Extracellular vesicles from prostate tumors reshape the pre-metastatic bone environment in an mTOR/RAB1A-dependent manner

Tingting LvYawen GuoYuehua ZhangJing CaoXing LiDehua WangXiaokuan ZhangDongwei HeXiaojin GuoChunwang YangZhiyu Wang^*

• Fourth Hospital of Hebei Medical University, Shijiazhuang, China

Background: Bone is the most common metastatic site in prostate cancer (PCa) patients and serves as a key contributing factor to the poor prognosis observed in advanced-stage patients. Mammalian target of rapamycin (mTOR) inhibition has limited clinical efficacy, potentially due to pathway complexity. Prior to the colonization by tumor cells, primary PCa cells actively remodel the bone microenvironment through the secretion of mediators including extracellular vesicles (EVs). The objective of this research is to investigate the regulatory mechanisms of EV biogenesis and the effects of EVs on the bone pre-metastatic niche (PMN), offering a novel therapeutic strategy against bone metastasis. Methods: PCa cell lines were employed to detect mTOR and Ras-related protein Rab-1A (RAB1A) protein expression levels via Western blotting (WB). Functional assays (invasion and proliferation) were used to validate the impact of RAB1A expression on biological behavior. The biological characteristics of EVs were characterized using WB, nanoparticle tracking analysis, and transmission electron microscopy. Bone marrow cell subpopulation alterations were analyzed based on the GSE143791 single-cell dataset. Cells and animal models were treated with EVs to assess their effects on the bone marrow microenvironment, survival time, and bone metastatic burden. Finally, peripheral blood routine parameters were compared in patients with or without bone metastasis. Results: Utilizing PCa cell lines, we demonstrated that mTOR activation inhibits the ubiquitination activity of the oncogenic factor RAB1A, thereby stabilizing its expression. The EVs derived from tumor promoted bone immunosuppression via B-cell dysfunction and myeloid cell expansion, highlighting their role in PMN formation. In RAB1A-overexpressing PCa animal models, GW4869-mediated inhibition of EV secretion prolonged mice survival, ameliorated bone marrow abnormalities, enhanced B-cell activation capacity, and reduced regulatory B-cell proportions. Conclusions: Our findings elucidated the detailed mechanism by which mTOR/RAB1A regulates EV secretion, providing new insight into cellular changes involved in PMN formation and a theoretical basis for the inhibition of the PMN in the development of targeted therapies for PCa. RAB1A represents a therapeutic target to reverse tEV-mediated immunosuppression, while peripheral B-cell dynamics provide diagnostic biomarkers for early metastasis detection.