RIPPLY1 suppresses cancer cell stemness via targeting TBX19 in CTNNB1-mutated hepatocellular carcinoma

Background & Aims CTNNB1-mutated HCCs exhibit a relatively low stem-like and well-differentiated phenotype. However, the mechanism remains unclear. Ripply transcriptional repressor 1 (RIPPLY1), a transcriptional repressor required for somite segmentation, has hardly been studied in cancer. Here, we aim to unveil the role of RIPPLY1 in the regulation of cancer cell stemness in CTNNB1-mutated HCCs. Approach and Results RIPPLY1 was found to be transactivated by the Wnt/ß-Catenin signal pathway. Human sample analysis confirmed that RIPPLY1 was significantly upregulated in CTNNB1-mutated HCC tissues and positively correlated with better prognosis of HCC patients. Hepatocyte-specific deletion of RIPPLY1 promoted tumorigenesis and progression in DEN/PB-induced CTNNB1-mutated HCC mouse model and hydrodynamic tail-vein injection (HDTVi)-induced CTNNB1-mutated HCC mouse model. RIPPLY1 knockout tumor cells displayed upregulated levels of stem cell makers and enhanced cancer stem cell properties. Co-IP and MS identified TBX19 as the target protein of RIPPLY1. RIPPLY1 suppressed the transcriptional activity of TBX19 via recruiting TLE1 and promoting proteasome-dependent degradation of TBX19. TBX19 deficiency abolished the effect of RIPPLY1 loss on cancer cell stemness in CTNNB1-mutated HCCs. Conclusion: Loss of RIPPLY1 promotes cancer cell stemness via facilitating the TBX19 transcriptional activity in CTNNB1-mutated HCCs. Overall design: Ripply1 exons 1 to 3 were flanked with loxP sites to generate floxed Ripply1 (Ripply1F/F) mice. Ripply1F/F mice were further crossed with Alb-Cre transgenic mice to generate mice with hepatocyte-specific deletion of RIPPLY1 (Ripply1?Hep) .The male Ripply1F/F and Ripply1?Hep mice were intraperitoneally injected with a single dose of DEN (90 µg/g) at 6 weeks of age. After a treatment-free interval of 3 weeks, mice were kept on a diet containing 0.05% PB for 27 weeks. The primary hepatocytes were isolated from DEN/PB induced liver tumors of Ripply1F/F and Ripply1?Hep mice 30 weeks after DEN injection and subjected to RNA-sequencing.