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

SIMPLE SUMMARY: Prostate cancer frequently metastasizes to the bone, where it forms primarily osteoblastic lesions. Currently there is no real therapeutic option for this late stage of disease, and understanding prostate cancer-bone interaction and communication is vital. Using a simple in vitro model of os-teoblast differentiation and mineralization, we studied this interaction and observed that prostate cancer cells secreted large quantities of extracellular vesicles containing microRNAs. When ex-posed to the extracellular vesicles, increased osteoblast differentiation and mineralization could be observed, and upon RNA-seq several of these microRNAs were implicated as upstream regulators of the mineralization process. These microRNAs also correlated with poor survival in online analysis of patient datasets. We characterized and validated four genes known to be targeted by microRNA-16, and found that extracellular vesicles could deliver miR-16, and increase minerali-zation. ABSTRACT: Skeletal metastases are the most common form of secondary tumour associated with prostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to the devel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in bone envi-ronments governs both the formation/development of the associated lesion, and growth of the secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and their conditioned medium could stimulate and increase mineralisation and osteoblasts’ differentiation. Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included small extracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, we profiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 was subsequently 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 of gene downregulation and an increased mineralisation. Ingenuity® Pathway Analysis (IPA(®)) revealed that 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 sEV miR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression of PLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16 is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. The mechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa.