MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis

Abstract

Bone metastasis is a frequent and incurable consequence of advanced prostate cancer (PC). An interplay between disseminated tumor cells and heterogeneous bone resident cells in the metastatic niche initiates this process. Melanoma differentiation associated gene-9 (mda-9/Syntenin/syndecan binding protein) is a prometastatic gene expressed in multiple organs, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), under both physiological and pathological conditions. We demonstrate that PDGF-AA secreted by tumor cells induces CXCL5 expression in BM-MSCs by suppressing MDA-9-dependent YAP/MST signaling. CXCL5-derived tumor cell proliferation and immune suppression are consequences of the MDA-9/CXCL5 signaling axis, promoting PC disease progression. mda-9 knockout tumor cells express less PDGF-AA and do not develop bone metastases. Our data document a previously undefined role of MDA-9/Syntenin in the tumor and microenvironment in regulating PC bone metastasis. This study provides a framework for translational strategies to ameliorate health complications and morbidity associated with advanced PC.

Keywords: CXCL5; MDA-9/Syntenin; PDGF-AA; bone marrow–derived mesenchymal stromal cell; bone metastasis.

Fig. 1.

Global mda-9/syntenin knockout (mda-9^−/−) animals are innately resistant to bone metastasis. (A) A total of 3 × 10⁴ RM1-BM-Luc cells were injected by the intracardiac route (I.C.) into wild-type C57BL/6 (WT) and mda-9^−/− mice. BLI imaging was performed at day 14 to monitor bone metastasis. Representative BLI images from each experimental group are presented. Luciferase intensity was calculated using built-in Living Image Software v.2.50, and the average value for each group is graphically presented. Region of interest (ROI) was calculated and graphed based on imaging (Middle). Metastatic events in femurs were identified based on BLI imaging at a single time point, and percentage (n = 7) effects are presented (Left). (B and C) Representative ex vivo BLI photomicrographs and corresponding H/E stained sections of different organs from RM1-BM1-Luc implanted (I.C.) WT and mda-9^−/− animals. In C, the tumor is indicated by an arrow. (D) Kaplan–Meier curve showing overall survival of tumor-bearing animals from different genetic backgrounds. *P < 0.05. (E) Schematic diagram for the bone marrow transfusion protocol is described in the upper panel. Briefly, whole bone marrow cells were obtained from WT mice (BM^WT) and injected into age-matched prior irradiated (at 10 Gy) WT and mda-9^−/− mice. The “Sham Transfusion” group received bone marrow culture media only. After 8 wk, all mice were challenged with RM1-BM-Luc (I.C.). Representative ex vivo image (liver and bone) for each experimental group is presented in the lower panel. *Statistically significant (P < 0.05).

Fig. 2.

Lack of MDA-9/Syntenin expression inhibits tumor outgrowth in the bone niche. (A) RM1-BM-Luc cells were directly implanted (into the femur) in 2-mo-old WT and mda-9^−/−mice (Top panel: schematic presentation). Representative images at day 14 are presented (Middle). Luciferase intensity was calculated using built-in Living Image Software v.2.50, and the average value for each group at day 14 is graphically presented (Bottom). Error bars ± SD. (B) Quantification of mineralized tissue by µCT in the distal femur. Femurs were assessed for amount of trabecular bone volume over total volume, bone volume alone, and thickness of the trabeculae in the distal femur. (C) Prevalence of pathologic fracture as observed by µCT. (D) Tumor-implanted bones (from animals studied in panel A) were immunostained for KI-67 expression, and the representative photographs at lower (Left) and higher magnifications (Right) are presented. (E) Bone marrow cells from WT (BM^WT) and mda-9^−/− (BM^mda-9−/−) mice were isolated and cocultured with fluorescently labeled tumor cells (RM1-BM-Luc) and incubated for different time periods. FACS analyses were performed to detect tumor cells at different time points. *Statistically significant (P < 0.05).

Fig. 3.

