Current concepts in bone metastasis, contemporary therapeutic strategies and ongoing clinical trials

Tumor cells necessarily require interaction with the microenvironment of a specific host organ to create a metastatic lesion [3]. This concept was first described over 100 years ago by the English surgeon, Stephen Paget. Paget described the ‘seed and soil’ hypothesis in which he sought to explain why certain cancers favored developing metastasis in specific organs. In his research, he studied the autopsy results of patients who had various primary tumors and found that these patients had specific organ patterns where the metastases developed. For example, he found that women who had breast cancer had a much greater probability of having metastases to the bone than any other organ. He explained these results by proposing that the tumor cells acted as ‘seeds’ and have an affinity for particular organs or the ‘soil’. Thus, metastases will develop when the right combination of a compatible seed is planted in the right soil [4, 5] (Fig. 1).

This complicated process is precisely coordinated and the molecular basis underlying its orchestration from initiation to development of distant metastasis is a vigorous area of research. The initial step in metastasis necessitates that the cancer cells escape from the primary tumor and into systemic circulation. Cancer cells accomplish this through a process termed epithelial-to-mesenchymal transition (EMT). This transformation enables epithelial type cancer cells to undergo a phenotypic change to exhibit mesenchymal traits such as loss of cell surface intercellular adhesion proteins and loss of epithelial polarization [6]. The cancer cells also secrete extracellular proteolytic enzymes to dissolve the extracellular matrix and escape the physical environment of the tumor stroma [7]. The most prominent of these factors are the matrix metalloproteinase enzymes [8]. After an adequate amount of the extracellular matrix has been dissolved, the cancer cells become locally invasive and begin to migrate into surrounding tissue [9]. Cancer cells continue to migrate through the endothelial cells to gain access to systemic circulation through a process called intravasation [10]. This process is mediated at the vascular level by the tortuous and leaky tumor vasculature [11] as well as cell signaling aberrations in the cancer cells that increase cellular adhesion factors such as integrin B1, enabling the cancer cells to interact with the endothelium [12].

Once cancer cells invade blood vessels and get into systemic circulation, they are termed circulating tumor cells (CTC) and are presented with a new set of challenges. The circulatory system is an inhospitable environment but metastatic tumor cells have mechanisms to improve their chances of survival. [13] One example of how these cells survive is by inhibiting anoikis. Anoikis is normally an apoptotic process which cells undergo when there is loss of cell-matrix or cell-cell interactions. As such, the deregulation of anoikis in the context of metastasis is likely present before cancer cells intravasate and continues during the circulation process [14]. One specific example that has been linked to anoikis resistance is a tyrosine kinase receptor, TrkB. It has been shown that overexpression of this receptor on the membrane of cancer cells, results in activation of the phosphatidylinositol-4,5-bisphosphate 3 kinase (PI3K)-AKT pro-survival pathways [15]. Cancer cells also have mechanisms to escape destruction by immune cells, such as macrophages, by upregulating certain cell surface proteins like CD47 [16].

The two main factors impacting the location CTCs will develop a metastatic lesion are: blood flow and molecular signaling. This is particularly true for cancers that metastasize to the bone. Consider the example of breast cancers which have a preference to metastasize to the thoracic spine due to venous drainage of the breast from the azygos venous system communicating with the plexus of Batson in the thoracic region [17]. This is in comparison to lung cancers which show a more general skeletal distribution due to venous drainage from the pulmonary veins into the left side of the heart and from there dissemination to systemic circulation [18]. Alternatively, the majority of prostate cancer metastasis are seen in the axial skeleton in the lumbar spine, sacrum, and pelvis due to venous drainage of the prostate through the pelvic plexus [19]. Further, colon cancer is known to metastasize to the liver due to portal venous drainage [20]. However, blood flow patterns do not fully explain the distribution of metastatic lesions. In addition to blood flow, a plethora of other factors and signaling events are crucial in the dissemination of CTCs. One well documented process is CTC homing to the bone marrow microenvironment.

One of the signaling pathways regulating CTC homing to the bone is the CXCL12-CXC-chemokine receptor 4 (CXCR4) axis [21]. CXCL12, also called stromal derived factor-1 (SDF-1), is a chemokine factor that is made by bone marrow mesenchymal stem cells, endothelial cells, and osteoblasts. CXCL12 binds primarily to the g-protein coupled receptor, CXCR4, activating several divergent intracellular signaling pathways that are involved in cellular processes including: cell survival, gene transcription, chemotaxis, and expression of integrins such as integrin avB3 on the surface of the CTCs [22]. The increased expression of α_Vβ₃ on the surface of the metastatic prostate tumor cells has been shown to cause it to adhere to endothelial cells of the bone marrow [23]. The CXCL12-CXCR4 axis is not only important for CTC from solid tumors, but also plays a significant role in hematopoietic stem cells and leukemia cells homing to the bone marrow [24, 25]. Other molecules have shown importance in the adhesion process as well. These include other integrins such as α4β1 [26], annexin II [27], and E-cadherin [28].

