Fig 1.
Soluble OPN is produced in the bone microenvironment and enhances human breast cancer cell migration.
(A) Goat anti-mouse OPN primary antibody was incubated for 20 min at RT with DynaBeads® Protein G prior to incubation with BMCM for 30 min at RT. The beads-antibody-antigen complex was removed with a DynaMag™-2 magnet. Concentration of OPN in BMCM, BMCM with beads only (no antibody) and BMCM depleted of OPN (ΔOPN) was assessed by ELISA. Data are presented as mean ± SEM (N = 3). (B) MDA-MB-231 and (C) SUM-159 cells were subjected to transwell migration assays (5 x 104 cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito+), BMCM, BMCM depleted of OPN (ΔOPN) or BMCM ΔOPN rescued with GST-hOPN. Plates were incubated at 37°C, 5% CO2 for 18 hr, fixed and stained. Five high-powered fields of view (HPF) were captured per transwell and migrated cells were analyzed. Data are presented as mean ± SEM (N = 3; fold-change from negative control basal media). * = significantly different than basal media; ϕ = significantly different than BMCM (P≤0.05).
Fig 2.
Bone-derived OPN enhances the migration and stem-like behavior of ALDHhiCD44+CD24- breast cancer cells.
(A) ALDHhiCD44+CD24- and ALDHloCD44-CD24+ cell subpopulations were isolated from the MDA-MB-231 human breast cancer cell line by FACS and subjected to transwell migration assays (5 x 104 cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito+), BMCM, or BMCM depleted of OPN (ΔOPN). Plates were incubated at 37°C, 5% CO2 for 18 hr, fixed and stained. Five high-powered fields of view (HPF) were captured per transwell and migrated cells were analyzed. (B) Whole population or (C) ALDHhiCD44+CD24- and ALDHloCD44-CD24+ subpopulations isolated from the MDA-MB-231 breast cancer cell line were plated in a limiting dilution fashion on 96-well ultra-low attachment plates for 7 days in basal media, BMCM, or BMCM ΔOPN and subjected to sphere-forming assays. (D) Whole population MDA-MB-231 cells were plated in a limiting dilution fashion on normal 96-well plates for 7 days in basal media, BMCM, or BMCM ΔOPN and subjected to colony-forming assays. Data are presented as mean ± SEM (N = 3). * = significantly different than basal media; ϕ = significantly different than BMCM (P≤0.05).
Fig 3.
Bone-derived OPN interacts with CD44 and RGD-dependent integrins to enhance breast cancer cell migration.
MDA-MB-231 and SUM-159 human breast cancer cells were blocked with an anti-CD44 antibody (A, B) or an RGD sequence-specific blocking peptide (C, D) for 30 min and subjected to transwell migration assays (5 x 104 cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito+), BMCM, or BMCM depleted of OPN (ΔOPN). Plates were incubated at 37°C, 5% CO2 for 18 hr, fixed and stained. Five high-powered fields of view (HPF) were captured per transwell and migrated cells were analyzed. Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). * = significantly different than basal media; ϕ = significantly different than non-blocked BMCM, δ = significantly different than non-blocked BMCM ΔOPN (P≤0.05).
Fig 4.
Breast cancer cell stem-like behavior is mediated by bone-derived OPN through CD44 and RGD-dependent integrins.
MDA-MB-231 human breast cancer cells were blocked with (A) an anti-CD44 antibody or (B) an RGD sequence-specific blocking peptide for 30 minutes prior to plating in a limiting dilution fashion on ultra-low adhesion 96-well plates for 7 days in basal media (DMEM/F12 + Mito+), BMCM or BMCM ΔOPN in the sphere limiting dilution assay (SLDA). Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). * = significantly different than basal media; ϕ = significantly different than non-blocked BMCM (P≤0.05).
Fig 5.
Bone-derived OPN induces phosphorylation of WNK1 and PRAS40.
MDA-MB-231 human breast cancer cells were exposed to basal media, BMCM and BMCM depleted of OPN (BMCM ΔOPN) for 2 hours, and cell lysates were harvested. (A) Cell lysates were assessed using with Human Phospho-Kinase Array membranes (ARY003B, R&D Systems) overnight at 4°C. Biotinylated detection antibodies were applied and membranes were visualized using chemiluminescence. Densitometry analysis was performed using the Protein Array Analyzer for ImageJ (N = 3 for each media condition). * = significantly different than basal media; ϕ = significantly different than BMCM (P≤0.05). Only proteins with significantly different phosphorylation or expression between at least two treatments are shown. Proteins within the rectangular box demonstrated a similar pattern of phosphorylation to each other (increase in response to BMCM, with a subsequent decrease upon depletion of bone-derived OPN). (B) Phosphorylation patterns for PRAS40 and WNK1 were successfully validated using immunoblotting (N = 3).
Fig 6.
WNK1 and PRAS40-related pathways play a role in BMCM-mediated migration of breast cancer cells.
(A, B) MDA-MB-231 human breast cancer cells were subjected to siRNA knockdown of WNK1 (A) or PRAS40 (B). Knockdown was confirmed by immunoblotting (top panels) prior to carrying out transwell migration assays (bottom panels; 5 x 104 cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito+) or BMCM. (C) MDA-MB-231 human breast cancer cells were serum-starved and treated with 20μM of PI3K inhibitor (LY294002) or Akt inhibitor (Triciribine) or an equivalent concentration of vehicle (DMSO) for 1 hour before being subjected to transwell migration assays (5 x 104 cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito+) or BMCM. Plates were incubated at 37°C, 5% CO2 for 18 hr, fixed, and stained. Five high-powered fields of view (HPF) were captured per transwell and migrated cells were analyzed. Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). * = significantly different than basal media; ϕ = significantly different than BMCM + siCON (A, B) or BMCM + vehicle (C) (P≤0.05).