Multiple myeloma disrupts the TRANCE/ osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression

Abstract

Bone destruction, caused by aberrant production and activation of osteoclasts, is a prominent feature of multiple myeloma. We demonstrate that myeloma stimulates osteoclastogenesis by triggering a coordinated increase in the tumor necrosis factor-related activation-induced cytokine (TRANCE) and decrease in its decoy receptor, osteoprotegerin (OPG). Immunohistochemistry and in situ hybridization studies of bone marrow specimens indicate that in vivo, deregulation of the TRANCE-OPG cytokine axis occurs in myeloma, but not in the limited plasma cell disorder monoclonal gammopathy of unknown significance or in nonmyeloma hematologic malignancies. In coculture, myeloma cell lines stimulate expression of TRANCE and inhibit expression of OPG by stromal cells. Osteoclastogenesis, the functional consequence of increased TRANCE expression, is counteracted by addition of a recombinant TRANCE inhibitor, RANK-Fc, to marrow/myeloma cocultures. Myeloma-stroma interaction also has been postulated to support progression of the malignant clone. In the SCID-hu murine model of human myeloma, administration of RANK-Fc both prevents myeloma-induced bone destruction and interferes with myeloma progression. Our data identify TRANCE and OPG as key cytokines whose deregulation promotes bone destruction and supports myeloma growth.

Figure 1

Marrow infiltration by MM is associated with increased TRANCE and decreased OPG expression. (A) Representative images of MM and normal bone marrow following immunohistochemistry (IHC, light field ×600) and in situ hybridization (ISH, dark field ×200). Intense staining by a TRANCE-specific antibody and increased hybridization by a TRANCE-specific riboprobe are seen in MM, but not normal marrow. In contrast, staining by OPG-specific antibody is dramatically decreased in MM compared with normal marrow. (B) Graphic display of TRANCE and OPG protein expression. Bone marrow biopsies from 14 MM and 13 non-MM patients (three MGUS, two NHL, one chronic lymphocytic leukemia, one chronic myelogenous leukemia, one Hodgkin's disease, and five normal) were evaluated for expression of TRANCE and OPG by IHC and independently graded by two investigators without knowledge of the diagnosis. TRANCE staining was scored on a scale from 0 to 100 based on the percentage of positive cells in foci of increased staining. The mean scores for TRANCE expression in MM and non-MM samples were 26 and 4.5, respectively (P < 0.001, Mann–Whitney test). OPG expression was graded on a 0 (none) to 4 (heavy) scale based on the number and intensity of cells stained. The mean scores for OPG expression in MM and non-MM samples were 1.0 and 2.6, respectively (P < 0.001, Mann–Whitney test).

Figure 2

MM triggers OC development through deregulation of the TRANCE–OPG cytokine axis. (A) MM stimulates stromal expression of TRANCE. Primary murine stromal cells were cultured for 4 days without (lane 1) or with (lanes 2–6) each of five MM cell lines (10⁵ cells/ml). Stromal cell mRNA was isolated and TRANCE expression was determined by RT-PCR by using primers specific for murine TRANCE and β-actin. (B) Media conditioned by MM–stroma coculture stimulates stromal expression of TRANCE. Primary murine stromal cells were cultured for 4 days without (lane 1) or with (lanes 2–6) media condition by the coculture of stroma with each of five MM cell lines. Stromal TRANCE mRNA expression was determined as above. (C) MM inhibits stromal expression of OPG. MG63 cells were cultured without (lane 1) or with (lanes 2–6) five MM cell lines (10⁵/ml). After 4 days, stromal RNA was isolated and subjected to Northern analysis by using ribosomal and OPG-specific riboprobes. (D) MM inhibits TGF-β-induced expression of OPG. U2OS cells were stimulated with TGF-β1 (200 nM) in the absence (lane 2) or presence (lanes 3–6) of four MM cell lines (10⁵/ml). After 4 days, stromal mRNA was isolated and OPG expression determined by RT-PCR by using primers specific for OPG and β-actin. (E) MM subverts OPG function in vitro. TRANCE (1 μg/ml) triggers the development of OCs from precursors present in CSF-1-treated murine marrow (column 2). OPG (1 μg/ml) inhibits TRANCE-induced osteoclastogenesis (column 3). Human MM cell lines ARH-77, U266, H929, RPMI 8226, and ARP-1 partially overcome the suppressive effect of OPG (columns 4–8). Osteoclastogenesis is assessed semiquantitatively by using a colorimetric assay for TRAP with results expressed as OD_405 nm. (F) MM-induced osteoclastogenesis is TRANCE dependent. Coculture of primary stroma with MM (ARP-1) triggers the generation of OCs from CSF-1-treated murine marrow (BM) (column 2). OCs fail to develop in the absence of MM (column 1), in the presence of 1 μg/ml RANK-Fc (column 3), or if TRANCE-deficient mice (TRANCE −/−, column 4), rather than wild-type littermates, are used as the source of stromal cells. OCs do not develop in the absence of marrow or stroma (not shown).

