Runx2 transcriptional activation of Indian Hedgehog and a downstream bone metastatic pathway in breast cancer cells

Runx2, required for bone formation, is ectopically expressed in breast cancer cells. To address the mechanism by which Runx2 contributes to the osteolytic disease induced by MDA-MB-231 cells, we investigated the effect of Runx2 on key components of the "vicious cycle" of transforming growth factor beta (TGFbeta)-mediated tumor growth and osteolysis. We find that Runx2 directly up-regulates Indian Hedgehog (IHH) and colocalizes with Gli2, a Hedgehog signaling molecule. These events further activate parathyroid hormone-related protein (PTHrP). Furthermore, Runx2 directly regulates the TGFbeta-induced PTHrP levels. A subnuclear targeting deficient mutant Runx2, which disrupts TGFbeta-induced Runx2-Smad interactions, failed to induce IHH and downstream events. In addition, Runx2 knockdown in MDA-MB-231 inhibited IHH and PTHrP expression in the presence of TGFbeta. In vivo blockade of the Runx2-IHH pathway in MDA-MB-231 cells by Runx2 short hairpin RNA inhibition prevented the osteolytic disease. Thus, our studies define a novel role of Runx2 in up-regulating the vicious cycle of metastatic bone disease, in addition to Runx2 regulation of genes related to progression of tumor metastasis.     

Bone metastasis in breast cancer: The story of RANK-Ligand

The primary cellular mechanism responsible for osteolytic bone metastases is osteoclastic activation. Preclinical models have shown that breast cancer cells can produce parathyroid hormone-related protein (PTHrP), and other osteolytic molecules, which stimulate excessive osteoclastic bone resorption and establishment of osteolytic lesions. It has been shown that PTHrP by itself cannot directly induce osteoclastic activation, but it mediates its effect through the transactivation of RANK-ligand (RANKL) gene on stromal and osteoblastic cells. Accordingly RANKL up-regulation has been considered as a prerequisite in virtually all conditions of cancer induced bone destruction. Hence, therapeutic targeting of RANKL seems to be a rational approach to treat or even to prevent the process of bone metastases. In this review, we will focus on the unique patho-physiological aspects related to the evolution of bone metastases in breast cancer, emphasizing the pivotal role of RANKL and some other key molecules in osteoclastic bone resorption. We will discuss the therapeutic interventions using bisphosphonates and RANKL inhibitors in patients with bone metastases and the outcome of this novel approach.     

Parathyroid Hormone-Related Protein in Hypercalcemia Associated with Hematological Malignancy

Hypercalcemia is an important complication in multiple myeloma as well as T-cell leukemia/lymphoma, and is moderately common in high and intermediate grade non-Hodgkin's lymphoma. The underlying mechanism has been unclear because the neoplastic cells are usually present in the bone marrow, where they are in a position to produce short range effects on bone resorption which are difficult to identify. This contrasts with the situation in hypercalcemia associated with non-metastatic carcinoma, where it has been clearly demonstrated that the most common cause is release from the tumor of a humoral mediator, Parathyroid Hormone-related Protein (PTHrP). Roles have been advocated in multiple myeloma for release of a number of other cytokines with osteolytic capacity on the basis of their enhancement of osteolytic activity in cultured fetal rat bone, although a causal relationship in patients has not been established. PTHrP has more recently been implicated in the genesis of hypercalcemia in patients with hematological malignancies by the demonstration in a proportion of cases of increased circulating levels of PTHrP, comparable to those in hypercalcemia due to cancer. Immunohistochemical studies indicate neoplastic hemopoietic cells can contain PTHrP, and thus have the capacity to act in a paracrine manner to enhance local bone resorption and contribute to the development of hypercalcemia.     

Establishment of a transplantable rat pulmonary carcinoma-derived cell line (IP-B12) as a new model of humoral hypercalcemia of malignancy and bone metastasis.

A cloned cell line (IP-B12) derived from a transplantable rat pulmonary carcinoma (IP), of which neoplastic cells produce parathyroid hormone-related protein (PTHrP), was established. Tumors induced in syngeneic F344 rats by intraperitoneal injection of IP-B12 cells had features of pulmonary adenocarcinomas, consisting of neoplastic cells immunopositive to PTHrP. The IP-B12 tumor-bearing rats developed severe emaciation and hypercalcemia, with a marked elevation of plasma PTHrP level; there was an increase in osteoclastic areas of the femur and calcium depositions in systemic organs, indicating progression to humoral hypercalcemia of malignancy (HHM) in the tumor-bearing rats. In addition, the injection of IP-B12 cells into the left cardiac ventricle of syngeneic rats resulted in osteolytic skeletal metastases in the long bones and vertebrae. In the metastatic lesions, histologically, neoplastic cells showed an immunopositive reaction to PTHrP, and a prominent osteoclastic activity was seen; bone lesions, including osteolysis, fracture, and nerve compression as well as replacement of bone marrow cells by proliferated tumor cells were similar to those reported in human cancer patients with bone metastases. IP-B12 is a new animal model for HHM and osteolytic bone metastases, and will become a useful tool for studies on the pathogenesis and therapeutic strategies for such conditions.     