In addition to the significance of CXCL12-CXCR4 axis for cell adhesion in cancer cells, this signaling pathway has also been shown to be important in cancer cell survival. It has been demonstrated that in breast cancer cells that aberrantly express the non-receptor cytoplasmic tyrosine kinase, Src, have improved survival in the bone marrow. It was shown that Src mediates this improved survival through Akt signaling in response to CXCL12-CXCR4 stimulation and through increasing resistance to TNF-related apoptosis-inducing ligand (TRAIL) specifically in the bone marrow microenvironment [29].

Once the process of homing and extravasation have taken place, the metastatic cells encounter native bone microenvironment cells. These cells play a vital role in maintaining homeostasis of the bone and include: osteoclast, osteoblasts, osteocytes, endothelial cells, and cells of the bone marrow. The growth and dynamic turnover of bone is regulated through precise signaling between these cells. Alteration in the homeostasis of these native cells can have disastrous effects. When cancer cells Infiltrate the bone, the lesions that develop are traditionally classified as either osteolytic, in which bone is broken down, or osteoblastic, in which bone is formed [30]. These processes are not binary. Rather, both the osteoclastic and the osteoblastic activities are generally activated in all metastatic bone lesions [31]. However, depending on which process is dominant the radiological appearance of a bone metastasis is either lytic, sclerotic, or mixed. The cancers that conventionally cause osteolytic lesions are breast and multiple myeloma [32]. These types of lesions can be particularly dangerous and have the highest rates of fracture. Osteoblastic lesions are seen most often with metastases from prostate cancer [33] and have an increased risk of fracture due to the altered architecture of the bone but not to the same degree in osteolytic lesions.

The cells responsible for bone resorption are known as osteoclasts. These cells are monocyte-macrophage derived multinuclear cells that are initially inactive [34]. Osteoclasts generally are positioned in resorption pits and when activated secrete cathepsin K. This creates an acidic environment on the underside of the osteoclast where the cell maintains a sealed ruffled border [35]. Osteoclast activation is under the control of both systemic factors as well as locally secreted cytokines. Parathryroid hormone, 1,25-dihydroxyvitamin D_3, and prostaglandins cause upregulation of receptor activator of nuclear factor-κB ligand (RANKL) [36, 37]. RANKL is a family member of tumor necrosis factors (TNF) which is expressed on the membrane surface of both stromal cells and osteoblasts as well as released by active T cells. Structurally, RANKL is a homotrimeric type II membrane protein with three isoforms. [38] The full length version of RANKL is denoted RANKL1. RANKL2 is shorter due to a portion of the intracytoplasmic domain missing. While RANKL3 is the soluble isoform and has the N-terminal portion deleted [38]. RANKL activates osteoclasts by signaling though its receptor, RANK, with subsequent activation of nuclear factor-κB and Jun N-terminal kinase pathways. Locally, stromal cells and osteoblasts also activate osteoclasts by production of macrophage colony stimulating factor. Additional control over osteoclast activation is managed by osteoprotegerin, which is a decoy receptor for RANKL and is normally present in the marrow [39]. An altered ratio of osteoprotegerin to RANKL can result in osteopetrosis or osteopenia [40, 41].

In addition to the osteoclasts, osteoblasts have a major role in maintaining the bone structure. These cells originate from mesenchymal stem cells and are responsible for synthesizing new bone [42]. This is a critical function, not only during development but also throughout life. Several factors allow for successful differentiation of osteoblasts such as bone morphogenetic proteins (BMPs), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor β (TGF-β) [43, 44]. The differentiation of osteoblasts is not as well understood as the process in osteoclasts, but one factor that is known to drive the differentiation process is the transcription factor Runx-2, also called core-binding factor alpha 1 (CBFA1) [45]. As osteoblasts become more mature they secrete osteocalcin and calcified matrix, eventually becoming osteocytes as they are encapsulated within the bone [46].

Osteocytes make up approximately 90% of the bone cells in the adult human, however less is known about their role in bone metastasis than osteoblasts and osteoclasts [47]. Even though osteocytes are surrounded by the bone matrix, they communicate through an extensive lacunar-cannicular network which connects the osteocytes to other osteocytes, the bone surface, and marrow cells. They regulate osteoclast development through expression of: RANKL, macrophage colony stimulating factor (M-CSF) and osteoprotegerin (OPG). In addition, they can inhibit osteoblasts by expression of sclerostin [48]. Osteocytes have an interesting ability to respond to stress and pressure. In fact, increased pressure in the bone from prostate cancer metastasis can upregulate matrix metalloproteinases and CCL5 in osteocytes resulting in increased tumor growth [49]. IL-11 has been shown to be released from apoptotic osteocytes causing osteoclast differentiation [50]. Additionally, physical interactions and secreted factors from cancer cells such as multiple myeloma cells impact osteocyte function [51].

Endothelial cells comprise another component of the bone microenvironment that contribute to the bone metastatic process through a variety of mechanisms. Endothelial cells in the metaphysis of long bones are known to constitutively express P-selectin, E-selectin, vascular adhesion molecule 1 and intercellular adhesion molecule A which aid in CTC adhesion when they travel through the bone marrow [52]. The physical architecture of the bone vasculature also plays a role in the homing process. The large volume of sinusoids decreases blood flow velocity thus decreasing shear forces and increasing the favorability for attachment of cancer cells [53]. Additional mechanisms by which the endothelial cells promote bone metastatic lesions are through promotion of cell dormancy and neovascularization for metastatic growth [54]. Tumor cells can secrete angiogenetic factors such as vascular endothelial growth factor (VEGF) and IL-8 that can serve to increase survival of the tumor cells and neovascularization [55].

More recent evidence has demonstrated the importance of immune cells in the development of bone metastases. The bone marrow is a major reservoir for dendritic cells, macrophages, myeloid derived cells, and different subsets of T cells [56]. T cells have been shown to regulate bone resorption in both solid tumors bone metastasis and multiple myeloma [57, 58]. T cells and B cells also produce RANKL and can impact osteoclastogenesis. IL-7 is an important cytokine that mediates interactions between T cells and the proliferative bone metastatic environment [59]. Myeloid derived suppressor cells from the bone marrow have proven to be impactful in their ability to drive cancer progression through suppression of innate and adaptive immune responses, impairing T cell antigen recognition and promotion of T regulatory cells [60‐62]. In the microenvironment of multiple myeloma patients, dendritic cells and IL-6, IL-23 and IL-1 are involved in increased Th17 cells, which increase IL-17 and can promote osteoclast and myeloma proliferation [48]. Additionally, IL-17 has been shown to be a growth factor for both prostate and breast cancer cells [63, 64].

During development, the bone marrow changes from being predominately red or hematopoietic marrow and having very little adipocytes or yellow marrow to being composed of approximately 70% adipose tissue, by the age of twenty five [65]. These adipocytes were previously thought to be inert but now are considered to have a significant impact on the development of bone metastasis in the microenvironment. It has been proposed that adipocytes play a supporting role for cancer cell survival in the bone marrow as an energy source [66, 67]. Bone marrow adipocytes also secrete several pro-inflammatory mediators such as IL-1B, IL-6, leptin, adiponectin, vascular cell adhesion molecule 1 (VCAM-1), tumor necrosis factor alpha (TNF-alpha) and CXCL12 that increase bone tropism, proliferation, and survival of certain cancer cells [65, 68‐70].

Additionally, cancers cells that are already within the bone microenvironment play in impactful role on the further development of these metastatic lesions. Important activating factors expressed by the prostate cancer cells that create bone metastasis include: FGFs [71] and BMPs [72]. It has been shown that FGF can act through autocrine or paracrine signaling [73]. Binding of FGF to an FGF receptor results in activation of multiple signal transduction pathways beneficial for the tumor. These stimulated pathways include: phosphatidylinositol 3-kinase (PI3K), phospholipase Cγ (PLCγ), mitogen-activated protein kinase (MAPK), and signal transducers and activators of transcription (STAT) [31, 73]. The resulting stimulation of these pathways from multiple FGFs results in simulation of the cells in the bone microenvironment and the cancer cells during metastatic lesion development [31].

The mineral structure of the bone itself presents additional components that can serve to enhance bone metastatic lesions. Encased within the hydroxyapatite are a number of factors such as: bone morphogenetic proteins, insulin like growth factors I and II, platelet-derived growth factor, transforming growth factor-beta and fibroblast growth factor [74]. These factors become important when liberated from the mineralized hydroxyapatite by promoting growth and proliferative effects on tumor cells and worsening the metastatic lesion.

Therapeutic strategies for bone metastatic cancers rely on three main principles: 1.) The cancer cells should be treated. This is critical because the cancer cells are the initial insult which cause bone metastatic lesions to develop. If cancer cells continue to proliferate and divide, it should not be expected that survival time will be extended. This principle can be broken down further into therapies that are cytotoxic and kill the cells, hormonal deprivation, or targeted agents that inhibit specific signaling pathways; 2.) Targeting the bone microenvironment is impactful. As was discussed in the above sections on the bone microenvironment, the complex biological signaling between cancer cells and bone resident cells creates a vicious cycle. Disruption of these interactions represents a therapeutic opportunity; 3.) Palliative therapies focus on alleviating symptoms associated with bone metastasis. This becomes an area that can be very impactful on the quality of life for these cancer patients as bone metastasis can be extremely debilitating and painful.

Most of the following discussion on approved therapeutics will focus on prostate, breast, and multiple myeloma. These are the most common cancers which cause bone metastatic lesions and thus represent the bulk of research efforts to understand the mechanisms involved. Patients with other cancers such as kidney, thyroid, lung and melanoma can also present with metastasis to the bone. There are many treatment commonalities between the various cancers that metastasize to the bone and strategies appropriate for one type of cancer are often effective for others.

A review of current, open, interventional clinical trials for “bone metastasis” was performed using the clinical trials database at clinicaltrails.​gov and 445 trials were found. Relevant clinical trials on cancers involving prostate, breast, renal, thyroid, lung, multiple myeloma, or trials involving therapies for multiple types of cancers were included. This information is included in Table 2.