ABSTRACT: Bone biology and the molecular basis of normal bone remodeling are important for understanding bone health, particularly for medical professionals who are treating patients with skeletal complications. Key factors in bone remodeling include RANK ligand, which stimulates bone resorption, and osteoprotegerin (OPG), which inhibits bone resorption. The ratio between these two factors regulates osteoclast formation and activity. An imbalance in the expression of RANK ligand and OPG is an underlying mechanism in cancer treatment–induced bone loss, in bone metastasis in patients with solid tumors, and in osteolysis in patients with multiple myeloma. In cancer-induced bone disease, for example, RANK ligand overwhelms the effects of OPG, leading to imbalanced bone remodeling and the “vicious cycle” of metastatic disease. Experimental therapeutics that target RANK ligand are increasingly being studied and have shown promise for decreasing tumor-related bone disease.
Bone renewal is essential for bone strength. During childhood and early adulthood, bone formation prevails over bone resorption, as bones increase in size and strength. Peak bone mass is achieved during the third decade in life, with a higher peak bone mass being protective against osteoporosis later in life. Bone loss is most prominent in women at menopause due to the effects of a natural decline in estrogen levels. However, bone mass begins to decrease with age, and bone loss is most prominent in women at menopause due to the effects of a natural decline in estrogen levels.
Normal bone remodeling is dependent on osteoclasts and critical molecular mediators, including RANK ligand, a key stimulator of bone resorption, and osteoprotegerin (OPG), a key inhibitor of bone resorption. Bone renewal is successful when resorption of bone by osteoclasts is perfectly coupled with formation of bone by osteoblasts. The goals of this article are to highlight the RANK ligand pathway and its involvement in healthy bone remodeling, bone metastasis, cancer treatment–induced bone loss, and multiple myeloma. Now and in the future, this topic will become even more important as novel therapies targeted at reducing skeletal complications emerge.
The bone remodeling process replaces approximately 20% of bone tissue annually; this process occurs continuously, with each cycle lasting 4 to 8 months. In healthy adults, bone remodeling occurs in a balanced, highly regulated manner in five phases: activation, resorption, reversal, formation, and quiescence (Figure 1). In the first phase, osteoclast precursors are attracted to the remodeling sites. Once they have matured, osteoclasts release osteoclastic enzymes to form a resorption pit in spongy bone and burrow a tunnel in compact bone, and calcium and phosphorous ions are released into the bloodstream. Resorption, from the time of osteoclast adherence to bone until ion release into the bloodstream, is controlled by hormones and thus affected by hormonal changes that occur during life.
During reversal, the next stage, precursors to osteoblasts appear at the resorption site, where they proliferate and differentiate. Mature osteoblasts release osteoid to form a new soft matrix, which is then mineralized with calcium salts and phosphorous that precipitate from the blood. At the conclusion of the cycle, resting cells line the area and remain dormant until the next cycle.[3, 4]
Under both normal bone remodeling and pathologic conditions (such as metastasis), bone resorption is mediated by osteoclasts, which are large, multinucleated cells with abundant cytoplasm, Golgi apparatus, and mitochondria. They originate from myeloid stem cells that differentiate into CD14 monocytes, which are then recruited to the bone surface to undergo differentiation into multinucleated osteoclasts. The process of osteoclast formation and activation occurs over 5 to 8 days and requires two important factors that are produced by bone marrow stromal cells and osteoblasts: macrophage colony–stimulating factor (M-CSF) and RANK ligand. Activation of osteoclasts is dependent on cell-to-cell interactions between osteoblasts and osteoclast precursors; RANK ligand binds to the RANK receptor on osteoclasts and promotes osteoclast activity (Figure 2).
Other systemic factors such as parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3, and prostaglandins induce osteoclast activity indirectly by increasing expression of RANK ligand (Table 1). PTH stimulates bone resorption and regulates serum calcium concentration, while 1,25-dihydroxyvitamin D3 exerts its major effect in the intestinal tract, where it promotes the absorption of calcium and phosphorous. Before osteoclastic resorption of bone occurs, osteoblasts secrete collagenase to remove osteoid. Osteoblasts are cuboidal, mononuclear cells with abundant endoplasmic reticulum and Golgi apparatus that lay down bone matrix. Osteoblasts produce interleukin-6, interleukin-1, prostaglandins, and macrophage colony-stimulating factors (M-CSF) which induce formation of osteoclasts. After osteoclastic activity, osteoblasts produce and mineralize the bone matrix, eventually forming osteocytes. T cells produce cytokines (interleukin-4, interleukin-18, and interferon-gamma), which inhibit the formation of osteoclasts. Bone matrix also serves as a major source of growth factors (Figure 2).
