Downregulation of CXCR4 Expression and Functionality After Zoledronate Exposure in Canine Osteosarcoma.

BACKGROUND: The establishment and progression of metastases remains the life-limiting factor for dogs diagnosed with osteosarcoma (OS). The pattern of metastases is likely regulated through interactions between chemokine receptors and chemokines, and perturbations in these signaling cascades responsible for cytoskeletal organization and directional migration have the potential to alter metastatic cell trafficking behaviors. HYPOTHESIS: Zoledronate will impair directional migration of OS cells through downregulation of chemokine (C-X-C motif) receptor 4 (CXCR4) expression and functionality. SAMPLES: Nineteen archived tumor specimens and plasma from 20 dogs with OS. METHODS: Prospectively, the expressions of CXCR4 were studied in OS cell lines and spontaneous tumor samples. The effect of zoledronate on CXCR4 expression and functionality was investigated by characterizing responses in 3 OS cell lines. In 19 OS specimens and 20 dogs with OS, changes in CXCR4 expression and circulating CXCR4 concentrations were characterized in response to zoledronate therapy respectively. RESULTS: All canine OS cells express CXCR4, and zoledronate reduces CXCR4 expression and functionality by 27.7% (P < .0001), through augmented proteasome degradation and reduced prenylation of heterotrimeric G-proteins in 33% of tumor cell lines evaluated. In OS-bearing dogs, zoledronate reduces CXCR4 expressions by 40% within the primary tumor compared to untreated controls (P = .03) and also decreases the circulating concentrations of CXCR4 in 18 of 20 dogs with OS. CONCLUSIONS AND CLINICAL IMPORTANCE: Zoledronate can alter CXCR4 expression and functionality in OS cells, and consequent perturbations in CXCR4 intracellular signaling cascades might influence patterns of metastases.

The Association of Endothelin-1 Signaling with Bone Alkaline Phosphatase Expression and Protumorigenic Activities in Canine Osteosarcoma.

BACKGROUND: Canine osteosarcoma (OS) is an aggressive sarcoma characterized by pathologic skeletal resorption and pulmonary metastases. A number of negative prognostic factors, including bone alkaline phosphatase, have been identified in dogs with OS, but the underlying biologic factors responsible for such observations have not been thoroughly investigated. Endothelin-1-mediated signaling is active during bone repair, and is responsible for osteoblast migration, survival, proliferation, and bone alkaline phosphatase expression. HYPOTHESIS: The endothelin-1 signaling axis is active in canine OS cells, and this pathway is utilized by malignant osteoblasts for promoting cellular migration, survival, proliferation, and bone alkaline phosphatase activities. ANIMALS: 45 dogs with appendicular OS. METHODS: The expressions of endothelin-1 and endothelin A receptor were studied in OS cell lines and in samples from spontaneously occurring tumors. Activities mediated by endothelin-1 signaling were investigated by characterizing responses in 3 OS cell lines. In 45 dogs with OS, bone alkaline phosphatase concentrations were correlated with primary tumor osteoproductivity. RESULTS: Canine OS cells express endothelin-1 and endothelin A receptor, and this signaling axis mediates OS migration, survival, proliferation, and bone alkaline phosphatase activities. In OS-bearing dogs, circulating bone alkaline phosphatase activities were positively correlated with primary tumor relative bone mineral densities. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine OS cells express endothelin-1 and functional endothelin A receptors, with the potential for a protumorigenic signaling loop. Increases in bone alkaline phosphatase activity are associated with osteoblastic OS lesions, and might be an epiphenomenon of active endothelin-1 signaling or excessive osteoproduction within the localized bone microenvironment.

Expression of nociceptive ligands in canine osteosarcoma.

BACKGROUND: Canine osteosarcoma (OS) is associated with localized pain as a result of tissue injury from tumor infiltration and peritumoral inflammation. Malignant bone pain is caused by stimulation of peripheral pain receptors, termed nociceptors, which reside in the localized tumor microenvironment, including the periosteal and intramedullary bone cavities. Several nociceptive ligands have been determined to participate directly or indirectly in generating bone pain associated with diverse skeletal abnormalities. HYPOTHESIS: Canine OS cells actively produce nociceptive ligands with the capacity to directly or indirectly activate peripheral pain receptors residing in the bone tumor microenvironment. ANIMALS: Ten dogs with appendicular OS. METHODS: Expression of nerve growth factor, endothelin-1, and microsomal prostaglandin E synthase-1 was characterized in OS cell lines and naturally occurring OS samples. In 10 dogs with OS, circulating concentrations of nociceptive ligands were quantified and correlated with subjective pain scores and tumor volume in patients treated with standardized palliative therapies. RESULTS: Canine OS cells express and secrete nerve growth factor, endothelin-1, and prostaglandin E2. Naturally occurring OS samples uniformly express nociceptive ligands. In a subset of OS-bearing dogs, circulating nociceptive ligand concentrations were detectable but failed to correlate with pain status. Localized foci of nerve terminal proliferation were identified in a minority of primary bone tumor samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine OS cells express nociceptive ligands, potentially permitting active participation of OS cells in the generation of malignant bone pain. Specific inhibitors of nociceptive ligand signaling pathways might improve pain control in dogs with OS.

