Pharmacological Inhibition of the Skeletal IKKβ Reduces Breast Cancer-Induced Osteolysis

Abstract

IKKβ has previously been implicated in breast cancer bone metastasis and bone remodelling. However, the contribution of IKKβ expressed by bone cells of the tumour microenvironment to breast cancer-induced osteolysis has yet to be investigated. Here, we studied the effects of the verified selective IKKβ inhibitors IKKβ^III or IKKβ^V on osteoclast formation and osteoblast differentiation in vitro and in vivo, human and mouse breast cancer cells' support for osteoclast formation and signalling in vitro and osteolysis ex vivo and in immunocompetent mice after supracalvarial injection of human MDA-MB-231 conditioned medium or intra-cardiac injection of syngeneic 4T1 breast cancer cells. Pre-treatment with IKKβ^III or IKKβ^V prior to exposure to tumour-derived factors from human and mouse breast cancer cell lines protected against breast cancer-induced osteolysis in two independent immunocompetent mouse models of osteolysis and the ex vivo calvarial bone organ system. Detailed functional and mechanistic studies showed that direct inhibition of IKKβ kinase activity in osteoblasts and osteoclasts was associated with significant reduction of osteoclast formation, enhanced osteoclast apoptosis and reduced the ability of osteoblasts to support osteoclastogenesis in vitro. When combined with previous findings that suggest NFκB inhibition reduces breast cancer tumorigenesis and metastasis our present findings have an important clinical implication on raising the possibility that IKKβ inhibitors, as bone anabolics, osteoclast inhibitors as well as anti-metastatic agents, may have advantages over anti-osteoclasts agents in the treatment of both skeletal and non-skeletal complications associated with metastatic breast cancer.

Keywords: Bone; Breast cancer; IKKβ; Osteoclast; Osteoclastogenesis; Osteolysis.

Fig. 1

Inhibition of IKKβ reduces breast cancer—induced osteoclastogenesis and osteolysis. a Graphic representation of mouse calvaria organ co-culture system. b Total bone volume in mouse calvaria bone after exposure CM from human MDA-231 and mouse 4T1 breast cancer cells in the presence and absence of vehicle or the selective IKKβ inhibitors IKKβ^III or IKKβ^V (10 μM). c Representative photomicrographs of microCT scan of mouse calvaria bone from the experiment described in a, b. d Graphic representation of supracalvarial injection in adult immunocompetent mice. e Total bone volume (BV/TV, %) in mouse calvaria bone after exposure to CM from human MDA-231 breast cancer cells in the presence and absence of vehicle or the selective IKKβ inhibitor IKKβ^III (20 mg/kg/day) from the experiment described in panel d (n = 7). Data are mean ± SD, **p < 0.01 from vehicle

Fig. 2

Inhibition of IKKβ prevents RANKL and breast cancer cell-induced osteoclastogenesis. a Number of osteoclasts in mouse M-CSF-dependent pre-osteoclast cultures after exposure to RANKL (100 ng/ml) and/or CM from human MDA-231 breast cancer cells (20% v/v) in the presence of vehicle (0.01% DMSO) or the selective IKKβ inhibitors IKKβ^III or IKKβ^V (3 μM). b Representative photomicrographs of M-CSF-dependent pre-osteoclasts and TRAcP-positive multinucleated osteoclasts from the experiment described in panel a. c In vitro osteoclast apoptosis as evidenced by caspase-3/7 activation in M-CSF- and RANKL-generated mature osteoclast cultures treated with IKKβ^III or IKKβ^V (10 μM) after 6 h. d In vitro osteoclast apoptosis as evidenced by DNA fragmentation in M-CSF- and RANKL-generated mature osteoclast cultures treated with IKKβ^III (10 μM) in the presence or absence of the caspase inhibitor zVAD-fmk (10 μM) after 24 h. Values are mean ± SD; **p < 0.05 and ***p < 0.001 from vehicle; ⁺⁺⁺p < 0.001 from RANKL treated; ⁺p < 0.05 from IKKβ^III plus caspase inhibitor zVAD-fmk

Fig. 3

Inhibition of IKKβ enhances osteoblast maturation and reduces their support for osteoclastogenesis. a Number of osteoclasts in mouse osteoblasts—bone marrow cell cultures after exposure to CM from human MDA-231 breast cancer cells in the presence of vehicle (0.01% DMSO) or the selective IKKβ inhibitors IKKβ^III or IKKβ^V (3 μM). The average number of osteoclasts in vehicle-treated cultures in three independent experiments is 45 ± 6, 51 ± 12 and 72 ± 6 osteoclasts/well. b The mRNA expression of RANKL (left) and OPG (right) in mouse calvarial osteoblasts after exposure to CM from human MDA-231 breast cancer cells in the presence of vehicle (0.01% DMSO) or the selective IKKβ inhibitors IKKβ^III or IKKβ^V (10 μM). c, d Alkaline phosphatase activity (Alk Phos, c) and bone nodule formation (ALZ, d) in mouse calvarial osteoblasts after exposure to CM from human MDA-231 breast cancer cells in the presence of vehicle (0.01% DMSO) or the selective IKKβ inhibitors IKKβ^III or IKKβ^V (1 μM). Representative photomicrographs of osteoblast cultures from the experiment are shown in panel d, right. Data are mean ± SD, **p < 0.01 from vehicle

Fig. 4

The selective IKKβ inhibitors IKKβIII or IKKβV reduce breast cancer-induced IκBα phosphorylation in osteoclasts and osteoblasts. a Western blot analysis of total and phosphorylated of IκB and actin in M-CSF dependent pre-osteoclasts after exposure to CM from human MDA-231 (left) and 4T1 (middle) breast cancer cells (20% v/v) and RANKL (100 ng/ml) in the presence and absence of the selective IKKβ inhibitors IKKβ^III or IKKβ^V (10 µM). b Western blot analysis of total and phosphorylated IκBα and actin in mouse calvarial osteoblasts after exposure to CM from human MDA-231 breast cancer cells (20% v/v) in the presence and absence of the selective IKKβ inhibitors IKKβ^III or IKKβ^V (10 µM)

Fig. 5

Selective IKKβ inhibition reduces breast cancer-induced osteolysis in vivo. a Graphic representation of intra-tibial injection of the mouse breast cancer cells 4T1 in immune-competent BALB/c mice pre-treated with vehicle (DMSO/PBS: 0.01:10) or IKKβ^III (20 mg/kg/day) for 12 days (n = 7). b Total bone volume (BV/TV) in tibial metaphysis of mice from the experiment described in panel a. c Representative photomicrographs of microCT scans of bone from the experiment described in panels a, b. Arrowhead denotes osteolysis. d, e In vivo number of osteoclasts (Oc.N/BS, left) and osteoblasts (Ob.N/BS, right) per bone surface (BS) from the experiment described in panels a–c. Values are mean ± SD; *p < 0.05 and **p < 0.01 from vehicle plus breast cancer cells