Tumor-suppressive circRHOBTB3 is excreted out of cells via exosome to sustain colorectal cancer cell fitness

Background & Aims To clarify the biological roles, circularization process and secretion pathway of circRHOBTB3 in colorectal cancer (CRC) progression. Methods We performed a comprehensive analysis of circRNA levels in serum exosomes from multiple types of cancer patients in public databases and verified the higher level of circRHOBTB3 in CRC sera versus healthy donors by RT-qPCR. Then, the function of circRHOBTB3 in CRC was investigated in vitro and in vivo. RNA-seq and RNA pull-down assays together with mass spectrometry identified the downstream signals and the binding proteins of circRHOBTB3. Finally, Antisense oligonucleotides (ASOs) were designed to target circularization and secretion elements of circRHOBTB3 for CRC therapy. Results circRHOBTB3 levels were increased in the sera but was downregulated in tissue samples in CRC, and the downregulation was associated with poor prognosis. Furthermore, circRHOBTB3 acts a tumor-suppressive circRNA by repressing metabolic pathways, intracellular ROS production in CRC. Several key elements were discovered to regulate circRHOBTB3 circularization and exosomal secretion. Moreover, SNF8 was identified that sorts circRHOBTB3 into exosomes. Interestingly, we found that CRC cells could actively secrete more circRHOBTB3 than normal cells. According to the sequence of regulatory elements for circularization and exosomal secretion, we designed and synthesized ASOs, which increased circRHOBTB3 expression and blocked circRHOBTB3 exosomal secretion. More importantly, ASOs could inhibit CRC growth and metastasis in vitro and in vivo. Conclusions circRHOBTB3 plays a tumor-suppressive role in CRC and has to be excreted out of cells to sustain cancer cell fitness. ASOs targeting regulatory elements for circularization and exosomal secretion will become a novel antitumor strategy.

Automated summary

The study reveals that circRHOBTB3 is upregulated in serum and downregulated in tissue samples of CRC patients, with the downregulation associated with poor prognosis. CircRHOBTB3 acts as a tumor suppressor by repressing metabolic pathways and intracellular ROS production. SNF8 is identified as the protein responsible for sorting circRHOBTB3 into exosomes, and CRC cells secrete more circRHOBTB3 compared to normal cells. Antisense oligonucleotides targeting regulatory elements of circRHOBTB3 can increase its expression and inhibit its exosomal secretion, thereby inhibiting CRC growth and metastasis.