DNASE1L3 inhibits hepatocellular carcinoma by delaying cell cycle progression through CDK2

Purpose: Dysregulated cell cycle targeting is a well-established therapeutic strategy against hepatocellular carcinoma (HCC). Dissecting the underlying mechanism may improve the efficacy of HCC therapy.

Methods: HCC data from TCGA and new clinical samples were used for DNASE1L3 expression analysis and for assessing its correlation with HCC development. The in vitro function of DNASE1L3 in HCC cell proliferation, colony formation, migration and invasion was assessed using RTCA, CCK-8 and transwell assays and the in vivo function in subcutaneous tumor formation in a xenograft nude mouse model. The role of DNASE1L3 in HCC tumorigenesis was further verified in AKT/NRASV12-induced and DEN/CCl₄-induced primary liver cancers in wildtype and Dnase1l3^-/- mice. Finally, RNA-Seq analysis followed by biochemical methods including cell cycle, immunofluorescence, co-immunoprecipitation and Western blotting assays were employed to reveal the underlying mechanism.

Results: We found that DNASE1L3 was significantly downregulated and served as a favorable prognostic factor in HCC. DNASE1L3 dramatically attenuated HCC cell proliferation, colony formation, migration and invasion in vitro and reduced subcutaneous tumor formation in nude mice in vivo. Furthermore, DNASE1L3 overexpression dampened AKT/NRASV12-induced mouse liver cancer in wildtype mice and DNASE1L3 deficiency worsened DEN/CCl₄-induced liver cancer in Dnase1l3^-/- mice. Systemic analysis revealed that DNASE1L3 impaired HCC cell cycle progression by interacting with CDK2 and inhibiting CDK2-stimulated E2F1 activity. C-terminal deletion (DNASE1L3^ΔCT) diminished the interaction with CDK2 and abrogated the inhibitory function against HCC.

Conclusion: Our study unveils DNASE1L3 as a novel HCC cell cycle regulator and tumor suppressor. DNASE1L3 impairs HCC tumorigenesis by delaying cell cycle progression possibly through disrupting the positive E2F1-CDK2 regulatory loop. DNASE1L3 may serve as a target for the development of novel therapeutic strategies against HCC.