Several members of the tumor necrosis factor (TNF) superfamily of proteins— RANK, RANK ligand, and OPG—are key to enabling osteoclasts to resorb bone and maintain balanced bone remodeling. The importance of RANK ligand has been studied in experimental systems, in which knockout mice lack RANK ligand or the RANK gene; these mice have no osteoclasts and develop osteopetrosis (Figure 3).[5, 6] The interaction between the RANK receptor on osteoclast precursors and the RANK ligand expressed by immature osteoblasts and bone marrow stromal cells is critical for osteoclasts to form, function, and survive. RANK ligand binds to the RANK receptor on osteoclast precursors, promoting their differentiation into mature osteoclasts capable of osteoclastic activity.
OPG is produced by osteoblasts to maintain balance between bone resorption and formation. It is a soluble member of the TNF receptor superfamily and serves as a decoy receptor for RANK ligand. OPG binds to the RANK ligand and prevents it from binding to its intended receptor, RANK, on the surface of osteoclasts, thus inhibiting bone resorption (Figure 4). In experimental systems, bones of OPG-deficient mice are osteopenic. Bone resorption is thus intricately linked to the ratio between RANK ligand and OPG; osteolysis in patients with multiple myeloma and bone metastasis in patients with solid tumors often occur due to an altered ratio between RANK ligand and OPG.
Experimental therapeutics targeting RANK ligand are increasingly being studied and have shown promise for decreasing tumor-related bone disease. RANK ligand also has a role in cancer cell migration, whereas OPG has been shown to prevent bone metastases in experimental models.[8,9] A disparity in RANK ligand expression and OPG expression plays a key role in bone diseases that develop secondary to increased bone resorption. For instance, in cancer-induced bone disease, RANK ligand overwhelms the effects of OPG, leading to imbalanced bone remodeling and the “vicious cycle” of metastatic disease.
Bone metastases are a frequent complication of cancer and may lead to devastating consequences—severe pain, pathologic fractures, life-threatening hypercalcemia, and spinal cord compression. They are often seen in patients with advanced breast and prostate cancer. In metastatic disease, RANK ligand plays a key role in a continuous cycle of bone destruction and tumor growth. In this cycle, reciprocal feedback between tumor cells and the bone microenvironment leads to the release of growth factors from the bone matrix. The subsequent tumor growth causes further release of factors from the tumor itself, and bone destruction ensues. Metastases can be osteoblastic, osteolytic, or mixed; they result from increased osteoclastic activity due to an imbalance between RANK ligand expression and OPG expression. In breast cancer, osteolytic lesions are most common, whereas in prostate cancer, osteoblastic lesions predominate. Purely lytic lesions develop in multiple myeloma, and suppression of osteoblastic activity also has been implicated in the development of such lesions.
For a number of reasons, bone is fertile ground for tumor metastasis. Blood flow is highest in the red marrow. In addition, bone itself holds a number of growth factors that are released and/or activated during bone resorption. When tumor cells invade bone, the released factors induce RANK ligand expression in osteoblasts. Increased expression of RANK ligand in the tumor microenvironment enables osteoclasts to differentiate and survive. Greater osteoclast activity leads to more resorption and bone destruction, which in turn leads to the release of bone-derived growth factors and calcium, fueling further tumor growth.
The vicious cycle of metastatic bone disease has been further studied in breast cancer. Breast cancer cells, like cells of many other solid tumors, produce PTH-related peptide (PTHrP). Tumor cells can also produce other factors—interleukin-6, prostaglandin E2, TNF, and M-CSF. PTHrP binds the same receptor (PTHR1) as PTH and mimics its effects, leading to increased osteoclast activity. Expression of RANK ligand is increased, and osteoclasts are formed. Bone resorption leads to the release of factors such as transforming growth factor-beta (TGF-ß), insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, and bone morphogenic proteins. These increase production of PTHrP and other factors from tumor cells. PTHrP is a major mediator in the cycle of bone metastasis in breast cancer and other solid tumors (Figure 5).