The HNF-1β-USP28-Claspin pathway upregulates DNA damage-induced Chk1 activation in ovarian clear cell carcinoma

Transcription factor hepatocyte nuclear factor 1-beta (HNF-1β) enhances checkpoint kinase 1 (Chk1) activation and promotes G2/M cell cycle progression in ovarian clear cell carcinoma (CCC) following exposure to diverse genotoxic agents including bleomycin. However, the underlying mechanism leading to checkpoint activation of HNF-1β still remains largely unknown. To clarify the effects of HNF- 1β on cell cycle checkpoints, human CCC cell lines were transfected with siRNAs targeting HNF-1β, Claspin, USP28, or a control vector. Ubiquitination and stabilization of Claspin protein by HNF-1β was assessed by immunoprecipitation. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated the Claspin expression after treatment with a genotoxic agent bleomycin, resulting in accumulation of phosphorylated Chk1 (p-Chk1) and promotion of survival in CCC cell lines. This study showed for the first time that USP28, a de-ubiquitinase crucial for Claspin expression, is one target gene of HNF-1β. Knockdown of endogenous USP28 suppressed the Claspin expression and p-Chk1 activation and cell viability. Our findings identify a novel pathway of the HNF-1β-USP28-Claspin-Chk1 axis in checkpoint signal amplification in response to DNA damage. Targeting this pathway may represent a putative, novel, anticancer strategy in ovarian CCC.