Elsevier

Biochemical Pharmacology

Volume 131, 1 May 2017, Pages 29-39
Biochemical Pharmacology

USP7 inhibitor P5091 inhibits Wnt signaling and colorectal tumor growth

https://doi.org/10.1016/j.bcp.2017.02.011Get rights and content

Abstract

Aberrant activation of Wnt/β-catenin signaling is closely associated with the development of various human cancers, especially colorectal cancers (CRC). The ubiquitin proteasome system (UPS) is essential in the regulation of Wnt signaling and inhibitors targeting the UPS could have great potential in CRC therapy. Ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme, plays a significant role in neoplastic diseases due to its well-known function of regulating the MDM2-p53 complex. Inspired by our recent study identifying the positive role of USP7 in the Wnt signaling, we report here that USP7 is overexpressed in colorectal carcinoma cell lines and tissues, which is closely related with the poor prognosis. USP7 knockdown inhibits the proliferation of CRC cells with different p53 status, and USP7 inhibition by its inhibitor P5091 attenuates the activity of Wnt signaling via enhanced ubiquitination and the subsequent degradation of β-catenin. In vitro, P5091 inhibited the proliferation and induced apoptosis of CRC cells. P5091 also suppressed in vivo tumor growth in the HCT116 xenograft mouse model, which is consistently associated with reduced expression of β-catenin and Wnt target genes. In conclusion, our preclinical study indicated that USP7 could be a potential drug target and its inhibitor P5091 deserves further development as anticancer agent for Wnt hyper-activated CRC therapy.

Introduction

The ubiquitin proteasome system (UPS), consisting of ubiquitin ligases, deubiquitinating enzymes (DUBs) and proteasome, is essential for regulation of protein turnover and function [1]. The fundamental roles of UPS pathways are often altered in cancer progression, thereby offering inhibitors of UPS pathways as a novel therapeutic strategy [2], [3]. Targeting this pathway was validated as a strategy by the FDA approval of the proteasome inhibitor bortezomib/Velcade for the treatment of multiple myeloma [4].

Ubiquitin-specific protease 7 (USP7), a member of DUBs, is the most studied due to its pivotal roles in cancer progression [5]. USP7 preferentially deubiquitylates and stabilizes E3 ligase MDM2 (human ortholog HDM2), which negatively regulates the celebrated tumor suppressor p53 [6], [7]. Genetic ablation of USP7 via siRNA or somatic knockout prevents USP7 from deubiquitylating MDM2, resulting in subsequent stabilization of p53, which promotes cell cycle arrest and apoptosis [6], [8]. However, emerging evidences suggest that mechanism by which USP7 regulates the proliferation of tumor cells may be various, besides the regulation of the MDM2-p53 complex, consistent with the diverse nature of USP7 functions [5].

Dysregulation of the Wnt/β-catenin signaling pathway is closely associated with a range of human disorders, most notably cancer [9], [10]. UPS plays a crucial role in the regulation of the Wnt signaling [11]. Signaling events in the Wnt/β-catenin cascade converge on the regulation of the key transcriptional regulator β-catenin, a target of the ubiquitin-proteasome pathway [11], [12]. In the absence of Wnt ligands, β-catenin is phosphorylated by the destruction complex, which consists of the GSK3β, APC, Axin and CK1α. The phosphorylated β-catenin is then recognized by the E3 ligase β-Trcp, which induces β-catenin polyubiquitination and the subsequent proteasomal degradation [10]. Besides β-Trcp, other E3 ligases targeting β-catenin for degradation have also been detected, including Siah-1, Jade-1, c-Cb1 and TRIM33, which regulate different forms of β-catenin under different conditions [13], [14], [15], [16]. To be noted, atypical polyubiquitination of β-catenin mediated by the E3 ligase EDD was reported to stabilize it and enhance the activity of Wnt signaling [17].

Even though the ubiquitination of β-catenin is relatively well characterized, explorations about β-catenin deubiquitination make progress until recently [18], [19]. Our recent study showed that RNF220, an E3 ligase with RING domain, mediated the binding of USP7 and β-catenin, thereby leading to deubiquitination and stabilization of β-catenin. Knockdown of USP7 in colorectal cancer cell lines with hyperactivated Wnt signaling downregulates the activity of Wnt signaling and expression of Wnt target genes, indicating a potentially novel role of USP7 in Wnt-related carcinogenesis [20].

In the present study, therefore, we attempt to investigate the possibility of USP7 as a drug target in CRC therapy, and the effect of P5091, a small molecule inhibitor of USP7, on the Wnt signaling and growth of colorectal cancers. Our in vitro and in vivo data indicate the potential of P5091 in CRC therapy and provide evidences demonstrating the rationality for development of USP7 inhibitors as anti-CRC agents.

Section snippets

Cell culture

HEK293 and the human colon carcinoma cell lines (HCT116, SW480, Caco-2, SW620 and HT29) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Normal colonic epithelial cell line (CCD-841-CoN) was kindly gifted by Professor Lin Li (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China). Cells were cultured in medium (DMEM for HEK293, HCT116, SW480, Caco-2, CCD-841-CoN and SW620 cells,

USP7 is upregulated in CRCs and positively correlates with cell proliferation and poor CRC prognosis

To assess the USP7 expression in CRC, we first analyzed the expression level of USP7 in normal colonic epithelial cell line and five CRC cell lines by western blot and qRT-PCR analysis. Compared with the normal cells, USP7 upregulation was detected in CRC cells (Fig. 1A and B). We further examined the expression pattern of USP7 in human colorectal cancer tissues by analyzing Oncomine, a publicly accessible cancer informatics database. In accordance with cellular expression level of USP7, USP7

Discussion

Identification of small molecules inhibiting Wnt signaling is one of the most actively explored fields in cancer therapy [38], [39], especially remarkable for colorectal cancer therapy, in view of increased Wnt signaling pathway as a characteristic of colorectal cancer [40], [41]. Downregulation of β-catenin, the key transcription factor of Wnt signaling pathway, is one manner to suppress the over-activation of Wnt pathway. Benefited from the expounded mechanism of β-catenin regulation by

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. Aaron M Zorn (Cincinnati Children’s Hospital) and Dr. Siqing Zhang (Xiamen University) for providing the SuperTopflash, pCS-mWnt1, PLL3.7-shUSP7#1/#2 and Myc-Ub plasmids. We also thank Professor Lin Li (Institute of Biochemistry and Cell Biology) for the gift of normal colonic epithelial cell line (CCD-841-CoN). This work was supported financially by the project of science and technology of Yunnan Province (2013FA047) and the open project of State Key Laboratory of Genetic

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