Metastasis is defined as a very inefficient process, since less than 0.01% of cancer cells injected into the circulation will engraft in a distant organ, where they must acquire the ability to survive and proliferate inside a “foreign” environment. In bone metastases, the interaction with the host organ is much more favoured if tumour cells gain “osteomimicry”, that is the ability to resemble a resident bone cell (i.e. the osteoblast), thus intruding in the physiology of the bone. This is accomplished by the expression of osteoblast markers (e.g. alkaline phosphatase) and the production of bone matrix proteins and paracrine factors which deregulate the physiology of bone, fuelling the so-called “vicious cycle”. The main challenge of researchers is therefore to identify the genetic profile determining the osteotropism of tumour cells, which would eventually lead to bone colonisation. This could likely provide the answer to a quite intriguing question, that is why some cancers, such as prostate and breast, have a specific predilection to metastasise to the bone. Therefore, it is important to completely address the molecular mechanisms underlying this aspect of bone oncology, identifying relevant pathways, the targeting of which could make any type of bone metastasis curable or avoidable.
Metastasis is paradoxically defined as a very inefficient process, since it has been estimated that less than 0.01% of cancer cells injected into the circulation will take root in a distant organ . Understanding what allows this exiguous number of cells to survive in the circulatory bed, extravasate and colonise a secondary site is the great challenge for the researchers. Indeed, several steps must be completed by tumour cells in order to colonise a distant organ, which include also genetic and epigenetic changes of their profile. The Epithelial–Mesenchymal Transition (EMT) step allows tumour cells to acquire a motile phenotype, so that they detach from the primary site, migrate and invade the surrounding connective tissue and reach the circulatory system. Escape of host immune reaction and the ability to bear shear stress during the circulation represent another rate-limiting step crucial for tumour cell fate before reaching a distant organ, where they deal with the resident cells by producing soluble factors and/or establishing cell–cell interactions. As it will be discussed later in detail, in bone metastases these interactions are much more favoured if tumour cells have acquired osteomimicry properties, that is the ability to resemble a resident bone cell (i.e. the osteoblast), thus intruding in the physiology of the bone. This is assumed to be very likely because this way bone metastatic cells can better communicate with the host organ, thus increasing their chance of survival. The main challenge of researchers is therefore to identify the genetic profile determining the osteotropism of tumour cells eventually leading to a successful bone colonisation.
The second intriguing question is why and how some tumour cells show the predilection to metastasise a specific organ rather than another. This is an old question, which could have an old answer, moving back to the 1889 and referring to Steven Paget’s studies , which represent a milestone for scientists working on metastases. Paget proposed the so-called “seed and soil” theory, which points out that the occurrence of cancer metastasis in a specific organ is not due to chance but it depends on the affinity of tumour cells (the seeds) for the target organ (the soil). This means that the metastasis is formed thanks to a specific tumour cell tropism towards a secondary site, which in turns depends on the molecular profile of tumour cells. Therefore, the study of the “soil” could be of great relevance to understand the mechanisms of the “seed” inducing tumour colonisation of a specific distant organ.
Later on (1928), Ewing challenged the Paget’s theory, proposing that metastasis is mainly influenced by the vascular system’s anatomy . In particular, he postulated that those organs more sprayed and containing haematic filters are usually the preferential sites of metastases. Moreover, the anatomic proximity to a primary tumour is also another factor influencing cancer dissemination.
These two theories are not in opposition but quite complement each other. In fact, we should consider that the lung and liver represent the most affected sites for metastases (50 and 40%, respectively), followed by skeleton (20%), brain and adrenal gland (10%). However, in agreement with the seed and soil theory, it is well ascertained that some tumours have a predilection to selectively metastasise specific sites, thus suggesting the need for a sort of host-tumour “compatibility” for that metastasis to occur. This is the case of bone metastases, which are observed in 65–75% of advanced breast and even more prostate cancer patients, in which the prevalence reaches 90% [4, 5].
In this review, we will dissect the principal players involved in tumour cell homing to bone, with particular regard to the phenomenon of osteomimicry, which could be defined as a “Trojan horse”, exploited by tumour cells to survive and grow in the bone.
Biology of Bone Metastases
As already told, bone metastases are a frequent complication of breast and prostate cancers and, to a lesser extent, lung, kidney and melanoma. Once they occur, survival chances dramatically drop along with the quality of life, since patients usually experience bone pain, spinal cord compression, pathological fractures and hypercalcemia. Like other metastases, bone lesions are incurable and only palliative treatments are available so far, which can ameliorate the quality of life but do not improve the overall survival .
From a clinical point of view, bone metastases can be osteolytic, osteosclerotic and with mixed features, the former typically being observed in breast cancer patients, while about all the prostate cancer patients with bone metastases developing osteosclerotic lesions [5, 6].
