Overview of Wnt signalingThe Wnt family of glycoproteins signals through a "canonical" or "noncanonical" pathway ( Figure 1). In the absence of Wnt's, glycogen synthase kinase-␤ (GSK3␤), Axin, adenomatous polyposis coli (APC), and casein kinase I␣ (CKI␣) form a ␤-catenin destruction complex. 1 CKI␣ phosphorylates ␤-catenin at Ser45, and then GSK3␤ phosphorylates ␤-catenin at Ser33/Ser37/Thr41. Phosphorylated ␤-catenin is recognized by ␤-transducin repeat protein, ubiquitinated, and degraded by proteasomes. In contrast, activation of canonical Wnt signaling occurs when a Wnt protein binds to one of the 10 members of the frizzled (FZD) receptor family; Wnt-FZD then binds low-density lipoprotein-related protein-5 (LRP5) or LRP6. The resultant complex activates Dishevelled (Dvl), a protein that attracts Axin away from the destruction complex and antagonizes the ability of GSK3␤ to phosphorylate ␤-catenin, thereby preventing destruction complex formation. If ␤-catenin is not degraded, it translocates to the nucleus where it binds to the transcription factor T-cell factor-4 (TCF4) and enhances target gene expression. 1 Canonical Wnt's promote caveolin-dependent LRP internalization and facilitate its interaction with Axin. 2 In contrast, DKK1 binds to LRP5/6 causing the receptor to attract a Kremen; this interaction promotes clathrin-mediated internalization, thereby inactivating LRP, though some data suggest that Kremen may not be essential for DKK1-mediated Wnt inhibition. 3,4 Moreover, R-Spondins amplify the activity of canonical Wnt's while antagonizing DKK1-mediated interaction with LRP and Kremen. 5 Wise and SOST are also secreted Wnt inhibitors that bind to and inactivate LRP. 6,7 Wnt inhibitory factor (WIF) proteins, which are structurally similar to the extracellular portion of the Derailed/Ryk class of transmembrane Wnt receptors, and secreted forms of frizzled proteins (sFRP, sizzled, and FrzB) act by directly binding Wnt molecules and can function as Wnt inhibitors, but may also stabilize Wnt's and facilitate Wnt signaling. 8,9 DKK1 regulates bone development and accrual and maintenance of bone mass Bone marrow-derived mesenchymal stem cells (MSC) can differentiate into adipocytes, chondrocytes, or osteoblasts. Although the precise orchestration of Wnt signaling during bone development is dependent on complex microenvironmental cues, data from several groups suggest that Wnt3a, Wnt5a, Wnt7b, and Wnt10b are central to osteoblast differentiation (Figure 2). 10-14 Increased ␤-catenin is found in cells committed to the osteoblast lineage, and loss of ␤-catenin in osteoblast precursor cells results in reduced bone deposition. 15 In addition to promoting osteoblast commitment, canonical Wnt signaling inhibits adipocyte differentiation, primarily through accrual of stabilized ␤-catenin and subsequent transactivation of TCF-responsive genes. 16,17 However, noncanonical Wnt signaling through Wnt5a inactivates peroxisome proliferatoractivated receptor-␥ (PPAR␥), a key adipogenic transcription factor and activates Ru...

Background Osteoblastic bone metastasis is the predominant phenotype observed in prostate cancer patients and is associated with high patient mortality and morbidity. However, the mechanisms determining the development of this phenotype are not well understood. Prostate cancer cells secrete several osteogenic factors including Wnt proteins, which are not only osteoinductive but also oncogenic. Therefore, the purpose of the study was to investigate the contribution of the Wnt signaling pathway in prostate cancer growth, incidence of bone metastases and osteoblastic phenotype of bone metastases. The strategy involved overexpressing the Wnt antagonist, DKK-1, in the mixed osteoblastic and osteolytic Ace-1 prostate cancer cells. Methods Ace-1 prostate cancer cells stably expressing human DKK-1 or empty vector were established and transduced with lentiviral yellow fluorescent protein (YFP)-luciferase (Luc). The Ace-1/vectorYFP-LUC and Ace-1/DKK-1YFP-LUC cells were injected subcutaneously, intratibially, or in the left cardiac ventricle in athymic mice. Results Unexpectedly, DKK-1 significantly increased Ace-1 subcutaneous tumor mass and the incidence of bone metastases after intracardiac injection of Ace-1 cells. DKK-1 increased Ace-1 tumor growth associated with increased phospho46 JNK by the Wnt noncanonical pathway. As expected, DKK-1 decreased the Ace-1 osteoblastic phenotype of bone metastases, as confirmed by radiographic, histopathological, and microcomputer tomographic analysis. DKK-1 decreased osteoblastic activity via the Wnt canonical pathway evidenced by an inhibition of T-cell factor (TCF) activity in murine osteoblast precursor ST2 cells. Conclusion The present study showed that DKK-1 is a potent inhibitor of bone growth in prostate cancer-induced osteoblastic metastases.

The Ace-1 xenograft is a useful model for investigating the pathogenesis of prostate cancer invasion and mixed osteoblastic/osteolytic bone metastases.

Expression of parathyroid hormone-related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer-derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor-derived PTHrP on bone was investigated using a vossicle co-implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro-osteoclastic and pro-osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro-angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer-derived PTHrP is actively involved in osteoblastic skeletal progression.

Objectives Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Materials and Methods Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Results Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor κB ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Conclusion Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL : OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL.

Squamous cell carcinoma (SCC) is the most common form of oral cancer. Destruction and invasion of mandibular and maxillary bone frequently occurs and contributes to morbidity and mortality. We hypothesized that the bisphosphonate drug zoledronic acid (ZOL) would inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a murine xenograft model of bone-invasive oral SCC (OSCC) derived from an osteolytic feline OSCC. Luciferase-expressing OSCC cells (SCCF2Luc) were injected into the perimaxillary subgingiva of nude mice, which were then treated with 100 μg/kg ZOL or vehicle. ZOL treatment reduced tumor growth and prevented loss of bone volume and surface area but had no effect on tumor invasion. Effects on bone were associated with reduced osteolysis and increased periosteal new bone formation. ZOL-mediated inhibition of tumor-induced osteolysis was characterized by reduced numbers of tartrate-resistant acid phosphatasepositive osteoclasts at the tumor-bone interface, where it was associated with osteoclast vacuolar degeneration. The ratio of eroded to total bone surface was not affected by treatment, arguing that ZOL-mediated inhibition of osteolysis was independent of effects on osteoclast activation or initiation of bone resorption. In summary, our results establish that ZOL can reduce OSCC-induced osteolysis and may be valuable as an adjuvant therapy in OSCC to preserve mandibular and maxillary bone volume and function.

BACKGROUND-Bone metastasis is the most common cause of morbidity and mortality in patients with advanced prostate cancer and is manifested primarily as mixed osteoblastic and osteolytic lesions. However, the mechanisms responsible for bone metastases in prostate cancer are not clearly understood, in part due to the lack of relevant in vivo models that mimic the clinical presentation of the disease in humans. We previously established a nude mouse model with mixed bone metastases using intracardiac injection of canine prostate cancer cells (Ace-1). In this study, we hypothesized that tumor-induced osteolysis promoted the incidence of bone metastases and osteoblastic activity.