province, P.R. China.
6. School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, 453003, Henan Province, P.R. China.
7. State Key Laboratory of Esophageal Cancer Prevention & Treatment and Henan Key Laboratory for Esophageal Cancer Research of The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China.
8. Department of General Surgery, The Second Hospital, Cheeloo College of Medicine, Shandong University, Shandong Province, P.R. China.
*These authors contributed equally to this study.

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Citation:
Zhou X, Li Y, Wang W, Wang S, Hou J, Zhang A, Lv B, Gao C, Yan Z, Pang D, Lu K, Ahmad NH, Wang L, Zhu J, Zhang L, Zhuang T, Li X. Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin ligase PARK2. Theranostics 2020; 10(21):9443-9457. doi:10.7150/thno.46078. Available from http://www.thno.org/v10p9443.htm

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Abstract

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.

Keywords: PARK2, Hippo, YAP, ESCC, Ubiquitin