Inside a Metastatic Fracture: Molecular Bases and New Potential Therapeutic Targets
Orthopedic and Traumatology Unit IRCCS Azienda Ospedaliero‐Universitaria di Bologna Bologna Italy
Department of Biomedical and Neuromotor Sciences University of Bologna Bologna Italy
Department of Medical and Surgical Sciences Alma Mater Studiorum University of Bologna Bologna Italy
Department of Radiology IRCCS Azienda Ospedaliero‐Universitaria di Bologna Bologna Italy
* Correspondence:Andrea Sambri (andrea.sambri@aosp.bo.it)
ABSTRACT
Introduction
Bone metastases and pathological fractures significantly impact the prognosis and quality of life in cancer patients. However, clinical and radiological features alone have been shown to fail to predict skeletal related events of a bone metastasis (SREs).
Aim
This study focuses on key molecular players including Matrix Metalloproteinases (MMPs), Integrins, Bone Morphogenetic Proteins (BMPs), Parathormone‐related Protein (PTHrP).
Results
The RANK/RANKL/Osteoprotegerin (OPG) pathway, and N‐terminal peptide (NTx), involved in the metastatic process and bone integrity disruption. Elevated levels of these molecules have been pointed out as potential biomarkers for predicting SREs, but they have been poorly investigated. Moreover, batimastat, marimastat, tanomastat, andecaliximab, and HIV protease targeting MMPs; Volociximab/M200, cilengitide, abituzumab, and FAK inhibitors targeting integrins; LDN193189, DMH1, and ISLR modulators targeting BMPs; and PTH (7–33)‐CBD targeting PTHrP have shown promising results antagonizing these molecules, but no effect on preventing and managing metastatic fractures has been assessed yet.
Conclusions
This paper underscores the importance of advanced molecular biology and transcriptomics in identifying novel therapeutic targets. The integration of these biomarkers with clinical and radiological assessments using artificial intelligence tools could revolutionize the diagnostics and treatment strategies for patients with bone metastases.
Article notes
Footnote Group
1Introduction
Bone is the third most common metastatic site after lungs and liver [1]. Primary cancers most frequently causing bone metastases include breast cancer (bone metastasis prevalence of 25%–68%), lung cancer (5%–15%), kidney cancer (5%–18%), prostate cancer (8%–15%), and thyroid cancer (6%) [1, 2]. However, recent advancements in cancer treatment have led to improved survival rates, resulting in a higher incidence of bone metastatic disease. Consequently, bone metastases are now also reported in colon cancer (1.1% of 5479 in the study published by Baek et al. [3]), pancreatic cancer (2.2% as stated by Borad et al. [4]), melanoma (over 20% according to Wilson et al. [5]), urothelial cancer (32%–47% according to Shinagare et al. [6]) and hepatocellular carcinoma (around 2% as reported by Bhatia et al. [7]).
In bone metastatic process, cancer cells spread into the bone marrow space, creating secondary lesions by determining microenvironmental changes [8, 9]. Bone metastases can be classified as osteolytic, osteosclerotic, or mixed lesions [9]. Osteolytic metastases are more frequently seen due to metastatic involvement by breast, lung and kidney cancer. They are caused by tumor‐derived factors activating osteclasts, leading to increased bone resorption. Radiographically, these lesions usually appear as areas of bone lysis with cortical resorption. Histologically, tumor cells in the bone marrow cavity are surrounded by active osteoclasts. As cortical wall is destroyed by the osteolytic process, tumor cells can also infiltrate surrounding soft tissues [9, 10]. These areas with bone cortical weakening are prone to fractures even without trauma [10].
On the other side, osteosclerotic metastases result from cancer‐derived factors that stimulate osteoblasts to produce bone matrix. These are more commonly seen in prostate cancer; however, breast cancer can form osteosclerotic metastases as well. Radiographically, these lesions appear sclerotic, typically in vertebral bodies and pelvis [8, 9]. Histologically, tumor cells are surrounded by osteoblasts forming wide trabeculae of woven bone. However, the microstructure of this tumor‐associated woven bone is poorly organized, making the bone prone to pathological fracture as well [9, 10].
Bone resorption and formation are usually coupled processes. However, cancer disrupts this balance, leading to osteolytic or osteoblastic skeletal lesions. Nevertheless, many bone metastases are mixed lesions [9]. It is reported that a bone metastasis can transit from an osteoblastic to an osteolytic pattern through a continuous process, though this is only captured statically during radiographic or histological assessment [9, 11].