Pro-tumorigenic effects of transforming growth factor beta 1 in canine osteosarcoma.

BACKGROUND: Transforming growth factor beta 1 (TGFß1) is a pleiotropic cytokine that contributes to reparative skeletal remodeling by inducing osteoblast proliferation, migration, and angiogenesis. Organic bone matrix is the largest bodily reservoir for latent TGFß1, and active osteoblasts express cognate receptors for TGFß1 (TGFßRI and TGFßRII). During malignant osteolysis, TGFß1 is liberated from eroded bone matrix and promotes local progression of osteotropic solid tumors by its mitogenic and prosurvival activities. HYPOTHESIS: Canine osteosarcoma (OS) cells will possess TGFß1 signaling machinery. Blockade of TGFß1 signaling will attenuate pro-tumorigenic activities in OS cells. Naturally occurring primary OS samples will express cognate TGFß1 receptors; and in dogs with OS, focal malignant osteolysis will contribute to circulating TGFß1 concentrations. ANIMALS: Thirty-three dogs with appendicular OS. METHODS: Expression of TGFß1 and its cognate receptors, as well as the biologic effects of TGFß1 blockade, was characterized in OS cells. Ten spontaneous OS samples were characterized for TGFßRI/II expressions by immunohistochemistry. In 33 dogs with OS, plasma TGFß1 concentrations were quantified and correlated with bone resorption. RESULTS: Canine OS cells secrete TGFß1, express cognate receptors, and TGFß1 signaling blockade decreases proliferation, migration, and vascular endothelial growth factor secretion. Naturally occurring OS samples abundantly and uniformly express TGFßRI/II, and in OS-bearing dogs, circulating TGFß1 concentrations correlate with urine N-telopeptide excretion. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine OS cells possess TGFß1 signaling machinery, potentially allowing for the establishment of an autocrine and paracrine pro-tumorigenic signaling loop. As such, TGFß1 inhibitors might impede localized OS progression in dogs.

Association between absolute tumor burden and serum bone-specific alkaline phosphatase in canine appendicular osteosarcoma.

BACKGROUND: In dogs with appendicular osteosarcoma (OSA), increased pretreatment serum bone-specific alkaline phosphatase (BALP) activity is a negative prognostic factor, associated with shorter disease-free intervals and survival times, but a biologic basis for observed differential serum BALP activities in canine OSA patients remains incompletely defined. OBJECTIVE: Serum BALP activity will correlate with absolute tumor burden in dogs with OSA. ANIMALS: This study included 96 client-owned dogs with appendicular OSA. METHODS: In canine OSA cell lines, the expression and membranous release of BALP was evaluated in vitro. The correlation between serum BALP activity and radiographic primary tumor size was evaluated in OSA-bearing dogs. In dogs developing visceral OSA metastases, serial changes in serum BALP activities were evaluated in relation to progression of macroscopic metastases, and visceral metastatic OSA cells were evaluated for BALP expression. RESULTS: In vitro, BALP expression was not associated with either tumorigenic or metastatic phenotype, rather the quantity of membranous BALP released was proportional with cell density. In dogs devoid of macroscopic metastases, there was a positive correlation between serum BALP activity and absolute primary tumor size. In dogs with progressive OSA metastases, serum BALP activity increased and coincided with the development of macroscopic metastases. OSA cells derived from visceral metastatic lesions retained BALP expression. CONCLUSIONS AND CLINICAL IMPORTANCE: Tumor burden is a determinant of serum BALP activity in dogs with appendicular OSA. The association between increased pretreatment BALP activity and negative clinical prognosis may simply be attributed to greater initial tumor burden, and consequently more advanced tumor stage.

Cathepsin K expression and activity in canine osteosarcoma.

BACKGROUND: Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations. HYPOTHESIS: Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption. ANIMALS: Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs. METHODS: In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents. RESULTS: Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFß1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03. CONCLUSIONS AND CLINICAL IMPORTANCE: Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis.