The Signaling Axis of β1 integrins in Prostate Cancer Extracellular Vesicle Biology

Intercellular communication has emerged as a crucial mediator of both normal and pathophysiological processes, such as cancer. Extracellular vesicles (EVs) carry many different molecules such as proteins and nucleic acids and play a large role in intercellular communication. EVs have emerged as very important regulators of disease progression through the transfer of biological materials from cancer cells to other cell types. There are many different types of EVs and in this thesis we examined a subset of small EVs (sEVs) isolated from both cell culture media and mouse blood plasma via ultracentrifugation and density gradient separation. The samples isolated only by ultracentrifugation are called EVs, while the samples further purified by density gradient separation are referred to as sEVs, as they are free of any larger, denser vesicles and non-vesicular debris. These sEVs are about 30-150 nm in size, as confirmed by nanoparticle tracking analysis and transmission electron microscopy, and are enriched in tetraspanins such as CD63, CD81, and CD9 and other endosomal-related proteins such as TSG101 and Alix, as confirmed by western blotting. These sEVs are also known as exosomes. Our laboratory has previously demonstrated that sEVs are enriched in integrins, transmembrane receptors, which have previously been implicated in prostate cancer (PrCa), the disease model for all studies in this dissertation. We show here that there are multiple signaling proteins implicated in PrCa progression and the integrin signaling axis, such as Src (and activated SrcpY416), focal adhesion kinase (FAK, and activated FAKpY861), insulin-like growth factor receptor 1 enriched in PrCa sEVs. This study expands on the function of β1 integrins, which contribute to PrCa tumor growth and resistance to radiation therapy. We demonstrated that sEVs from the plasma of a PrCa mouse model (Transgenic Adenocarcinoma of the mouse prostate, TRAMP) and PrCa cell lines stimulate anchorage-independent growth, and that β1 is essential for this function. This was examined in PrCa cells using a β1 knockdown, followed by genetic rescue, and also in TRAMP mice with a β1 genetic ablation specific to the prostate tumor epithelium (β1pc–/– /TRAMP). Upon treatment with β1-positive and FAK-positive (but not β1-negative, FAK-low) EVs, recipient cells also exhibited an increase in FAK, a nonreceptor tyrosine kinase downstream of β1 associated with increased cell migration, growth and anoikis. Upon further examination of the TRAMP mice, we also discovered that β1 also plays a role in sEV secretion, as the β1pc–/– /TRAMP mice had less circulating sEVs in the bloodstream. In sEVs from both β1pc–/– /TRAMP and Wild-Type mice, β1 is also undetectable, along with CD63 and CD9. This indicates that the β1-postive prostate tumor is the source of sEVs with pro-anchorage-independent functions, with the potential to affect tumor growth at metastatic sites as well as the primary. Radiation therapy, a common therapeutic strategy for primary tumors, also decreased the tumorsupportive functions of PrCa-derived EVs. This study sheds light on the importance of β1 in sEV biogenesis and secretion as well as in sEV effects on recipient cells, both at primary and future metastatic tumor sites