Small extracellular vesicle-mediated ITGB6 siRNA delivery downregulates the alpha V beta 6 integrin and inhibits adhesion and migration of recipient prostate cancer cells

The alpha V beta 6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the beta 6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the alpha V beta 6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro, the androgen receptor-negative PrCa cells express high levels of the alpha V beta 6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the beta 6 but not alpha V subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the alpha V beta 6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6-targeting siRNAs into PC3 cells specifically downregulates expression of the beta 6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an alpha V beta 6-specific substrate, LAP-TGF beta 1. Our results demonstrate an approach for specific targeting of the alpha V beta 6 integrin in PrCa cells using sEVs encapsulating ITGB6-specific siRNAs.