CXCL5 expression correlates positively with bone metastasis progression. (A) Serum samples were isolated from tumor-bearing mice [14 d after intracardiac (I.C.) injection, 3 × 10⁴ cells/mouse], and protein-based arrays were analyzed as described by R&D Biosystems. Heatmap is presented. Cropped blot for CXCL5 is presented in the bottom panel. (B) CXCL5 level in serum from mice under different experimental conditions (sham/basal level vs. 14-d post-I.C. or postorthotopic implantation (1 × 10⁴ cells/mouse). A mouse-specific CXCL5 enzyme-linked immunosorbent assay (ELISA) kit (R&D Biosystems) was used for determining CXCL5 levels. (C) Bone marrow (BM) cells were isolated from WT (BM^WT) and mda-9^−/− (BM^KO) mice and stimulated with tumor cell–derived conditioned media for 12 h. Total cellular RNA was extracted, and qPCR was performed to detect mouse CXCL5 mRNA. Data are presented as fold-change relative to the unstimulated wild-type (BM^WT) group. Different letters in two variables are statistically significant (P < 0.05). (D) CXCL5 levels in serum from tumor-bearing mice receiving “sham” (represented as “−“) or “WT” (represented as “+”) bone marrow (experiments described in Fig. 1E). *P < 0.01. (E) A total of 3 × 10⁴ RM1-BM-Luc cells were injected by the intracardiac (I.C.) route into WT and mda-9^−/− mice (schematically presented in the left panel). Fourteen days postinjection, total BM cells were isolated and stained for lineage-specific cell surface markers and intracellular CXCL5 expression to identify the specific cell population(s) that differentially respond to tumor cells. Percentages of CXCL5 expression in bone marrow–derived mesenchymal stromal cells: (CD45⁻CD90⁺) are presented. *P < 0.05. (F) Tumor-implanted bones (from animals in Fig. 2A) were immunostained with different antibodies as indicated, and representative photographs are presented.

Fig. 4.

MDA-9 expression in tumor cells nonautonomously activates the Hippo pathway and induces CXCL5 in HS5 cells. (A) Bone metastasis development in athymic nude mice following intracardiac injection of PC-3ML control and mda-9 KO (PC-3ML^{mda-9 KO}) clone (1 × 10⁵ cells/mouse). Representative images 36 d after tumor cell implantation (Left). Bone metastases detected by BLI imaging. The percentage of mice with BLI-positive lesions is shown (Right). (B) HS5 cells were incubated with normalized (equal amount of total protein) tumor cell–derived condition media for 12 h. HS5-derived conditioned media were analyzed for CXCL5 levels using ELISA. (C) HS5 cells were transfected with a 1.5-kb CXCL5-Prom using a standard transfection protocol. Twelve hours posttransfection, cells were stimulated with conditioned media for an additional 12 h. Luciferase activity was measured and presented after normalizing with Renilla luciferase. (D) HS5 cells were incubated with tumor cell–derived conditioned media for 12 h and stained with fluorescently labeled anti-YAP. DAPI was used for nuclear staining. Translocation of YAP into the nucleus was monitored under a fluorescence microscope. (E) HS5 cells were incubated with tumor cell– (parental and its mda-9 knockout variant) derived conditioned media for 12 h, and cell lysates were analyzed for different proteins, as indicated. (F) MDA-9/Syntenin expression was knocked down in HS5 cells using an Adenovirus expressing shmda-9. Twenty-four hours postinfection, culture media were replaced with fresh media containing PC-3ML CM for an additional 12 h. Cell lysates were analyzed for the indicated proteins. Different letters in two variables are statistically significant (P < 0.05).

Fig. 5.

Hippo pathway activates CXCL5 in HS5 cells. (A) Panel i Schematic representation of the experimental protocol used in panels ii–iv. Different overexpression/siRNAs (GOF/LOF) constructs were transfected into the indicated cells, and after 24 h, tumor-derived condition medium was used to stimulate HS5 cells for an additional 12 h. Panels ii and iii represent CXCL5 expression in HS5-derived conditioned media, determined by ELISA. Panels iv and v show CXCL5 promoter activity. In this experiment, HS5 cells were cotransfected with both CXCL5-Prom and either shYAP1 or YAP1 OE vectors before treating with conditioned media. *=P <0.05. (B) YAP expression was analyzed in paraffin sections (tumor-bearing bones from the experiment performed in Fig. 2A). (C) HS5 cells were incubated with different fractions of PC-3ML-derived conditioned media for 24 h after initial transfection with a CXCL5-Prom. Luciferase activity was measured and presented after normalizing with Renilla luciferase. *P <0.05.

