Functional siRNA Screen Links Ras/MAPK and Wnt Pathway to EV Secretion in HCT-116 Colorectal Cancer Cells

Background: Extracellular vesicles (EVs) play an important role in tumor progression and intercellular communication, yet the contribution of specific cancer-related genes to EV secretion remains incompletely defined. Methods: To address this, we performed an siRNA-based loss-of-function screen targeting 30 frequently altered (proto-)oncogenes and tumor suppressor genes in the colorectal carcinoma cell line HCT-116 to assess their impact on EV release. EVs were isolated by sequential ultracentrifugation to obtain P14 and P100 fractions pelleting at 14,000× g or 100,000× g, respectively, and were characterized by nanoparticle tracking analysis, EV marker expression, and total protein quantification. Cell viability was assessed to control for potential apoptosis-related effects. Results: With few exceptions, knockdown of the investigated genes led to an increase in EV secretion. Silencing of KRAS and BRAF resulted in significantly elevated P14 EV levels, increased EV marker expression, and higher total protein content, while KRAS knockdown was additionally associated with a shift toward larger particle sizes. Downregulation of CTNNB1 increased P14 and decreased P100 EV secretion, whereas CDH1 knockdown reduced P14 EV levels and slightly increased P100 EVs. No general distinction between tumor suppressor genes and (proto-)oncogenes regarding their effects on EV secretion was observed, and cell viability was not significantly altered under the experimental conditions. Conclusions: These findings suggest that components of the Ras/Raf/MAPK and Wnt signaling pathways may contribute to the regulation of EV secretion in colorectal cancer cells.