Citations

AbstractDeletion of TGFβ inducible early gene‐1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl‐Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl‐Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl‐Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl‐Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl‐Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX‐1 serum levels were unaffected by Scl‐Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl‐Ab therapy elicits potent bone‐forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice.

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage–specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.

Abstract Background: Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We and others have previously demonstrated that endoxifen is the most active tamoxifen metabolite responsible for eliciting the anti-cancer effects of this drug and that endoxifen concentrations are an important factor with regard to tamoxifen efficacy. These studies have led to the development of endoxifen as a novel anti-breast cancer drug for which phase I clinical trials are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast gene expression profiles were compared to that of estrogen, tamoxifen, raloxifene and lasofoxifene by microarray and RT-PCR analyses in both estrogen receptor alpha (ERα) and ERβ expressing cell lines. The in vivo effects of an anti-cancer dose of endoxifen (50mg/kg/day) on the skeleton were first analyzed in 3-month-old ovariectomized C57BL/6 mice using Dual-energy X-ray absorptiometry, peripheral Quantitative Computed Tomography, micro-Computed Tomography and histomorphometry. In a second set of studies, a pre-clinical rat model was used to determine the effects of endoxifen (10mg/kg/day) on the skeleton in both a pre- and post-menopausal setting. Results: Endoxifen treatment of ERα and ERβ expressing mouse osteoblast cells led to dramatically different gene expression profiles when compared to that of estrogen and other anti-estrogens. In ovariectomized mice, daily administration of endoxifen led to significant increases in bone mineral density and content throughout the skeleton relative to vehicle control treated animals. The numbers and activity of both osteoblasts and osteoclasts were also found to be significantly higher in endoxifen treated mice. In the pre-clinical model system, endoxifen treatment of 4 month-old ovariectomized Sprague-Dawley rats significantly protected against bone loss following estrogen depletion primarily due to suppression of osteoclast mediated bone resorption. Importantly, in sham operated rats (thus retaining ovarian function), endoxifen treatment enhanced bone volume and trabecular thickness and did not suppress osteoclast activity. Conclusions: These data are the first to examine the effects of the novel breast cancer therapy, endoxifen, on bone and reveal that the molecular mechanisms of action of this compound are substantially different than that of other SERMs. Endoxifen was shown to protect against bone loss following estrogen depletion in both mice and rats and interestingly, enhanced bone mass in ovary intact rats, an observation that is in stark contrast to the known effects of tamoxifen which induces bone loss in the “pre-menopausal” setting. These studies suggest that endoxifen may have superior bone-beneficial effects compared to tamoxifen, and if efficacy is confirmed in later phase trials, endoxifen may represent a better drug of choice for a sub-set of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-01.

Abstract Background Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We have recently demonstrated that endoxifen is the most active tamoxifen metabolite with regard to inhibiting the growth of ERα+ breast cancer cells and these studies have led to the development of endoxifen as a novel anti-breast cancer drug for which first-in-human studies are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast (OB) and osteoclast (OC) maturation and gene expression were monitored by cell differentiation assays and real-time PCR. Dual-energy X-ray absorptiometry (DXA), peripheral Quantitative Computed Tomography (pQCT) and micro-Computed Tomography (μCT) were used to determine changes in bone density, mass and architecture following 45 days of oral endoxifen administration (50mg/kg/day) to 3-month-old ovariectomized (OVX) C57BL/6 mice relative to vehicle control treated animals. Alterations in the numbers and activity of OBs and OCs were determined by histomorphometry and serum levels of P1NP and CTX-1 respectively. Results: Endoxifen treatment of mouse derived bone marrow stromal cells and human OBs led to significant increases in the expression of critical bone marker genes such as Runx2, osterix, osteocalcin, osteoprotegerin and alkaline phosphatase in a dose dependent manner. Daily administration of endoxifen to OVX mice led to significant increases in total body bone mineral density (BMD) (6%) and content (BMC) (9%), which was accompanied by a 50% decrease in fat tissue mass as determined by DXA. pQCT analysis of the tibial metaphysis revealed dramatic increases in BMD (35%) and BMC (20%), as well as trabecular density (52%), cortical content (62%), cortical area (60%) and cortical thickness (78%). μCT analysis of the femoral metaphysis revealed increases in bone volume/total volume (200%), trabecular number (38%) and trabecular thickness (18%), as well as decreased trabecular spacing (29%). Interestingly, there was nearly a 50% increase in the numbers of OCs derived from endoxifen treated mice which was associated with elevated expression of OC marker genes such as NFATcl, RANK, c-fms and cathepsin-K compared to control treated animals. Approximately 4 times as many OBs and OCs were observed on the bone surfaces of endoxifen treated mice which correlated with nearly 2-fold increases in serum levels of the bone formation (P1NP) and resorption (CTX-1) markers. Conclusions: These data are the first to demonstrate that endoxifen has anabolic effects on the mouse skeleton which are similar to that of estrogen. Additionally, these data reveal that endoxifen's mechanism of action in bone is different than that reported for tamoxifen and other selective estrogen receptor modulators in mice as it increases, rather than decreases, bone formation and remodeling. Therefore, the use of endoxifen for the treatment of endocrine responsive breast cancer may avoid the detrimental skeletal effects of many conventional endocrine therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-09.

Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with β-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

Abstract 2-Methoxyestradiol (2ME2), a physiologic metabolite of 17β-estradiol (estrogen), has emerged as a promising cancer therapy because of its potent growth-inhibitory and proapoptotic effects on both endothelial and tumor cells. 2ME2 also suppresses osteoclast differentiation and induces apoptosis of mature osteoclasts, and has been shown to effectively repress bone loss in an animal model of postmenopausal osteoporosis. Given these observations, we have examined whether 2ME2 could effectively target metastasis to bone, osteolytic tumors, and soft tissue tumors. A 4T1 murine metastatic breast cancer cell line was generated that stably expressed Far Red fluorescence protein (4T1/Red) to visualize tumor development and metastasis to bone. In an intervention study, 4T1/Red cells were injected into bone marrow of the left femur and the mammary pad. In the latter study, 2ME2 (10, 25, and 50 mg/kg/d) treatment began on the same day as surgery and was continued for the 16-day duration of study. Tumor cell growth and metastasis to bone were monitored and bone volume was determined by micro-computed tomography. 2ME2 inhibited tumor growth in soft tissue, metastasis to bone, osteolysis, and tumor growth in bone, with maximum effects at 50 mg/kg/d. Furthermore, tumor-induced osteolysis was significantly reduced in mice receiving 2ME2. In vitro, 2ME2 repressed osteoclast number by inducing apoptosis of osteoclast precursors as well as mature osteoclasts. Our data support the conclusion that 2ME2 could be an important new therapy in the arsenal to fight metastatic breast cancer. [Cancer Res 2007;67(21):10106–11]