The destruction of bone by metastatic disease weakens its load‐bearing capabilities, initially causing microfractures that result in pain. This eventually leads to fractures, most commonly occurring in the ribs and vertebrae [11]. Fractures frequently occur in lytic lesions within weight‐bearing bones, where damage to both cortical and trabecular bone is structurally significant. Certain radiological features can predict an imminent fracture; these include large lesions, predominantly lytic lesions, and those that erode the cortex. Mirels proposed a scoring system based on the site, nature, size, and symptoms of a metastasis. Lesions scoring more than 7 typically require prophylactic surgical intervention, while those scoring 10 or higher have an estimated fracture risk of over 50% [12]. However, the predictive value of Mirels's score has been criticized by many studies for the high rates of unpredicted fractures, for the high inter‐/intra‐observer variation in the score and for the lack of reproducibility [2, 13, 14, 15, 16]. To date, no biomarker is used to predict skeletal related events (SREs, such as pathological fracture, spinal cord compression, bone pain, prophylactic surgical procedures, malignant hypercalcemia). Therefore, the aim of this study is to review the Literature to outline which biomarkers can integrate clinical and radiological assessment to predict metastatic bone involvement and metastatic fracture. This could be useful to prevent metastatic fracture and to define potential targets of medical treatment to reduce metastatic bone involvement.
2Molecules Involved in Metastatic Bone Disease and Potential Therapeutic Antagonists
The Literature has identified many key molecules implicated in the process of bone metastasis, and they have emerged as promising candidates for predicting the risk of bone metastatic involvement and metastatic fractures [9, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28] (Figure 1).
Some of these molecules have also been identified as potential targets for medical therapies [29, 30, 31, 32, 33, 34, 35, 36, 37, 38]. In this article we present some of the most reported ones; however, they may represent only a portion of a broader spectrum of biomarkers that need to be identified to increase the accuracy in the prediction of SREs and to serve as potential pharmacological targets for patients with bone metastases. These molecules exhibit a predominantly osteolytic pattern, like Metalloproteinases (MMPs), integrins, the RANK‐RANKL pathway, Parathormone related peptide (PTH‐rP), or a predominantly osteoblastic pattern like Bone Morphogenetic Proteins (BMPs), but their role can shift from osteolytic to osteoblastic and vice versa in particular cases depending on the specific microenvironment in which they are embedded [39, 40, 41, 42, 43]. Further research is therefore essential to expand our understanding of these biomarkers and to develop targeted therapies to effectively manage bone metastatic disease.
2.2Integrins
Integrins play a role in cancer cell adhesion to the bone matrix and subsequent invasion, in activating osteoclasts and promoting MMPs activity [20, 21]. They also display a pivotal role in avoiding anoikis (programmed cell death due to the detachment from the surrounding ECM) by promoting cancer cell adhesion [7]. In 2021, Pantano et al. used in silico transcriptomic analyses of primary tumors and metastases to identify potential target genes involved in the spread of breast cancer to distant organs. Their study showed that integrin alpha5 (ITGA5) is highly expressed in bone metastases compared to non‐bone metastases. Additionally, multivariate analysis demonstrated that ITGA5 expression in primary breast tumors is an independent prognostic factor for bone relapse; their study identifies ITGA5 as a contributor to breast cancer metastasis to bone and suggests that volociximab/M200 might be repurposed for treating breast cancer patients with bone metastases who are ITGA5‐positive [65]. This outlines the importance of transcriptomic analysis to define gene expression in bone metastases and potential targets to be addressed by medical treatments [65, 66, 67]. Moreover, studies in mouse mammary tumor models and lung cancer show that integrin signaling promotes resistance to various therapies [68, 69, 70]. Integrins are attractive drug targets due to their accessible cell surface sites, but clinical trials have had limited success [71]. Notably, inhibitors targeting integrin‐ligand interactions, such as cilengitide and abituzumab, have failed to improve patient survival in trials in glioblastoma patients, but no data on skeletal involvement in other types of cancer is available [72]. New strategies may focus on tumor‐specific integrin profiles or downstream effectors: FAK, a protein activated by integrins, is linked to drug resistance and immunosuppression [32, 33, 73]. Current trials are investigating FAK inhibitors in combination therapies [7, 32, 74]. Integrins also modulate the tumor stroma, with LOX‐mediated matrix stiffening and bulky glycocalyx structures promoting integrin signaling and tumor survival [32]. Finally, no study has investigated the dosage of integrins in predicting metastatic fractures.
