ROBO receptors function as tumor suppressors in cholangiocarcinoma

Introduction Cholangiocarcinoma (CCA) is a rare but highly aggressive malignancy of the bile duct with inadequate therapy options and poor prognosis. Genetic alterations in ROBO receptors were identified exclusively in the invasive form of CCA (Goeppert et.al, 2022). ROBO receptors act as axon-guidance-cues during neuronal development, but are deregulated in many cancers in a context-dependent manner. The aim of this project is to functionally characterize the ROBO receptors in CCA.

Methods ROBO1 and ROBO2 were introduced into intrahepatic-CCA cells with a doxycycline-inducible expression system by using lentiviral transduction. Functional assays were performed such as viability, migration, and colony formation. RNA-seq analysis was performed to detect ROBO downstream signaling. BioID, a proximity-based labelling method was used to identify interacting partners of ROBOs. Hydrodynamic-tail-vein injections (HDTV) served to assess the function of ROBO1 and ROBO2 in vivo.

Results ROBO1 and ROBO2 restricted cell viability, migration, and clonogenicity of cells in vitro. RNA-seq revealed a decrease in cell cycle and DNA-replication whereas an increase in interferon response. BioID identified candidate interaction partners of which the majority is involved in MAPK signaling. In-vivo experiments with HDTV showed striking tumor suppressor features of ROBO1 and ROBO2.

Conclusion ROBO receptors restrict tumorgenicity in vitro and in vivo. Additionally, they may play a role in anti-tumor immunity or anti-inflammatory responses. Further experiments may reveal interaction partners, downstream signaling pathways and their involvement in anti-tumor/anti-inflammatory response. Therefore, our research on ROBO1/2 might be useful to develop better therapy options.

Publication History

Article published online:
20 January 2025