BHLHE22, targeted by deubiquitinating enzyme OTUD3, exerts an antitumor role in triple-negative breast cancer progression via transcriptionally inhibiting CDT1.

BACKGROUND: Basic helix-loop-helix family, member e22 (BHLHE22), a basic helix loop helix transcription factor family member, functions vary in different types of cancer. Currently, its function in triple-negative breast cancer (TNBC) is unclear. METHODS: The GSE45827 and GSE113865 datasets were used to screen potential TNBC marker. Biological websites were used to analyze BHLHE22 expression in TNBC, its relationship with the prognosis of patients with TNBC, and its potential function. A series of in vitro and in vivo experiments was performed to investigate the function of BHLHE22 in TNBC. The regulatory relationship between OTU domain-containing protein 3 (OTUD3) and BHLHE22 was verified by Co-Immunoprecipitation, ubiquitination assay, and site-specific mutation experiments. The mRNA-seq analysis was performed to identify potential genes for the anti-cancer role of BHLHE22. The transcriptional regulation of DNA replication factor 1 (CDT1) by BHLHE22 was confirmed by dual luciferase reporter assay. RESULTS: We identified the downregulation of BHLHE22 in TNBC tissues based on the GSE45827 and GSE113865 datasets. The expression of BHLHE22 in TBNC patients was lower than that in non-TNBC patients, and patients at stages 3 and 4 tended to express lower BHLHE22 expression than patients at stages 1 and 2. Patients with high expression of BHLHE22 had better survival prognosis than those with BHLHE22 low expression. Functional studies revealed that BHLHE22 overexpression impaired cell growth in vitro and in vivo. However, BHLHE22 silencing enhanced the malignant behaviors of cancer cells. OTUD3, a deubiquitinase known to suppress TNBC progression, was found to increase the stability of BHLHE22 protein through deubiquitination regulation. The C76 site mutation of OTUD3 eliminated the catalytic activity of OTUD3 and failed to regulate the protein stability of BHLHE22. Furthermore, BHLHE22 medicated the antitumor effect of OTUD3 in TNBC. The mRNA-seq analysis identified potential genes for the anti-cancer role of BHLHE22, involving CDT1. BHLHE22 was proved to reduce CDT1 RNA expression by blocking CDT1 transcription. The anti-proliferative effect of BHLHE22 overexpression was reversed by CDT1 overexpression. CONCLUSIONS: Our observations demonstrate that BHLHE22 may be a promising target for TNBC therapy.