It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes tumor growth and angiogenesis. In this study, we demonstrate that c-Src, IGF-IR and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (SrcpY416) is co-expressed in Exo with phosphorylated FAK (FAKpY861), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, SrcpY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo.

Summary The β1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs). We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether β1 integrins are required for this effect. Specifically using a cell-line-based genetic rescue and an in vivo PrCa model, we show that gradient-purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP (transgenic adenocarcinoma of the mouse prostate) mice promote anchorage-independent growth of PrCa cells. In contrast, sEVs from cultured PrCa cells harboring a short hairpin RNA to β1, from wild-type mice or from TRAMP mice carrying a β1 conditional ablation in the prostatic epithelium (β1 pc−/− ), do not. We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express β1 and sEV markers; in contrast, sEVs from β1 pc−/− /TRAMP or wild-type mice lack β1 and sEV markers. Our results demonstrate that β1 integrins in tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth.

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