Heparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL

Abstract

Excessive bone destruction is a major cause of morbidity in myeloma patients. However, the biological mechanisms involved in the pathogenesis of myeloma-induced bone disease are not fully understood. Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors, including multiple myeloma. We previously showed that heparanase promotes robust myeloma tumor growth and supports spontaneous metastasis of tumor cells to bone. In the present study, we show, for the first time, that the expression of heparanase by myeloma tumor cells remarkably enhances bone destruction locally within the tumor microenvironment. In addition, enhanced heparanase expression in the primary tumor also stimulated systemic osteoclastogenesis and osteolysis, thus mimicking the systemic osteoporosis often seen in myeloma patients. These effects occur, at least in part, as the result of a significant elevation in the expression and secretion of receptor activator of NF-κB ligand (RANKL) by heparanase-expressing myeloma cells. Moreover, analysis of bone marrow biopsies from myeloma patients reveals a positive correlation between the level of expression of heparanase and RANKL. Together, these discoveries reveal a novel and key role for heparanase in promoting tumor osteolysis and show that RANKL is central to the mechanism of heparanase-mediated osteolysis in myeloma.

Figure 1. Elevated expression of heparanase by myeloma cells dramatically enhances osteolysis locally and systemically in animal models

A. Imaging of human bones harvested from the SCID-hu model (7 mice per group). Left panel: X-ray image showing three most highly degraded bones from each group that were injected with either HPSE-low or HPSE-high cells. Numbers below the images are the ng/ml of human kappa light chain present in the murine serum, which reflects the whole animal tumor burden. Middle panel: MicroCT imaging of the same bones shown in the x-ray. Right panel: human bones contralateral to those shown in the left panel. B. Left panel: MicroCT imaging of a primary murine tibiae injected with tumor cells (HPSE-low and HPSE-high) Significant bone destruction is observed only in the tibia bearing HPSE-high tumor cells (red arrow, note loss of trabecular architecture) (5 mice per group). Right panel: MicroCT microarchitectural analyses of contralateral tibiae of mice injected with either HPSE-low or HPSE-high tumor cells. Significant decreases in bone volume/total volume (BV/TV), trabecular number, connectivity density, and increased trabecular spacing are observed n animals bearing HPSE-high tumors, as compared to animals bearing HPSE-low tumors.

Figure 2. Osteoclastogenesis is enhanced in mice bearing HPSE-high tumors

A. TRAP staining was performed on the primary and contralateral bones harvested from SCID-hu mice. The purple-red stained cells are osteoclasts (indicated by arrows; original magnification 200x). B. Numbers of TRAP-positive osteoclasts in human bones harvest from the SCID-hu model were determined. ** p<0.01 for HPSE-high compared with HPSE-low. C. TRAP 5b present in the murine sera is significantly higher in animals bearing HPSE-high tumors compared with HPSE-low tumors.

Figure 3. RANKL expression is elevated in tumors formed by HPSE-high cells

A. Human RANKL staining within human bones from SCID-hu mice bearing tumors formed by HPSE-low or HPSE-high cells. The red asterisks denote bone and inserts are controls lacking addition of primary antibody (original magnification 400x). B. Primary myeloma tumors formed by HPSE-low or HPSE-high cells growing subcutaneously in SCID mice were extracted with detergent and western blotting was performed for detection of RANKL, OPG, and β-actin. Each lane of the blot represents tumor from a different animal. C. The bands from western blots were quantified relative to β-actin using ImageJ software. The bars represent means ± SD.

Figure 4. Heparanase upregulates RANKL expression and secretion in myeloma cells

A. Real-time PCR for human RANKL. The graph is representative of three individual experiments performed in triplicate. B. CAG cells from two different heparanase transfections using different vectors (pIRES2 or pcDNA3) or from heparanase knock-down (HPSE k/d) cells were lysed and probed for human RANKL by Western blotting((HPSE-low (L), HPSE-high (H), HPSE knock-down (k/d)). C. Quantification of the RANKL from Western blots. Each bar graph represents an average reading number of RANKL/β-actin from 3 independent experiments. D. RANKL levels in conditioned medium were determined by ELISA. The bar graph shows the changes in RANKL levels in HPSE-high cells or HPSE k/d cells as the percentage of control. The results (representative of 3 independent experiments) are means ± SD, **p < 0.01 compared to controls.

Figure 5. Recombinant heparanase (rHPSE) induces RANKL expression and secretion in myeloma cells

Equal numbers of human myeloma U266 or MM.1S cells were grown in complete medium in the absence or presence of rHPSE (100 ng/ml, added every 12 hours) for 48 h. The cells were lysed for western blotting, and the conditioned media were harvested for ELISA. A. Western blots were probed with antibody to human RANKL or β-actin. Saos-2 cell lysate was used as a positive control for human RANKL. B. RANKL bands from western blots were quantified using ImageJ and normalized to the corresponding β-actin bands. Each bar graph represents an average quantitative result of two independent experiments with two western blots. C. RANKL ELISA of conditioned media. The graph shows the percentage increase in RANKL present in medium conditioned by cells treated with rHPSE compared to control treated cells. The results are means ± SD from 2 independent experiments, *** p<0.001 to the controls.

Figure 6. Medium conditioned by CAG HPSE-high cells induces osteoclastogenesis via increased RANKL

Human PBMCs were cultured in conditioned medium from CAG cells expressing either low or high levels of heparanase (HPSE-low or HPSE-high) in the presence or absence of OPG. Addition of recombinant human (rh) RANKL was used as a positive control. The negative control was cells cultivated only with DMEM and mCSF. *** p< 0.001 compared to “no OPG” group.

Figure 7. Myeloma tumor cell expression of heparanase correlates with the level of RANKL in the bone marrow of myeloma patients

Heparanase and RANKL staining were performed on 20 of bone marrow biopsy specimens from myeloma patients. Myeloma cells expressing high level of heparanase show elevated RANKL expression (original magnification 400x).