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

<b>Background</b>: 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. <b>Methods</b>: 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× <i>g</i> or 100,000× <i>g</i>, 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. <b>Results</b>: With few exceptions, knockdown of the investigated genes led to an increase in EV secretion. Silencing of <i>KRAS</i> and <i>BRAF</i> resulted in significantly elevated P14 EV levels, increased EV marker expression, and higher total protein content, while <i>KRAS</i> knockdown was additionally associated with a shift toward larger particle sizes. Downregulation of <i>CTNNB1</i> increased P14 and decreased P100 EV secretion, whereas <i>CDH1</i> 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. <b>Conclusions</b>: 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.