USP7 facilitates SMAD3 autoregulation to repress cancer progression in p53-deficient lung cancer

Yu-Ting Huang,Greg G Wang,An-Chieh Cheng,Guo-Cheng Huang,Ping-Hui Tseng,Hui-Chi Tang,Kou-Juey Wu,Ta-Hsien Lin,Ling Cai,Wei-Yi Chen

doi:10.1038/s41419-021-04176-8

Abstract

USP7, one of the most abundant ubiquitin-specific proteases (USP), plays multifaceted roles in many cellular events, including oncogenic pathways. Accumulated studies have suggested that USP7, through modulating the MDM2/MDMX-p53 pathway, is a promising target for cancer treatment; however, little is known about the function of USP7 in p53-deficient tumors. Here we report that USP7 regulates the autoregulation of SMAD3, a key regulator of transforming growth factor β (TGFβ) signaling, that represses the cell progression of p53-deficient lung cancer. CRISPR/Cas9-mediated inactivation of USP7 in p53-deficient lung cancer H1299 line resulted in advanced cell proliferation in vitro and in xenograft tumor in vivo. Genome-wide analyses (ChIP-seq and RNA-seq) of USP7 KO H1299 cells reveal a dramatic reduction of SMAD3 autoregulation, including decreased gene expression and blunted function of associated super-enhancer (SE). Furthermore, biochemical assays show that SMAD3 is conjugated by mono-ubiquitin, which negatively regulates the DNA-binding function of SMAD3, in USP7 KO cells. In addition, cell-free and cell-based analyses further demonstrate that the deubiquitinase activity of USP7 mediates the removal of mono-ubiquitin from SMAD3 and facilitates the DNA-binding of SMAD3-SMAD4 dimer at SMAD3 locus, and thus enhance the autoregulation of SMAD3. Collectively, our study identified a novel mechanism by which USP7, through catalyzing the SMAD3 de-monoubiquitination, facilitates the positive autoregulation of SMAD3, and represses the cancer progression of p53-deficient lung cancer.

Highlights

Ubiquitination is a type of posttranslational modification in that ubiquitin is covalently conjugated to target proteins through a process catalyzed by multiple enzymes, including the E1 ubiquitinactivating enzymes, the E2 ubiquitin conjugation enzymes, and the E3 ubiquitin ligases [1]
ubiquitin-specific protease 7 (USP7) inhibition has become a promising strategy for cancer loading scores for peaks identified in USP7 KO cells, we identified a larger number of SEs in USP7 KO cells than that in WT H1299 cells (Fig. 2C and Dataset 3)
USP7 inactivation decreased cell progression of p53-positive but enhanced that of p53-negative, lung cancer A549 cell line many previous reports showed that inactivation of USP7 using inhibitory compounds or RNAi-mediated gene silencing hampered the growth of cancer cells, our results clearly demonstrated that USP7 KO impaired the SMAD3 autoregulation and resulted in enhanced growth of p53-null H1299 cells

Summary

Introduction

Ubiquitination is a type of posttranslational modification in that ubiquitin is covalently conjugated to target proteins through a process catalyzed by multiple enzymes, including the E1 ubiquitinactivating enzymes, the E2 ubiquitin conjugation enzymes, and the E3 ubiquitin ligases [1]. The addition of a single (mono-) or non-Lys-48-linked polyubiquitin to protein substrates may serve a non-degradative function, such as trafficking, protein–protein interaction, and modulating the activity of enzymes. The Lys-48 polyubiquitinated proteins are often recognized and subjected to degradative processes mediated by the 26 S proteasome pathway [2, 3]. Deubiquitination, through the action of deubiquitinating enzymes (DUBs), are responsible for the removal of the ubiquitin/polyubiquitin chain from protein substrates and reverses the functions of ubiquitination [4]. Components in ubiquitin pathways have been proposed as potential targets for therapeutic strategies against diseases and cancers [6]

Methods

Results

Conclusion