2.3Bone Morphogenetic Proteins (BMPs)
BMPs are members of the transforming growth factor‐beta (TGF‐β) superfamily and play a role in bone formation and homeostasis [22]. BMP signaling demonstrates context‐dependent behavior, ranging from inhibition to tumor promotion. As the BMP pathway regulates the normal balance of stem cells, it often undergoes abnormal activation or repression depending on the microenvironment in which cancer stem cells (CSCs) reside [75]. CSCs, a small subset of tumor cells with stem‐like properties, play a critical role in driving tumor growth, managing therapy resistance, and contributing to recurrence [76]. Fully understanding the complex interactions between CSCs and their surrounding environment is crucial for developing effective treatments. Among the various signaling pathways that control CSCs' cellular behavior, BMP signaling stands out as a key regulator, guiding CSC self‐renewal, differentiation, and the complex interactions within the tumor microenvironment [77]. Additionally, BMP signaling's role in cancer extends beyond CSCs, intricately controlling cellular migration, invasion, and metastasis [34]. BMP signaling exhibits context‐dependent pleiotropic effects in different cancers. For instance, in lung, colorectal, prostate, and breast cancers, BMP signaling can drive tumorigenesis and metastasis, while in others, such as Glioblastoma Multiforme (GBM), BMP signaling suppresses tumor stemness by promoting the differentiation of Glioblastoma stem cells (GSCs) into an astroglial fate, thus inhibiting tumor growth progression [78]. A study has demonstrated that the BMP antagonist, COCO, plays a crucial role in modulating the reawakening of dormant metastatic breast tumors linked to CSCs in the lung, while BMP signaling itself exerts suppressive effects [79]. On the other hand, regarding the promotion of organ‐specific tumor metastasis, the role of BMPs in bone metastasis is particularly well‐documented in prostate cancer. In vitro studies have highlighted the combined effect of BMP4 and SHH in supporting the survival of prostate cancer cells and promoting the differentiation of bone stromal cells, which may lead to the osteoblastic metastasis typical of prostate cancer [80]. Furthermore, in vivo research has emphasized BMP4's role in bone formation within a xenograft model of prostate cancer bone metastasis. Importantly, blocking BMP receptors with LDN193189 has been shown to hinder osteoblast differentiation and reduce tumor growth [43].
Recent studies have highlighted the potential of inhibiting systemic BMP signaling as a strategy to stop tumor progression and metastasis, targeting both the tumor and its surrounding microenvironment. A significant example is the use of dorsomorphin (DMH1), a BMP antagonist, which acts by blocking the activation of BMP type I receptors, preventing their activation. These receptors are essential for BMP signal transduction in cells by promoting phosphorylation of SMAD proteins (particularly SMAD1, SMAD5, and SMAD8) [35]. In this way, DMH1 prevents transcription of genes that regulate cell differentiation, growth, and bone formation. DMH1 has demonstrated promising results: DMH1 treatment has been effective in reducing lung metastases in breast cancer and in human xenograft lung cancer models [34, 35, 81]. Additionally, in vivo studies showed a decrease in tumor proliferation and an increase in apoptosis, underscoring the therapeutic potential of targeting BMP signaling [81]. In this 2021 study, the Authors tested a modulator of the effect of BMPs on the metastatic niche (ISLR) obtaining a prolonged survival of mice in which the drug was tested compared to the control group [82]. ISLR was on the other side evaluated as a potential target for osteoporosis in mice in this 2024 study, where ISLR knockout mice showed enhanced bone formation [83]. Actually, ISLR knockout led to increased osteogenic differentiation and mineral accumulation; ISLR acts as a negative regulator of osteogenic differentiation via the BMP4‐Smad‐ColIα1/Ocn pathway; ISLR interacts with BMP4 to influence osteogenic differentiation, facilitating the proteasomal breakdown of BMP4; targeted deletion of ISLR in osteoblasts resulted in higher bone density in Ocn‐cre/flox mice, whereas mice with overexpressed ISLR exhibited reduced bone mass; therefore, ISLR was identified as a new regulatory factor in osteogenic differentiation and bone growth [83]. However, the effect on skeletal metastases was not specifically assessed in any study as well as the potential use of BMPs in the prediction of metastatic fractures.
2.5 RANK/RANKL/Osteoprotegerin (OPG)
Receptor Activator of Nuclear Factor Kappa‐B (RANK) and its molecular signaling cascade play a critical role in the development of bone metastases and the resulting pathological fractures observed in advanced cancers [25, 26, 27]. RANK is a receptor expressed on the surface of osteoclast precursors and mature osteoclasts, which are the cells responsible for bone resorption. Its primary ligand, RANKL, is produced by osteoblasts, stromal cells, and even cancer cells in the bone microenvironment [25, 91]. The binding of RANKL to RANK is a critical step in osteoclast differentiation, activation, and survival [91]. This interaction promotes the maturation of osteoclasts from their precursors and stimulates bone resorption, a process essential for normal bone remodeling but detrimental when dysregulated in the context of bone metastasis [25, 91].
The RANK/RANKL/OPG (osteoprotegerin) axis is tightly regulated under normal physiological conditions, where OPG acts as a decoy receptor for RANKL, preventing its interaction with RANK and thereby inhibiting osteoclastogenesis [91]. However, in cancer, the balance of this axis is often disrupted [26]. Tumor cells can produce RANKL or induce stromal cells to increase RANKL production while simultaneously decreasing OPG levels, leading to enhanced osteoclast activity and bone resorption [91, 92]. This creates an environment conducive to bone metastasis, as the resorption process releases growth factors from the bone matrix that further promote tumor cell proliferation and survival.
The RANK/RANKL/OPG pathway is central to the vicious cycle of bone metastasis and metastatic fractures: as tumor cells invade the bone, they disrupt normal bone homeostasis by increasing RANKL expression, which in turn enhances osteoclast‐mediated bone destruction; this not only facilitates the growth of the metastatic tumor within the bone but also leads to the deterioration of bone integrity, making it prone to fractures even under normal stress [93].
Therapeutically, targeting the RANK/RANKL pathway has shown significant promise in treating bone metastases and preventing SREs. Denosumab, a monoclonal antibody that binds to RANKL, effectively inhibits its interaction with RANK, thus preventing osteoclast activation [29, 38, 94]. Clinical trials have demonstrated that denosumab reduces the incidence of SREs, including pathological fractures, in patients with bone metastases from solid tumors [95, 96]. This highlights the critical role of the RANK/RANKL pathway in the pathophysiology of bone metastasis and underscores the potential of targeting this pathway to improve outcomes in patients with metastatic bone disease [29, 38, 94]. As well as denosumab, bisphosphonates represent a cornerstone in the management of metastatic bone involvement, showing reduced SREs [86, 87, 97, 98]. Despite not directly targeting RANKL, they interfere with the RANK/RANKL/OPG chain by inhibiting osteoclast function by two main mechanisms: (1) Non‐Nitrogen‐Containing Bisphosphonates (N‐NBPs): these compounds, such as etidronate and clodronate, are metabolized by osteoclasts into non‐hydrolyzable ATP analogs, which accumulate within the cell and lead to apoptosis. This disrupts the energy metabolism of osteoclasts, effectively reducing bone resorption; (2) Nitrogen‐Containing Bisphosphonates (N‐BPs): drugs like alendronate, zoledronate, and risedronate belong to this group. They inhibit the mevalonate pathway, a crucial metabolic pathway in osteoclasts, which leads to the disruption of protein prenylation—a process necessary for the function of small GTPase signaling proteins. This disruption causes osteoclast apoptosis, thereby inhibiting bone resorption more effectively than N‐NBPs [97, 98]. Interestingly, prostate cancer bone metastases also have a lytic component underlying their osteoblastic phenotype, with RANK/RANKL/OPG playing an important role [86]; actually, denosumab and bisphosphonates have been positively used also to reduce SREs in prostate cancer [86, 99].
Therefore, the RANK signaling cascade is crucial to the process of bone metastasis and the development of pathological fractures. By promoting osteoclast‐mediated bone resorption, the RANK/RANKL interaction not only facilitates tumor growth within the bone but also leads to the structural weakening of bones, resulting in fractures. Targeting this pathway is the gold standard approach to manage bone metastases and reduce the incidence of SREs in cancer patients. However, both denosumab and bisphosphonates are burdened by side effects like jaw osteonecrosis, hypocalcemia, urinary tract infections, dermatitis, hypercholesterolemia, and atypical fractures [87, 100]; moreover, RANK, as shown in this article, is not the only pivotal factor in the bone metastatic process; thus, molecular biology and transcriptomic analysis may be crucial in defining new molecular targets other than the RANK/RANKL/OPG pathway, optimizing bone metastases treatment [65, 66].
2.6N‐Terminal Peptide (NTx)
NTx, a byproduct of type I collagen degradation, serves as a biomarker for osteoclastic activity and bone resorption. Its serum and/or urine dosage can play a significant role in the context of bone metastases and pathological fractures, providing critical insights into the processes of bone resorption and turnover [101].
In patients with bone metastases, elevated levels of NTx are indicative of increased osteoclastic activity [102]. This heightened resorption activity can weaken the bone structure, predisposing patients to pathological fractures [17, 28]. Monitoring NTx levels can thus provide valuable prognostic and diagnostic information in the management of patients with cancers that have a high propensity to metastasize to bone [17, 101, 103, 104].
