RANKL Targeting Aims Beyond Bone Health

Two decades ago, researchers identified numerous members of the tumor necrosis factor (TNF) superfamily of cytokine receptors. Among them, the receptor activator of nuclear factor kappa B (RANK) has since emerged as a central player in bone physiology and beyond.

Inhibiting RANK signaling has proved a useful strategy in preventing the debilitating skeletalrelated events (SREs) that are associated with bone metastases in many patients with cancer.

Researchers are now uncovering hints of the importance of the RANK pathway at different stages of tumor development, from the early steps of tumorigenesis to the establishment and maintenance of the metastatic niche, in addition to its role in other physiological processes linked to cancer development, such as inflammation and immunity.

Maintaining the Bone Niche

As a result, investigators are turning their focus to the study of patient outcomes beyond bone health, and early results have raised the possibility that exploiting this strategy can help treat and possibly even prevent the development of certain cancers. Thus far, denosumab (Xgeva) is the only FDA-approved drug that directly inhibits RANK pathway activity. Ongoing studies are exploring a wider role for the agent as part of therapeutic regimens in a number of tumor types (Table).In order to repair and maintain the skeleton and ensure adequate levels of calcium in the body, bone is constantly being synthesized and destroyed. This complex process of remodeling is orchestrated by 2 major cell types: the osteoblasts (which build up the bone) and the osteoclasts (which break it down).

Osteoblasts are derived from mesenchymal stem cells and they synthesize and secrete type I collagen, which is the main structural component of the bone matrix, while osteoclasts originate from hematopoietic stem cells and produce hydrogen ions and protease enzymes that digest the bone matrix.

Table. Selected Clinical Trials of Denosumab in Cancer

^aTrial ongoing but not recruiting participants.

^bTrial not yet open for recruitment.

A plethora of different molecules produced by the bone and its surrounding environment maintain a tight balance of osteoblast and osteoclast function. As researchers sought to tease out the underlying molecular mechanisms, the RANK receptor, a member of the TNF superfamily, and its only known receptor RANKL, emerged front and center in the bone remodeling process through their effects on osteoclasts.

RANKL, in its membrane-bound form, is found on the surface of osteoblasts and various cells within the bone microenvironment. The ligand can also be cleaved from the membrane by proteases and found in a soluble form. Both forms of the ligand are able to activate the receptor and it is unclear whether the 2 have different functions, although it is suspected their roles may be dependent upon the context in which RANK activation is required.

RANK is expressed on the surface of osteoclast precursors and mature osteoclasts. Similar to other members of this family of receptors, RANK itself does not possess any enzymatic activity.

Instead, upon RANKL binding, the receptor recruits a range of adaptor molecules that propagate the signal—central among them, the TNF receptor associated factors (TRAFs)—by activating downstream signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen- activated protein kinase (MAPK) cascades.

Master of Bone Metastasis

Ultimately, this initiates a transcriptional program within the nucleus that coordinates the differentiation, activation, and survival of osteoclasts. RANK and RANKL are 2 members of a triad of molecules that keep the process of osteoclastogenesis tightly in check. The third member is osteoprotegerin (OPG), a natural decoy receptor for RANKL. It competes with RANKL for binding to RANK and thus helps to propagate a feedback loop that balances osteoclast activation and inhibition (Figure).Tumor metastases are responsible for the vast majority of all cancer-related deaths. Although the bone is not the most common site of metastasis, some tumor types show a striking preference for metastasizing to the skeletal system; in particular, bone metastases occur in more than 70% of breast and prostate cancers. Bone metastasis is associated with particularly poor quality of life for patients because of its painful effects on the skeletal and nervous systems.

High levels of RANK and RANKL expression have been noted in a variety of different cancers, including breast, prostate, and lung cancer, as well as multiple myeloma and melanoma. RANK is frequently found to be expressed by cancer cells, while RANKL is often upregulated in the tumor microenvironment. It is now widely thought that RANK signaling may, in part, explain the tendency of some tumors to metastasize to the bone.

Metastasis is a challenging process for cancer cells, and very few cancer cells typically go on to fuel secondary tumor growths. Success depends upon the interaction between the disseminated tumor cells and the local microenvironment at the site of metastasis. The English surgeon Stephen Paget dubbed this the “seed and soil” hypothesis.

RANK signaling may play a role in priming both the seed and the soil in the formation of bone metastases. It has been implicated in changes cancer cells undergo as they metastasize, including the epithelial to mesenchymal transition (EMT).

Meanwhile, RANKL expression in the bone microenvironment can help to make the metastatic niche a fertile environment for the tumor cells. It is thought to represent an important chemoattractant that drives the tumor cells to metastasize to the bone, at least in certain types of cancer.

Once the tumor is seeded in the bone, a vicious circle is established between the cancer cells and bone microenvironment that is driven in large part by RANK signaling. The tumor secretes several factors that further promote RANKL expression by the cells of the bone microenvironment.

This stimulates osteoclastogenesis and drives breakdown of the bone. Since the bone matrix is an important storage site for growth factors and calcium, bone breakdown sets them free in the bone microenvironment, where they can further promote tumor cell growth.

Beyond the Bone

Tumor-driven dysregulation of RANK signaling can also tip the scales in the other direction and lead to abnormal buildup of the bone, producing more osteoblastic bone metastases. Indeed, numerous tumor types have been shown to express higher levels of the RANK inhibitor OPG.More recently, it has become clear that the role of RANK signaling is not limited to the bone. In fact, some of the highest levels of RANKL expression have been noted in the lung and other tissues around the body, with relatively low levels in the bone and bone marrow.

