Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms

Abstract

Incidence of skeletal metastases and death from prostate cancer greatly increases with age and obesity, conditions which increase marrow adiposity. Bone marrow adipocytes are metabolically active components of bone metastatic niche that modulate the function of neighboring cells; yet the mechanisms of their involvement in tumor behavior in bone have not been explored. In this study, using experimental models of intraosseous tumor growth and diet-induced obesity, we demonstrate the promoting effects of marrow fat on growth and progression of skeletal prostate tumors. We reveal that exposure to lipids supplied by marrow adipocytes induces expression of lipid chaperone FABP4, pro-inflammatory interleukin IL-1β, and oxidative stress protein HMOX-1 in metastatic tumor cells and stimulates their growth and invasiveness. We show that FABP4 is highly overexpressed in prostate skeletal tumors from obese mice and in bone metastasis samples from prostate cancer patients. In addition, we provide results suggestive of bi-directional interaction between FABP4 and PPARγ pathways that may be driving aggressive tumor cell behavior in bone. Together, our data provide evidence for functional relationship between bone marrow adiposity and metastatic prostate cancers and unravel the FABP4/IL-1β axis as a potential therapeutic target for this presently incurable disease.

Figure 1. Diet-induced marrow adiposity accelerates progression of PC3 tumors in bone

FVB/N/N5 Rag-1^−/− mice were fed normal (LFD, A-F) and high fat (HFD, G-L) diets for 8 weeks followed by intratibial injections of PC3-DsRed cells. The x-ray (A, D, G, J) and 600nm RFP fluorescence (B, E, H, K) imaging 6 weeks post injection (n=6 mice/group). C, F, I, L: H&E staining of tumor-bearing tibiae. M: Box and whisker plot showing percentage of tissue occupied by tumors. Results were analyzed by Mann-Whitney test.

Figure 2. Bone marrow adipocyte-supplied lipids stimulate proliferation and invasion of prostate tumor cells and upregulate genes involved in fatty acid transport

A: DNA assay results for cells grown in collagen I gels in the absence or presence of Adipo CM. B: Images of invasion filters coated with reconstituted basement membrane; cells in the absence (Control) or presence of media conditioned by Bone Marrow Mesenchymal cells (BMMC CM) or adipocytes (Adipo CM) were allowed to invade toward DMEM containing 10% FBS for 48 hours C: Quantitation results of invaded cells shown as % control ± SD. D: BODIPY 493/503 staining of lipid droplets (green) for control and Adipo CM-treated PC3 cells. E: Quantitation of lipid fluorescence (Metamorph). F: Taqman RT-PCR analysis (Life Technologies) of lipid droplet-associated genes: CD36, FABP4 and Perilipin 2 expression in PC3 cells +/− Adipo CM. Data are normalized to 18S. G: BODIPY 493/503 staining of lipid droplets (green) in adipocytes cultured alone (left panels) or in transwell with PC3 cells (right panels). H: Taqman RT-PCR analysis (Life Technologies) of adipocyte-specific gene (FABP4, Adiponectin and Resistin expression) in bone marrow adipocytes cultured alone or in transwell with PC3 cells. Data are normalized to HPRT1.

Figure 3. FABP4, IL-1β, and HMOX-1 are expressed in tumor cells exposed to adipocyte-derived factors in vitro and in bone tumors in vivo

A: Genes upregulated in PC3 cells exposed to Adipo CM in vitro as detected by Taqman RT PCR Human Inflammation Array (Life Technologies). Data are normalized to HPRT1 and GUSB and shown as fold increase relative to control. B, C: Taqman RT-PCR of FABP4, IL-1β, and HMOX-1 in PC3 bone tumors (B) and PC3 subcutaneous tumors (C) from LFD and HFD mice. Data are normalized to HPRT1 and GUSB and shown as fold increase relative to LFD.