As it will be discussed in more details in the next paragraph, osteolytic lesions represent areas in bone districts (e.g. vertebrae, sternum, femur) where there is no more bone, being completely reabsorbed by an exacerbated activity of osteoclasts, triggered by tumour cells . Conversely, osteosclerotic or osteoblastic metastases appear dense in radiographic analyses and, in fact, they are characterised by an excessive bone deposition, which however is preceded, and likely triggered, by an exacerbated osteoclast activity, eventually creating a physical space, inside a “hard tissue” like the bone, in which tumour cells can home. Therefore, after this bone erosion, osteoblasts are stimulated to lay down new bone, which however is of poor quality, being comparable to a woven bone, with the final result that patients carrying these metastases may experience bone fractures. Whatever is the morphological feature of a bone metastasis, this is always the result of a dramatic deregulation of bone remodelling orchestrated by tumour cells .
Bone Remodelling: The Virtuous Versus the Vicious Cycle
Bone health relies on a perfect balance between the osteogenic functions of osteoblasts and the resorptive activity of osteoclasts, which accomplishes the bone remodelling process. This allows a continuous renewal of bone throughout the life of each individual as well as a correct calcium homeostasis and repair of microfractured bone.
Several factors, systemic and paracrine, regulate bone remodelling by influencing osteoblast and osteoclast activity. Moreover, these cells interact with each other to reciprocally regulate their functions. Osteoblasts are the source of two key regulators of bone resorption, the production of which must be perfectly balanced to keep a proper remodelling: receptor activator of nuclear factor kappa B (NFκB) ligand (RANKL) and osteoprotegerin (OPG) [7, 8]. RANKL is mainly produced as a transmembrane and to a lesser extent as a soluble cytokine, and interacts with the receptor RANK expressed by osteoclast precursors, which in turn activates an NFκB-mediated signalling, thus promoting osteoclast differentiation. The synthesis of RANKL is not exclusive of osteoblasts in the bone, since also osteocytes and bone marrow stromal cells contribute to its production .
Besides its role in bone homeostasis, RANKL has been shown to be implicated in the mammary gland development, in the immune system regulation and as a survival factor for dendritic cells [10,11,12]. Looking at the pathologic side, its role as a pro-tumour and pro-metastatic factor in breast cancer has also been clearly ascertained. As it will be described later, tumour cells produce RANKL, which acts in an autocrine manner and in support of the osteomimicry profile. Considering the pro-osteoclastogenic and pro-tumoural role of RANKL, this finding makes this cytokine even more attractive as a therapeutic target in breast cancer malignancies.
OPG is the other side of the coin of osteoclast regulation. This is a secreted glycoprotein member of the Tumour Necrosis Factor Receptor (TNFR) superfamily, expressed in bone, bone marrow, heart, kidney, liver and spleen and produced by osteoblasts, endothelial cells and vascular smooth muscle cells . It shares the same extracellular structure of RANK but, lacking the transmembrane domain, it works as a decoy receptor, which binds RANKL and prevents its interaction with RANK expressed by osteoclast precursor, inhibiting osteoclastogenesis . Therefore, a controlled osteoclast differentiation relies on an optimal RANKL/OPG ratio, which in turn guarantees a correct bone remodelling in a balanced “virtuous cycle”.
When tumour cells finally reach the bone, they wreck the balance between osteoblast and osteoclast functions, thus disrupting the bone remodelling process and diverting bone environment in their favour. A “vicious cycle” is therefore established, fuelled by tumour cells through the production of factors that destroy the osteoblast–osteoclast function coupling (Fig. 1) . Among them, there is the parathyroid hormone-related peptide (PTHrP), a paracrine regulator of bone remodelling produced by osteoblasts. Breast cancer cells that switch towards an osteomimicry phenotype produce this factor, which, in turn, increases the RANKL while reducing the OPG synthesised by the osteoblasts, eventually leading to an exacerbated bone resorption . Tumour cells can also directly stimulate osteoclastogenesis producing TNF α, Interleukin (IL) 1β, IL-6, IL-8 and IL-11 (Fig. 1). Consistently, chronic inflammation is usually linked to tumour progression, as well as to the ability of tumour cells to awake from a dormant state leading to bone metastasis relapse. Finally, it has been demonstrated that breast cancer cells also exert pro-apoptotic effect on osteoblasts, thus further compromising the bone mass.
In this scenario, what perpetuates the vicious cycle is that not only the excessive bone resorption dramatically compromises the bone, but it further promotes tumour cell growth, since it allows the release in the bone milieu of growth factors usually embedded in the bone matrix. These include platelet-derived growth factor (PDGF), transforming growth factor (TGF) β, insulin-like growth factors (IGFs), some bone morphogenetic proteins (BMPs) and fibroblast growth factor (FGF), which exert pro-survival effects on tumour cells (Fig. 1). Likewise, calcium released by osteoclast bone resorption is another enemy responsible for hypercalcaemia, a serious and potentially lethal condition in bone metastatic patient. Moreover, it has been demonstrated that in tumour cells expressing the calcium-sensing receptor this ion promotes their proliferation as well as PTHrP production, which in turn inhibits nuclear accumulation of the pro-apoptotic factors, thus promoting tumour cell survival .