Fig. 6.

Tumor cell–derived PDGF-AA induces CXCL5 expression in HS5 cells. (A) Normalized (based on equal amounts of total protein) conditioned media, isolated from parental and knockout variants, were subjected to protein-based growth factor arrays as described by Ray Biotech. Heatmap is presented. Densitometry analyses (n = 2, for corresponding proteins) were performed for each growth factor, and the data are presented only for three. (B) HS5 cells were treated for the indicated times with PDGF-AA, and CXCL5 protein expression was determined. (C) HS5 cells were transfected with a 1.5-kb CXCL5-Prom using a standard transfection protocol. Twelve hours posttransfection, cells were treated with PDGF-AA for an additional 12 h. Luciferase activity was measured and presented after normalizing with Renilla luciferase. (D) PC-3ML-derived conditioned media were incubated with control IgG or anti-PDGF-AA before treatment of HS5 cells for 12 h, and CXCL5 levels were measured in media. (E) Expression of PDGF-AA was determined in the indicated tumor cells and corresponding mda-9 knockout clones. (F) Cells were infected with an Adenovirus expressing control or mda-9 overexpressing construct for 24 h, and PDGF-AA expression in media was determined using ELISA. (G) HS5 cells were treated with PDGF-AA and analyzed for expression of the indicated proteins. (H) Tumor cells were cultured in the presence or absence of IKK2i for 12 h and analyzed for expression of the indicated proteins. Different letters in two variables are statistically significant (P < 0.05). (I) MDA-9 was knocked-into PC-3ML^{mda-9 KO} cells and cultured for an additional 12 h in the presence or absence of IKK2i. PDGF-AA was determined using ELISA. *=P < 0.05.

Fig. 7.

MDA-9 expression in HS5 regulates PDGF/PDGFRα signaling. (A) mda-9 expression was blunted, and 24 h postinfection, HS5 cells were stimulated with PDGF-AA for an additional 12 h. mRNA for CXCL5 expression was analyzed using qPCR. Different letters in two variables are statistically significant (P < 0.05). (B) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media for 12 h, and western blotting was done for phosho-PDGFRα and total PDGFR. (C) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media and PDGF-AA for 12 h, and western blotting was done for the indicated proteins. (D) BM-MSCs from WT and mda-9^−/− mice were stimulated with mouse PDGF-AA, and CXCL5 expression was determined in the culture media. (E) phospho-PDGFR expression was analyzed in paraffin sections (tumor-bearing bone from the animal experiment, described in Fig. 2A). Different letters in two variables are statistically significant (P < 0.05).

Fig. 8.

CXCL5 is expressed in both stroma and tumor cells. (A) Paraffin-embedded section from a patient with metastatic PC bone metastasis was immunostained with anti-CXCL5, and the expression in the stroma and tumor compartment is presented in the Inset. (B) H-Score was determined by an anatomic pathologist based on staining intensity, and values are presented. (C) PC3-ML cells were stained with CytoTrack™ Red and cocultured with of HS5 cells. After 24 h, cells were stained with CXCL5-FITC and analyzed by FACS [the left panel represents double staining (tumor cell-Red and CXCL5-FITC)]; the middle panel is for only CXCL5-FITC (only HS5). The right panel represents conditioned media that were collected from the above indicated PC3-ML and HS5 cocultured cells, and secretion of CXCL5 was measured by ELISA. (D) Human serum samples from the indicated patient groups were analyzed for PDGF-AA and CXCL5 (n = 20). Different letters in two variables are statistically significant (P < 0.05). *Statistically significant. **P < 0.001.

Fig. 9.

Schematic of the proposed role of MDA-9 in BM-MSCs in creating a favorable environment for metastatic outgrowth through secretion of CXCL5 in response to tumor cell–derived growth factor, PDGF-AA.