Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an In Vitro Model

Skeletal metastases are the most common form of secondary tumour associated withprostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to thedevel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in boneenvi-ronments governs both the formation/development of the associated lesion, and growth ofthe secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and theirconditioned medium could stimulate and increase mineralisation and osteoblasts’ differentiation.Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included smallextracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, weprofiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 wassubsequently higher in EV-treated 7F2 cells and a miR-16 mimic could also stimulate mineralisation.Next, using RNA-seq of extracellular vesicle (EV)-treated 7F2 cells, we observed a large degree ofgene downregulation and an increased mineralisation. Ingenuity®Pathway Analysis (IPA®) revealedthat miR-16-5p (and other miRs) was a likely upstream effec-tor. MiR-16-5p targets in 7F2 cells,possibly involved in osteoblastogenesis, were included for val-idation, namely AXIN2, PLSCR4,ADRB2 and DLL1. We then confirmed the targeting and dow-regulation of these genes by sEVmiR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression ofPLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. Themechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa