Identification of Fibulin-1 as a Human Bone Marrow Stromal (HS-5) Cell-Derived Factor That Induces Human Prostate Cancer Cell Death

BACKGROUND: Previous studies showed that human bone marrow stromal HS-5 cells secreted unidentified factor(s) inducing PCa cell death. Herein, the HS-5-derived factor (HS-5 DF) was characterized and identified. METHODS: Conditioned media from confluent HS-5 cells were collected and modified for biochemical characteristic testing of HS-5 DF. Cell survival was measured by apoptosis assay and live/dead assay. Fibulin-1 was identified from gel electrophoresis and mass spectrometry. The validation of Fibulin-1 as a HS-5 DF was done by immunoprecipitation (IP) and genetic knockdown by CRISPR/Cas9 system. RESULTS: HS-5 DF was trypsin and heat sensitive, but pH stable. The tentative size of the factor fell between 30 kDa and 100 kDa. TGF-β1 treatment led to a suppression of HS-5 DF activity, a property consistent with bone metastasis in prostate cancer. Examination of TGF-β1 down regulated proteins led to identification of fibulin-1 as a candidate for the DF. IP of Fibulin-1 from HS-5 CM and CRISPR knockdown of Fibulin-1 showed a significant reduction of HS-5 CM-derived PCa cell death. These results strongly support a role for fibulin-1 in HS-5 bone marrow stromal cell induction of PCa cell death. CONCLUSION: Our data indicate that Fibulin-1 functions as a HS-5 bone marrow stromal cell-derived factor inducing prostate cancer cell death. Prostate 77:729–742, 2017.