Skeletal metastasis: Established and emerging roles of parathyroid hormone related protein (PTHrP)

Parathyroid hormone related protein (PTHrP) is a well characterized tumor derived product that also has integral functions in normal development and homeostasis. PTHrP is produced by virtually all tumor types that metastasize to bone and numerous studies have demonstrated a correlation between PTHrP expression and skeletal localization of tumors. PTHrP has prominent effects in bone via its interaction with the PTH-1 receptor on osteoblastic cells. Through indirect means, PTHrP supports osteoclastogenesis by upregulating the receptor activator of NFκB ligand (RANKL) in osteoblasts. PTHrP also regulates osteoblast proliferation and differentiation in manners that are temporal and dose dependent. Bone turnover has been implicated in the localization of tumors to bone and PTHrP increases bone turnover. Bone turnover results in the release of growth factors such as TGFβ and minerals such as calcium, both of which impact tumor cell growth and contribute to continued PTHrP production. PTHrP also has anabolic properties and could be in part responsible for osteoblastic type reactions in prostate cancer. Finally, emerging roles of PTH and PTHrP in the support of hematopoietic stem cell development in the bone marrow microenvironment suggest that an interaction between hematopoietic cells and tumor cells warrants further investigation.     

Breast cancer bone metastasis and current small therapeutics

Patients with advanced breast cancer frequently develop metastasis to bone. Bone metastasis results in intractable pain and a high risk of fractures due to tumor-driven bone loss (osteolysis), which is caused by increased osteoclast activity. Osteolysis releases bone-bound growth factors including transforming growth factor beta (TGF-β). The widely accepted model of osteolytic bone metastasis in breast cancer is based on the hypothesis that the TGF-β released during osteolytic lesion development stimulates tumor cell parathyroid hormone related protein (PTHrP), causing stromal cells to secrete receptor activator of NFκB ligand (RANKL), thus increasing osteoclast differentiation. Elevated osteoclast numbers results in increased bone resorption, leading to more TGF-β being released from bone. This interaction between tumor cells and the bone microenvironment results in a vicious cycle of bone destruction and tumor growth. Bisphosphonates are commonly prescribed small molecule therapeutics that target tumor-driven osteoclastic activity in osteolytic breast cancers. In addition to bisphosphonate therapies, steroidal and non-steroidal antiestrogen and adjuvant therapies with aromatase inhibitors are additional small molecule therapies that may add to the arsenal for treatment of osteolytic breast cancer. This review focuses on a brief discussion of tumor-driven osteolysis and the effects of small molecule therapies in reducing osteolytic tumor progression.     

TGF-beta promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.     

Ipriflavone inhibits osteolytic bone metastasis of human breast cancer cells in a nude mouse model

Osteolytic bone metastasis is a frequent problem in the treatment of cancer. Ipriflavone, a synthetic isoflavone that inhibits osteoclastic bone resorption, has been used for the treatment of osteoporosis in some countries. Some other isoflavones also exhibit an antitumor effect in vitro and in vivo. Here, we studied the effects of ipriflavone on osteolytic bone metastasis of MDA-231 human breast cancer cells injected intracardially into athymic nude mice (ICR-nu/nu). Daily oral administration of ipriflavone at 12 mg/mouse significantly inhibited the development of new osteolytic bone metastases (p < 0.05) and the progression of established osteolytic lesions (p = 0.01), prolonging the life of tumor-bearing mice (p = 0.01 vs. control). In addition, ipriflavone reduced the number of osteoclasts at the bone-cancer interface with no severe adverse effects on the host. In vitro, ipriflavone inhibited the proliferation and DNA synthesis of MDA-231 cells and blocked the ligand-induced phosphorylation of Tyr(845) of the EGFR. Ipriflavone did not promote apoptosis of MDA-231 cells. Our results show that ipriflavone not only directly inhibits the growth of cancer cells but also reduces osteoclasts to prevent the soft tissue tumor burden and osteolytic bone metastases. These findings raise the possibility that ipriflavone may be of use as a therapeutic agent against osteolytic bone metastasis.     

Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma

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. 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. 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. 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.     

Prostate carcinoma skeletal metastases: cross-talk between tumor and bone

The majority of men with progressive prostate cancer develop metastases with the skeleton being the most prevalent metastatic site. Unlike many other tumors that metastasize to bone and form osteolytic lesions, prostate carcinomas form osteoblastic lesions. However, histological evaluation of these lesions reveals the presence of underlying osteoclastic activity. These lesions are painful, resulting in diminished quality of life of the patient. There is emerging evidence that prostate carcinomas establish and thrive in the skeleton due to cross-talk between the bone microenvironment and tumor cells. Bone provides chemotactic factors, adhesion factors, and growth factors that allow the prostate carcinoma cells to target and proliferate in the skeleton. The prostate carcinoma cells reciprocate through production of osteoblastic and osteolytic factors that modulate bone remodeling. The prostate carcinoma-induced osteolysis promotes release of the many growth factors within the bone extracellular matrix thus further enhancing the progression of the metastases. This review focuses on the interaction between the bone and the prostate carcinoma cells that allow for development and progression of prostate carcinoma skeletal metastases.     

Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme of prostaglandin synthesis, has been implicated in invasiveness and distant metastases of cancer. Bone is one of the most common target sites of cancer metastasis. However, the role of COX-2 in bone metastasis is unclear. We examined the surgical specimens of bone metastases from patients with various types of cancers by using immunohistochemistry and observed evident COX-2 expression in these bone metastases. In a nude mouse model of bone metastasis, the MDA-MB-231 human breast cancer cells showed no COX-2 expression at orthotopic sites, whereas these cells, when metastasized to bone, intensely expressed COX-2, suggesting that the bone microenvironment induced COX-2 expression. Consistent with this notion, inhibition of bone resorption by the bisphosphonate ibandronate reduced COX-2 expression in MDA-MB-231 cells in bone. Transforming growth factor-beta (TGFbeta), one of the most abundant growth factors stored in bone, increased COX-2 expression and prostaglandin E2 production in MDA-MB-231 cells in culture. MDA-MB-231 cells overexpressing dominant-negative TGFbeta type II receptors showed decreased bone metastases and reduced osteoclastic bone resorption with impaired COX-2 expression. The COX-2 inhibitors, NS-398 and nimesulide, significantly suppressed bone metastases with decreased osteoclast number and increased apoptosis in MDA-MB-231 cells. These results suggest that bone-derived TGFbeta up-regulates COX-2 expression in breast cancer cells, thereby increasing prostaglandin E2 production, which in turn, stimulates osteoclastic bone destruction, leading to the progression of bone metastases. Our results also suggest that COX-2 is a potential therapeutic target for bone metastases in breast cancer.     

Pathophysiology of bone metastases

Normal bone remodeling maintains an appropriate balance between the action of osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells). Skeletal malignancies, including bone metastases, disrupt the OPG-RANKL-RANK signal transduction pathway and promote enhanced osteoclast formation, thereby accelerating bone resorption and inducing bone loss. This osteolysis in turn leads to the release of bone-derived growth factors, contributing to a "vicious cycle" in which interactions between tumor cells and osteoclasts not only lead to increased osteoclastogenesis and osteolytic activity, but also aggressive growth and behavior of the tumor cells. The osteolytic complications associated with bone metastases are caused by tumor-induced alterations of the OPG-RANKL-RANK system, which are accompanied by enhanced bone resorption and disassociated from counterbalancing bone formation by osteoblasts.     

The RANK/RANKL/OPG triad in cancer-induced bone diseases

The maintenance of skeletal integrity in a healthy individual requires a balanced regulation of the processes of bone formation, mediated by osteoblasts, and bone resorption, mediated by osteoclasts. This balanced process of bone remodeling becomes co-opted in the skeleton by tumor cells and this dramatically accelerates the process of remodeling and disrupts the normal equilibrium resulting in a spectrum of osteolytic to osteoblastic bone lesions. Certain tumor types, such as breast and prostate, frequently metastasize to the bone. It is now widely understood that the molecular triad—receptor activator of NF-κB ligand (RANKL), its receptor RANK, and the endogenous soluble RANKL inhibitor, osteoprotegerin (OPG)—play direct and essential roles in the formation, function, and survival of osteoclasts. Osteoclastic bone resorption contributes to the majority of skeletal sequelae, or skeletal-related events (SREs), in patients with bone metastases. In addition, osteoclastic bone resorption also contributes to the establishment of tumors in the skeleton. Therefore, blocking osteoclast activity and differentiation via RANKL inhibition may not only provide a beneficial treatment for skeletal complications of malignancy, but may also prevent bone metastases. In this review, we will first describe the operative role of osteoclasts and the RANK/RANKL/OPG triad in the pathophysiology of cancer-induced bone diseases, specifically solid tumor metastases to the bone. Secondly, we will describe a therapeutic approach that specifically targets the RANKL molecule.     

Malignancy-associated hypercalcemia

Hypercalcemia is the most frequent paraneoplastic syndrome observed in cancer patients. This morbidity can be divided into two categories: one is hypercalcemia induced by severe bone metastases; the other the elaboration of hypercalcemic factors by solid tumors, termed humoral hypercalcemia of malignancy (HHM). With regard to humoral factors responsible for HHM, a protein with parathyroid hormone (PTH)-like activity, designated PTH-related protein (PTHrP), was isolated from a cancer cell line established from a hypercalcemic patient's lung cancer tissue, and the structure of PTHrP mRNA was identified. Since the biological activity of PTHrP explained most of the clinical and laboratory findings of HHM patients and recent clinical studies indicated the very close relationship between the development of HHM and the production of PTHrP by tumor, PTHrP is now regarded to be the primary candidate for the actual factor responsible for HHM.