Abstract
It has been well-established that the deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) supports cancer growth by up-regulating multiple cellular pathways, including Wnt/β-catenin signaling. Therefore, considerable efforts are directed at identifying and developing USP7 inhibitors. Here, we report that sesquiterpene lactone parthenolide (PTL) inhibits USP7 activity, assessed with deubiquitinating enzyme activity assays, including fluorogenic Ub-AMC/Ub-Rho110, Ub-VME/PA labeling, and Di-Ub hydrolysis assays. Further investigations using cellular thermal shift (CETSA), surface plasmon resonance (SPR), and mass spectrum (MS) assays revealed that PTL directly interacts with USP7. Consistent with the role of USP7 in stimulating Wnt signaling and carcinogenesis, PTL treatment inhibited the activity of Wnt signaling partly by destabilizing β-catenin. Moreover, using cell viability assays, we found that PTL suppresses the proliferation of colorectal cancer cells and induces apoptosis in these cells. Additionally, we examined the effects of two other sesquiterpene lactones (costunolide and α-santonin) on USP7 and Wnt signaling and found that α-methylene-γ-butyrolactone may provide a scaffold for future USP7 inhibitors. In summary, our findings reveal that PTL inhibits USP7 activity, identifying a potential mechanism by which PTL suppresses Wnt/β-catenin signaling. We further suggest that sesquiterpene lactones might represent a suitable scaffold for developing USP7 inhibitors and indicate that PTL holds promise as an anticancer agent targeting aberrant USP7/Wnt signaling.
Highlights
It has been well established that the deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) supports cancer growth by up-regulating multiple cellular pathways, including Wnt/β-catenin signaling
We further suggest that sesquiterpene lactones might represent a suitable scaffold for developing USP7 inhibitors and indicate that PTL holds promise as an anticancer agent targeting aberrant
The USP7 activity towards K48-linked diubiquitin was investigated, and the results showed that PTL could decrease the amount of Ub hydrolyzed from di-Ub in a concentration-dependent manner (Fig. 1H)
Summary
In an effort to identify USP7 inhibitors, we performed an in vitro high-throughput screening (HTS) assay against a library of natural chemicals using Ub-AMC as a substrate [30], and the small molecule PTL was discovered (Fig. 1A). In line with the above-mentioned data, the results of cytoplasmic and nuclear fraction and immunofluorescence assays further indicated that PTL treatment downregulated β-catenin levels of the nuclear and cytoplasmic compartments in both HCT116 and SW480 cells (Fig. 3G and 3H). These data suggested that PTL decreased the levels of β-catenin via increasing its ubiquitination
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