Figure 4. Induced expression of FABP4, HMOX-1 and IL-1β and nuclear translocation of FABP4 and HMOX-1 upon exposure of PC3 cells to Adipo CM

A: Western blot for FABP4 (top), and HMOX-1 (middle) expression in PC3 cells grown in the absence or presence of Adipo CM. β-Actin was used as loading control (bottom). B: Cytoplasmic and nuclear fractions of PC3 cells probed for FABP4 (top) and HMOX-1 (top middle), cytoplasmic marker tubulin (bottom middle) and nuclear Lamin A/C (bottom). C: Immunofluorescence staining for FABP4 (top panels) and HMOX-1 (bottom panels) localization. Strong nuclear staining observed in Adipo CM-treated cells. D: Left panel: Immunoblot of intracellular IL-1β expression in PC3 cells. Pro-IL-1β (35 kDa) and active IL-1β (17 kDa) are detected upon exposure to Adipo CM. Right panel: IL-1β ELISA (R&D Systems) analysis of media conditioned by control and Adipo CM-treated PC3 cells. Data are normalized to DNA concentration in corresponding cell lysates and shown as fold increase in pg/ml relative to control.

Figure 5. Chronic exposure to low dose Adipo CM increases invasiveness and induces FABP4, HMOX-1 and IL-1β expression

A: Schematic of long-term culture conditions. Cells are exposed to gradually increasing concentration of Adipo CM (gradient of 5-25%) and maintained in 25% Adipo CM over multiple passages. B: DIC images showing invasive morphology of PC3 cells after chronic Adipo CM exposure. C: Taqman RT PCR for FABP4, IL-1β, and HMOX-1 expression of PC3 cells. Data are normalized to 18S and shown as increase relative to control cultures. D: Invasion assay of PC3 cells treated long-term with control or Adipo CM media for 12 passages. Prior to assay cells were serum starved, seeded in serum free media on top of rBM-coated filter and allowed to invade towards DMEM with 10% FBS for 48 hours. Top panels: Diff-Quik stained invasion filters. Bottom panels: Quantification results showing numbers of invaded cells.

Figure 6. Blocking FABP4/IL-1β axis reduces invasiveness induced by Adipo CM

Diff-Quik stained filters of Control (A), Adipo CM-treated (B), Adipo CM + IL-1β (C), Adipo CM+ FABP4 Inhibitor (D), and Adipo CM + Combination treatments (E). F: Numbers of invaded cells in response to each treatment. Data are representative of at least 3 experiments. P value < 0.05 is considered statistically significant.

Figure 7. FABP4 is strongly expressed in experimental PC3 bone tumors from HFD mice and in bone metastatic tissues from prostate cancer patients

Immunohistochemical analysis of FABP4 expression (Nova Red). A-B: Tumor sections from LFD mice; areas of the tumor with FABP4 positivity indicated by dotted line; C: No primary antibody control; D-F: sections from HFD mice. FABP4 expressed in tumor blood vessels (D, orange arrows); and tumor cells (E), especially in the areas neighboring adipocytes (F, blue arrows); 20 x images. FABP4 immunostaining of TMA sections from normal prostate (G), primary prostate tumor (H), and bone metastatic lesions (J-L). FABP4 expression detected in tumor cells, bone marrow cells and particularly strongly in blood vessels surrounding tumor cells (orange arrows). I: no antibody control. M, N: sections of adipocyte-rich bone marrow strongly positive for FABP4 (yellow arrows).

Figure 8. PPARγ is involved in Adipo CM-induced expression of FABP4, IL-1β, and HMOX-1

A: Western blot analysis of intracellular FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (ROSI, 1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). B: Densitometric analysis of FABP4 (top panel), IL-1β (middle panel) and HMOX-1 (bottom panel) bands normalized to β-actin. C: Taqman RT PCR analysis of FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). Similar increase in FABP4, IL-1β and HMOX-1 expression is observed with ROSI and Adipo CM and expression of FABP4 and IL-1β is reduced with GW9662. D: PPARγ gene expression (Taqman RT PCR) in PC3 cells exposed to Adipo CM. Data are normalized to 18S and GUSB and show a decrease in PPARγ levels with Adipo CM; E: DNA binding assay in nuclear fractions from control and Adipo CM-treated PC3 cells showing reduced PPARγ DNA binding upon Adipo CM treatment. Adipo CM-suppressed gene expression (F) and DNA-binding activity (G) of PPARγ can be restored by GW9662 antagonist or FABP4 inhibitor; Values * p <0.05; ** p <0.01 are considered statistically significant.