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

Ben Lanning; Jason Webber; Pinar Uysal-Onganer; Wen Guo Jiang; Aled Clayton; Dafydd Alwyn Dart

doi:10.3390/biology10040318

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

Biology (Basel). 2021 Apr 10;10(4):318. doi: 10.3390/biology10040318.

Authors

Ben Lanning^{1

2}, Jason Webber^{2

3}, Pinar Uysal-Onganer⁴, Wen Guo Jiang¹, Aled Clayton², Dafydd Alwyn Dart^{1

5}

Affiliations

¹ Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff CF14 4YS, UK.
² Tissue Microenvironment Group, Division of Cancer & Genetics, School of Medicine, Cardiff University, Cardiff CF14 4YS, UK.
³ Institute of Life Science, Swansea University Medical School, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
⁴ School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK.
⁵ Institute of Medical and Biomedical Education, St George's University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK.

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.

Keywords: bone; cancer; metastasis; prostate.