Özge Uluçkan

Skeletal Complications of Breast Cancer Therapies

Nonsurgical treatment options, such as hormonal therapy, chemotherapy, radiation, and bisphosphonate therapy, are undoubtedly improving outcomes for women with breast cancer; however, these therapies also carry significant skeletal side effects. For example, adjuvant hormonal treatments, such as aromatase inhibitors that disrupt the estrogen-skeleton axis, have the potential to cause decreased bone mineral density. Similarly, chemotherapy often induces primary ovarian failure in premenopausal women, resulting in decreased levels of circulating estrogen and subsequent osteopenia. In both cases, women receiving these therapies are at an increased risk for the development of osteoporosis and skeletal fracture. Furthermore, women undergoing radiation therapy to the upper body may have an increased incidence of rib fracture, and those receiving bisphosphonates may be vulnerable to the development of osteonecrosis of the jaw. Therefore, women with breast cancer who are undergoing any of these therapies should be closely monitored for bone mineral loss and advised of skeletal health maintenance strategies.

CD47 Regulates Bone Mass and Tumor Metastasis to Bone

CD47, also called integrin-associated protein, plays a critical role in the innate immune response and is an atypical member of the immunoglobulin superfamily that interacts with and activates beta3 integrins. beta3 integrin(-/-) mice have defective platelet and osteoclast function and are protected from bone metastasis. The role of CD47 in skeletal homeostasis and bone metastasis has not been described. CD47(-/-) mice had increased bone mass and defective osteoclast function in vivo. Although the number of functional osteoclasts formed by differentiating CD47(-/-) bone marrow macrophages was decreased, high doses of RANKL rescued differentiation and function of CD47(-/-) osteoclasts ex vivo and rescued the osteoclast defect in CD47(-/-) mice. Inhibition of nitric oxide (NO) synthase, which is expressed at higher levels in CD47(-/-) osteoclasts, also rescued the osteoclast defect in CD47(-/-) cells. We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47(-/-) mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47(-/-) mice. There was decreased tumor-associated bone destruction in the CD47(-/-) mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone.

Disruption of CXCR4 enhances osteoclastogenesis and tumor growth in bone

CXCR4 regulates hematopoietic and tumor cell homing to bone, but its role during osteoclast (OC) development is unknown. We investigated the role of CXCR4 in osteoclastogenesis and in a model of bone metastasis. Compared with controls, mice reconstituted with CXCR4 null hematopoietic cells exhibited elevated markers of bone resorption, increased OC perimeter along bone, and increased bone loss. CXCR4−/− OCs demonstrated accelerated differentiation and enhanced bone resorption in vitro. Furthermore, tumor growth specifically in bone was significantly increased in mice reconstituted with CXCR4−/− hematopoietic cells. Finally, enhancement of bone tumor growth in the absence of CXCR4 was abrogated with the OC inhibitor, zoledronic acid. These data demonstrate that disruption of CXCR4 enhances osteoclastogenesis and suggest that inhibition of CXCR4 may enhance established skeletal tumor burden by increasing OC activity.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

APT102, a novel adpase, cooperates with aspirin to disrupt bone metastasis in mice

Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti‐platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet αIIbβ3 activators, such as ADP and thromboxane A2 (TXA2). Inhibitors of platelet β3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ADPase, APT102, and an inhibitor of TXA2 synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with αIIbβ3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in β3−/− mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti‐platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases. J. Cell. Biochem. 104: 1311–1323, 2008.

Interferon-γ Targets Cancer Cells and Osteoclasts to Prevent Tumor-associated Bone Loss and Bone Metastases

Interferon-γ (IFN-γ) has been shown to enhance anti-tumor immunity and inhibit the formation of bone-resorbing osteoclasts. We evaluated the role of IFN-γ in bone metastases, tumor-associated bone destruction, and hypercalcemia in human T cell lymphotrophic virus type 1-Tax transgenic mice. Compared with Tax+IFN-γ+/+ mice, Tax+IFN-γ-/- mice developed increased osteolytic bone lesions and soft tissue tumors, as well as increased osteoclast formation and activity. In vivo administration of IFN-γ to tumor-bearing Tax+IFN-γ-/- mice prevented new tumor development and resulted in decreased bromodeoxyuridine uptake by established tumors. In vitro, IFN-γ directly decreased the viability of Tax+ tumor cells through inhibition of proliferation, suppression of ERK phosphorylation, and induction of apoptosis and caspase 3 cleavage. IFN-γ also inhibited macrophage colonystimulating factor-mediated proliferation and survival of osteoclast progenitors in vitro. Administration of IFN-γ to C57BL/6 mice decreased Tax+ tumor growth and prevented tumor-associated bone loss and hypercalcemia. In contrast, IFN-γ treatment failed to protect IFN-γR1-/- mice from Tax+ tumor-induced skeletal complications, despite decreasing tumor growth. These data demonstrate that IFN-γ suppressed tumor-induced bone loss and hypercalcemia in Tax+ mice by inhibiting both Tax+ tumor cell growth and host-induced osteolysis. These data suggest a protective role for IFN-γ in patients with bone metastases and hypercalcemia of malignancy.

Dissection of platelet and myeloid cell defects by conditional targeting of the β3-integrin subunit

The purpose of this work was to determine platelet and myeloid cell‐specific requirements for β3‐containing integrins in hemostasis, bone resorption, and tumor growth. LoxP‐flanked mice were generated to study the conditional deletion of β3‐integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the β3KOP and β3KOM strains of mice, we studied the role of β3‐integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue‐specific deletion of platelet β3‐integrins in β3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in β3KOP mice, in contrast to the defects observed in germline β3−/− mice. Conditional deletion of myeloid β3‐integrins in β3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in β3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet β3‐integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid‐specific β3‐integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that β3‐integrins have cell‐specific roles in complex biological processes.—Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, Ö., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica‐Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the β3‐integrin subunit. FASEB J. 24,1117–1127 (2010). www.fasebj.org

Thrombospondin-1 regulates bone homeostasis through effects on bone matrix integrity and nitric oxide signaling in osteoclasts.

Thrombospondin‐1 (TSP1), an endogenous antiangiogenic, is a widely expressed secreted ligand with roles in migration, adhesion, and proliferation and is a target for new therapeutics. While TSP1 is present in the bone matrix and several TSP1 receptors play roles in bone biology, the role of TSP1 in bone remodeling has not been fully elucidated. Bone turnover is characterized by coordinated activity of bone‐forming osteoblasts (OB) and bone‐resorbing osteoclasts (OC). TSP1−/− mice had increased bone mass and increased cortical bone size and thickness compared to wild type (WT). However, despite increased size, TSP1−/− femurs showed less resistance to bending than expected, indicative of diminished bone quality and a bone material defect. Additionally, we found that TSP1 deficiency resulted in decreased OC activity in vivo and reduced OC differentiation. TSP1 was critical during early osteoclastogenesis, and TSP1 deficiency resulted in a substantial overexpression of inducible nitric oxide synthase (iNOS). Importantly, administration of a NOS inhibitor rescued the OC function defects of TSP1−/− mice in vivo. To investigate the role of bone‐derived TSP1 in osteoclastogenesis, we found that WT pre‐OCs had defective iNOS expression when cultured on TSP1−/− bone compared to WT bone, suggesting that TSP1 in bone plays a critical role in iNOS signaling during OC development. These data implicate a new role for TSP1 in bone homeostasis with roles in maintaining bone matrix integrity and regulating OC formation. It will be critical to monitor bone health of patients administered TSP1‐pathway directed therapeutics in clinical use and under development.