Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment
Ya-Ting Wang, … , Yi-Ching Wang, Ruey-Hwa Chen
Ya-Ting Wang, … , Yi-Ching Wang, Ruey-Hwa Chen
Published July 10, 2017
Citation Information: J Clin Invest. 2017;127(8):2982-2997. https://doi.org/10.1172/JCI89957.
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Research Article Oncology

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment

Abstract

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4–containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Authors

Ya-Ting Wang, Jocelyn Chen, Chou-Wei Chang, Jayu Jen, Tzu-Yu Huang, Chun-Ming Chen, Roger Shen, Suh-Yuen Liang, I-Cheng Cheng, Shuenn-Chen Yang, Wu-Wei Lai, Kuang-Hung Cheng, Tao-Shih Hsieh, Ming-Zong Lai, Hung-Chi Cheng, Yi-Ching Wang, Ruey-Hwa Chen

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Figure 1

PML is a substrate of CRL4WDR4.

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PML is a substrate of CRL4WDR4. (A and B) Immunoprecipitation analysis f...
(A and B) Immunoprecipitation analysis for PML ubiquitination in 293T cells transfected with the indicated constructs and/or siRNA or in H1299 lung cancer cells stably expressing WDR4 shRNA and transfected with the indicated constructs. (C) Ni–nitrilotriacetic acid (Ni-NTA) pull-down analysis for PML-I and PML-IV ubiquitination in 293T cells transfected with the indicated constructs. (D) Immunoprecipitation analysis of the interaction between WDR4 or its mutant (R219A) and endogenous DDB1 in 293T cells. (E) Reciprocal immunoprecipitation analysis of the interaction between endogenous WDR4 and endogenous PML in H1299 lung cancer cells. The asterisk marks a nonspecific band. (F) Immunoprecipitation analysis of the interaction between PML-I and endogenous DDB1, CUL4A, and CUL4B in 293T cells transfected with the PML-I construct and/or WDR4 siRNA. (G) GST pull-down analysis of the in vitro interaction between GST-WDR4 and baculovirally purified PML-I. (H) In vitro ubiquitination assay for baculovirally purified PML-I incubated with the WDR4-based CUL4A or CUL4B ubiquitin ligase. The equal input of the CUL4A and CUL4B complexes is shown on the right. exp., exposure; WB, Western blot.