Silencing CDCA8 Suppresses Hepatocellular Carcinoma Growth and Stemness via Restoration of ATF3 Tumor Suppressor and Inactivation of AKT/β-Catenin Signaling

Simple Summary Although the overexpression of CDCA8 is frequently observed in hepatocellular carcinoma (HCC) tissues, the functions of CDCA8 during HCC development remain to be clarified. The aim of our study was to investigate if targeting CDCA8 could affect liver tumor phenotypes in vitro and in vivo and to identify underlying molecular mechanisms to exert its therapeutic effect. We found that silencing of CDCA8 by siRNA inhibits the growth of parental cancer cell culture and mice tumors and suppresses stemness of CD133(+) cancer stem cell population through the common responses of the upregulation of the tumor suppressive ATF3/GADD34 functional pathway and inactivation of the Akt/beta-catenin signaling axis. These findings suggest CDCA8 as a novel therapeutic target for both primary HCC treatment and the prevention of metastasis or recurrence providing mode of action performed by a CDCA8 inhibitor. Big data analysis has revealed the upregulation of cell division cycle associated 8 (CDCA8) in human hepatocellular carcinoma (HCC) and its poorer survival outcome. However, the functions of CDCA8 during HCC development remain unknown. Here, we demonstrate in vitro that CDCA8 silencing inhibits HCC cell growth and long-term colony formation and migration through the accumulation of the G2/M phase cell population. Conversely, CDCA8 overexpression increases the ability to undergo long-term colony formation and migration. RNA sequencing and bioinformatic analysis revealed that CDCA8 knockdown led to the same directional regulation in 50 genes (25 down- and 25 upregulated). It was affirmed based on protein levels that CDCA8 silencing downregulates the levels of cyclin B1 and p-cdc2 and explains how it could induce G2/M arrest. The same condition increased the protein levels of tumor-suppressive ATF3 and GADD34 and inactivated AKT/beta-catenin signaling, which plays an important role in cell growth and stemness, reflecting a reduction in sphere-forming capacity. Importantly, it was demonstrated that the extent of CDCA8 expression is much greater in CD133(+) cancer stem cells than in CD133(-) cancer cells, and that CDCA8 knockdown decreases levels of CD133, p-Akt and beta-catenin and increases levels of ATF3 and GADD34 in the CD133(+) cancer stem cell (CSC) population. These molecular changes led to the inhibition of cell growth and sphere formation in the CD133(+) cell population. Targeting CDCA8 also effectively suppressed tumor growth in a murine xenograft model, showing consistent molecular alterations in tumors injected with CDCA8siRNA. Taken together, these findings indicate that silencing CDCA8 suppresses HCC growth and stemness via restoring the ATF3 tumor suppressor and inactivating oncogenic AKT/beta-catenin signaling, and that targeting CDCA8 may be the next molecular strategy for both primary HCC treatment and the prevention of metastasis or recurrence.