The role of NTx extends beyond mere indication of bone turnover. It also offers insights into the efficacy of treatments aimed at reducing skeletal‐related events (SREs) in cancer patients. Therapies that target bone resorption, such as bisphosphonates and denosumab, aim to mitigate the osteoclastic activity that leads to high NTx levels [105, 106]. By monitoring changes in NTx levels, clinicians can assess the therapeutic response, adjusting treatment plans to optimize patient outcomes and potentially prevent the occurrence of pathological fractures [42, 107, 108].
Furthermore, understanding the dynamics of NTx in the bone microenvironment helps in deciphering the mechanisms through which bone metastases facilitate tumor growth and progression. The interaction between cancer cells and bone cells can stimulate the production of factors that increase bone resorption, thereby elevating NTx levels [103, 104]. This interaction not only aids in the diagnosis and management of bone metastases but also enhances our understanding of the metastatic process itself, offering potential avenues for targeted therapeutic interventions [42, 107, 109].
In summary, NTx might serve as a crucial biomarker in the management of bone metastases and in the prevention of metastatic fractures. Its role in reflecting osteoclastic activity provides essential insights into bone health in patients with advanced cancers, guiding therapeutic strategies and contributing to a more comprehensive understanding of bone metastasis biology (Table 1).
| Molecule | Role in bone metastasis | Associated drugs | Clinical trial phase | Main limitations |
|---|---|---|---|---|
| MMP‐9 | Bone matrix degradation, osteoclast activation, bone resorption | MMP inhibitors (marimastat, batimastat); humanized anti‐MMP‐9 monoclonal antibodies; HIV‐protease inhibitors | Preclinical and clinical studies [53, 55, 56] | Toxicity, poor specificity, lack of trials on bone metastasis |
| Integrins | Tumor cell adhesion to bone matrix, osteoclast activation, promoting MMPs activity | Integrin inhibitors (cilengitide, abituzumab); FAK inhibitors | Clinical studies [33, 72, 73] | Limited efficacy, difficulty in specific targeting, lack of trials on bone metastasis |
| BMPs | Promoting the differentiation of bone stromal cells, sclerotic bone formation | BMP antagonist (DMH1); modulator of the effect of BMPs on metastatic niche (ISLR) | Preclinical studies [35, 39] | Dual roles of BMPs (tumor suppression vs. pro‐ metastatic signaling), lack of trials on bone metastasis |
| PTHrP | Stimulates osteoblasts to produce RANKL, promoting osteoclastogenesis, bone resorption | Denosumab (anti‐RANKL); PTHrP antagonist (PTH (7–33)‐CBD) | Denosumab: approved; [29, 38, 94, 95] preclinical study [90] | Side effects, lack of trials on bone metastasis |
| RANK/RANKL | Regulation of osteoclastogenesis, bone resorption | Denosumab (anti‐RANKL), bisphosphonates | Approved for clinical use [87] | Side effects: jaw osteonecrosis, hypocalcemia, urinary tract infections, dermatitis, hypercholesterolemia, and atypical fractures |
| NTx | Biomarker for osteoclastic activity and bone resorption. Monitors the efficacy of bisphosphonate or denosumab treatment | — | — | Limited to diagnostic/prognostic role |
3Conclusion
Nowadays, clinical and radiological features alone fail to predict skeletal related events of a bone metastasis. This study underscores the pivotal role of MMPs, integrins, BMPs, PTHrP, RANK, and NTx in bone metastatic involvement and metastatic fracture. Their dosage can be useful to integrate clinical and radiological features to predict skeletal related events. To date, batimastat, marimastat, tanomastat, andecaliximab, and HIV protease for MMPs, Volociximab/M200, cilengitide, abituzumab, and FAK inhibitors for integrins, LDN193189, DMH1, and ISLR modulators for BMPs, PTH (7–33)‐CBD for PTHrP have shown promising results in targeting these molecules, but no effect on preventing and managing metastatic fractures has been assessed yet. Therefore, studies outlining their effects on skeletal related events can be a turning point in the treatment of bone metastases, aside from denosumab and bisphosphonates that only target the RANK/RANKL/OPG cascade and have several side effects. Finally, molecular biology and transcriptomics are crucial in identifying new genes and potential therapeutic targets involved in bone metastases and metastatic fractures. Including these data in artificial intelligence tools considering clinical, radiological, and biomarkers features can offer more precise diagnostics and therapeutic possibilities to patients suffering from bone metastases.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgements
Open access funding provided by BIBLIOSAN.
Data Availability Statement
This study did not generate or analyze any datasets.