Figure 3

TRANCE inhibition by RANK-Fc blocks MM-induced bone destruction in two murine models. Irradiated (200 cGy) SCID mice were injected intravenously with 10⁶ cells of the human MM line ARH-77. Intravenous administration of either RANK-Fc or hIgG₁(λ), both at 200 μg three times weekly, began the following day. At 3 and 6 weeks, both groups had comparable titers of hIgG₁(κ), the antibody produced by the ARH-77 cell line. (A) Urinary excretion of crosslinked Dpd in SCID/ARH-77 mice. Bone turnover was assessed at 0, 3, and 6 weeks by measuring urinary excretion of Dpd. At 6 weeks, mice treated with RANK-Fc exhibited significantly less bone turnover (P < 0.01, Student's t test). (B) Osteolysis in SCID/ARH-77 mice. Osteolysis was evident in hIgG₁ but not RANK-Fc-treated animals. Representative radiographs taken following 6 weeks of therapy are shown. (C) Incidence of hind limb paralysis in SCID/ARH-77 mice. Over 7 weeks, four of the five mice that received hIgG₁ developed hind limb paralysis as a result of vertebral bone destruction, whereas none of the mice treated with RANK-Fc developed paralysis (P < 0.01, Student's t test). (D) Xenograft osteolysis in SCID-hu–MM mice. SCID-hu mice inoculated with primary MM were treated with either RANK-Fc or hIgG₁ (200 μg three times weekly). Injections began when titers of MM paraprotein were detected in both mice of a pair, the mouse with the higher titer receiving RANK-Fc. Osteolysis of the xenograft was evident in hIgG₁ but not RANK-Fc-treated animals. Radiographs taken before (PRE) and following (POST) 8 weeks of therapy are shown for one pair of mice. (E) Osteoclastogenesis in the xenografts of SCID-hu–MM mice. Xenografts from SCID-hu–MM mice were removed after treatment with either hIgG₁ or RANK-Fc and stained for TRAP. Xenografts taken from RANK-Fc recipients had significantly fewer TRAP+ multinucleated giant cells (OCs) per mm² than did xenografts taken from hIgG₁ recipients (P < 0.001, Student's t test).

Figure 4

Effect of RANK-Fc on primary MM in SCID-hu mice. (A) Reduction in MM paraprotein. SCID-hu–MM mice demonstrate significantly less MM paraprotein when treated with RANK-Fc (P < 0.01, Student's t test), whereas paraprotein levels in SCID/ARH-77 mice are not affected by RANK-Fc treatment. For comparison among animals inoculated with MM from different sources, paraprotein levels for RANK-Fc-treated animals are presented as the percentage (mean ± SE) of the paraprotein level measured in the hIgG₁-treated animals. (B) OPG and TRANCE expression in xenografts from SCID-hu–MM mice (IHC, ×600). Immunohistochemistry performed on xenografts harvested from SCID-hu–MM mice after 8 weeks of treatment demonstrates normalization of TRANCE and OPG expression in the xenografts taken from RANK-Fc-treated animals.