Fos-related-antigen-1 (Fra-1), a member of the activator protein-1 (AP-1) transcription factor superfamily, has an essential role in cancer progress and metastasis and Fra-1 is considered a therapeutic target in metastatic cancer including metastatic colorectal cancer (mCRC). However, its regulation at protein level has not yet been clearly elucidated. We found that ubiquitin-specific protease 21 (USP21) increases Fra-1 stability by deubiquitinating Fra-1 and enhances the expression of Fra-1 target genes in colon cancer cells. We also showed that USP21 controlled Fra-1-dependent migration and invasion activities. The oncogenic property of USP21 was confirmed by a significant reduction in liver metastasis when USP21-knockdown cancer cells were injected intrasplenically into mice. Consistently, clinicopathological analysis of colorectal cancer patients revealed a correlation of USP21 expression with high-grade carcinoma and life span. These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1. Therefore, USP21 is considered an attractive therapeutic target in mCRC with high Fra-1 expression.

Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of β-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of β-catenin, as shown by immunoblotting, and it affected the target gene expression of β-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased β-catenin stability and reduced expression of β-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.