Other important cellular processes in which RANK signaling has a hand include the regulation of immunity and inflammation. It has been implicated in multiple divergent effects in immunity and inflammation, including in lymph node development, lymphocyte differentiation, dendritic cell survival, and T-cell activation. The roles of RANK signaling beyond bone physiology are becoming an increasing focus of research as they have the potential to play an important role across all stages of cancer development, particularly in the case of breast cancer.

Figure. RANK Signaling Networks

RANK, its ligand RANKL, and the decoy receptor OPG are the three members of the TNF family that regulate the bone environment through complex cell-signaling networks.

Walsh MC, Choi Y. Biology of the RANKL-RANK-OPG System in immunity, bone, and beyond. Front Immunol. 2014;5:511. doi:10.3389/fimmu.2014.00511.

The Emergence of Denosumab

Preventing Skeletal-Related Events

Osteoclast-independent RANK signaling has been shown to have direct effects on mammary epithelial cells. Among other functions, it is thought to mediate the effects of progesterone on the survival and proliferation of these cells. Unsurprisingly, given that survival and proliferation are hallmarks of cancer, dysregulated RANK signaling has now also been linked to the early stages of tumorigenesis in breast cancer.Denosumab is a fully human monoclonal antibody that binds to RANKL and prevents it from binding to RANK, thus blocking the downstream effects of this pathway. Success in phase III trials led to its first regulatory approval in 2010 for the prevention of SREs in patients with bone metastases from solid tumors. SREs are defined as pathologic fractures, the need for radiation therapy or surgery to the bone, or spinal cord compression, which result from bone metastases.

Approval was based on the results of 3 trials in which denosumab was compared with the bisphosphonate zoledronic acid, a compound that counteracts bone resorption. The trials were performed in breast cancer, prostate cancer, and in other types of solid tumors and multiple myeloma.

In the breast and prostate cancer trials, denosumab treatment resulted in a statistically significant increase in the time to first on-study SRE compared with zoledronic acid. In other solid tumors, denosumab was not inferior to zoledronic acid in this measure.

Broader Treatment Role Explored

The incidence of adverse events (AEs) was similar following treatment with zoledronic acid or denosumab. However, in a subgroup analysis of patients with multiple myeloma, mortality appeared to be higher for denosumab-treated patients, and thus the drug was not approved for this indication. Following its initial approval, denosumab was subsequently approved in patients at high risk for fracture after treatment with androgen deprivation therapy for nonmetastatic prostate cancer or adjuvant aromatase inhibitor (AI) therapy for breast cancer. That approval was based on the effects of denosumab on lumbar spine bone mineral density over 1 or 2 years.At the current time, denosumab is predominantly used to assuage the damage induced by bone metastases and has proved highly effective in this regard. But the question of whether targeting RANK signaling might be used to treat the cancer itself remains open. Since its initial approval, denosumab has been approved for the treatment of giant cell tumors of the bone, a rare, highly aggressive and challenging, but mostly benign type of bone tumor. Approval in this indication was based on the demonstration of objective responses in 2 phase II studies. It also is indicated for patients with hypercalcemia of malignancy refractory to bisphosphonate therapy.

There have been hints from several studies in solid tumors, including breast and lung cancer, that denosumab treatment may improve overall survival (OS) and other clinical outcomes. An exploratory analysis of a subgroup of patients with lung cancer from the phase III trial of denosumab in solid tumors identified a small but statistically significant increase in median OS compared with zoledronic acid (8.9 months vs 7.7 months; HR, 0.80).

A follow-up to the Austrian Breast Cancer Study Group (ABCSG)-18 study, presented at the 2015 San Antonio Breast Cancer Symposium, showed that denosumab not only helped to prevent fractures in postmenopausal women with hormone receptor-positive breast cancer treated with an AI, but it also reduced the risk of recurrence.

In this phase III trial, a 19% relative survival improvement was seen among women who had denosumab added to their AI therapy, suggesting that the agent should be recommended in this setting regardless of the patient’s bone health status. Researchers are also examining the impact of denosumab on breast cancer outcomes in the D-CARE study (NCT01077154), which has completed enrollment and is awaiting results, in addition to its potential in the treatment of non—small cell lung cancer in the SPLENDOUR trial (NCT02129699).

Intriguingly, in a recent preclinical study, researchers from the Austrian Institute of Molecular Biotechnology and the University of Maryland School of Medicine, discovered that blocking RANKL signaling in BRCA1/2-mutant mice largely prevented the development of cancer. Invasive carcinomas were detected in the control group, but the group treated with a RANKL inhibitor had relatively normal mammary glands.

If these findings translate into human subjects, this raises the tantalizing possibility of using denosumab to prevent breast cancer in women at high-risk due to BRCA1/2 mutations.

Key Research

• Cheng ML, Fong L. Effects of RANKL-targeted therapy in immunity and cancer. Front Oncol. 2014;3(329):1-8. doi:10.3389/fonc.2013.00329.
• Chu GC, Chung LW. RANK-mediated signaling network and cancer metastasis. Cancer Metastasis Rev. 2014;33(2-3):497-509.
• González-Suárez E, Sanz-Moreno A. RANK as a therapeutic target in cancer. FEBS J. 2016;283(11):2018-2033.
• Gül G, Sendur MA, Aksoy S, et al. A comprehensive review of denosumab for bone metastasis in patients with solid tumors. Curr Med Res Opin. 2016;32(1):133-145.
• Kiesel L, Kohl A. Role of the RANK/RANKL pathway in breast cancer. Maturitas 2016;86:10-16.
• Renema N, Navet B, Heymann MF, et al. RANK-RANKL signalling in cancer. Biosci Rep. 2016;36(4). pii:e00366. doi:10.1042/BSR20160150.