With regard to the molecular mechanisms that orchestrate the development of osteosclerotic lesions, these likely rely also on the capacity of cancer cells to produce and release their own paracrine factors in the bone microenvironment, which in turn alter osteoblast differentiation and function (Fig. 2). Hence, there is an intense crosstalk between cancer cells and osteoblasts. As it will be described in the next paragraphs, the underlying factors include especially wingless-related integration site (Wnt)-1, IGF-1, BMPs and endothelin (ET)-1.
Osteomimicry: Tumour Cells Mirroring Bone Cells
Osteomimicry is defined as the ability of tumour cells that preferentially metastasise the bone to express a genetic profile similar to that of the resident cells, i.e. the osteoblasts, thus acquiring a more favourable phenotype for the survival in the bone. Since osteoblasts are of mesenchymal origin, these phenotypic changes likely occur in a tumour cell that has already undergone the EMT, a fundamental step for tumour cell migration and invasion of distant organs.
Before going to the core of the osteomimicry process, it would be useful to outline the main phases of osteoblastogenesis.
Osteoblasts originate from the mesenchymal stem cell (MSC) lineage. In the presence of proper stimuli, MSCs can give rise to different cell populations, such as muscle cells, fibroblasts, chondrocytes, adipocytes and osteoblasts. Commitment towards the latter cell type requires a fine-tuned balance between inhibitory and stimulatory factors. However, the primum movens is the activation of the Wnt pathway. Wnt is part of a large family of secreted glycoproteins (at least 19 in humans), involved in the regulation of many biological processes, such as embryonic development and stem cell regulation. From a general point of view, the intracellular signalling triggered by Wnt ligands after their interaction with LRP5/6 receptor can be β-catenin dependent (canonical pathway) or independent (noncanonical pathway). In bone metabolism, Wnt has a crucial role since it stimulates osteogenic differentiation. In particular, Wnt3A and Wnt10b cooperate to inhibit adipogenic differentiation of MSCs in favour of osteoblast commitment . Consistently, overexpression of Wnt10b increases trabecular bone thickness, while its deficiency leads to a decrease of bone density . Wnt5a is also a pro-osteogenic factor, since osteoblasts from Wnt5a−/− mice showed significantly lower level of the osteoblast markers alkaline phosphatase (Alp), osterix (Osx) and osteocalcin (OCN), while the adipogenic markers were increased.
Another crucial signalling pathway for osteoblast differentiation is mediated by BMPs. These molecules belong to the TGFβ family and act especially in the early phases, favouring the MSC commitment towards osteo/chondroprogenitors. Different isoforms are involved, such as BMP-2, 4, 6 and 7. Osteoblast conditional knockout of BMP-2 and BMP-4 dramatically impairs osteogenesis, while BMP-7 has been shown to increase their ALP activity and mineralisation.
Osteo/chondroprogenitors are committed cells expressing the following transcription factors: Runx2, also known as Osteoblast-specific factor 2 (Osf2) and Core-binding factor alpha 1(Cbfa1), Distal-less homeobox 5 (Dlx5) and Osx. The crucial role of Runx2 as a master gene in osteoblast differentiation is no doubtful and was clearly demonstrated by Ducy and colleagues, who found that Runx2 knockout mice lack bone formation due to the absence of osteoblasts . Runx2 in turn drives the transcriptional expression of ALP, Collagen 1α1 (Col1A1), Bone Sialoprotein (BSP) and BGLAP, the latter being the gene coding for OCN.
Another pathway called into question in osteoblast differentiation is mediated by the Hedgehog family, which includes three orthologues of secreted proteins named desert hedgehog (DHh), sonic hedgehog (SHh) and Indian hedgehog (IHh). It seems that the Hedgehog pathway exerts an inhibitory effect on adipogenesis coupled with a stimulatory effect on osteoblastogenesis .
If the osteo/chondroprogenitor expresses Runx2 and Collagen I, it can be considered an osteoprogenitor, which, after a phase of proliferation and acquisition of ALP activity, becomes a pre-osteoblast. Going towards a mature osteoblast, the cell changes morphology and molecular profile, becoming cuboidal, highly positive to ALP, producing large amounts of bone matrix proteins. Finally, in the late stage of osteoblast differentiation we have a very active cell, producing the highest amounts of bone matrix proteins, including OCN, which is usually considered a late marker of osteoblast differentiation, while ALP progressively drops. In histological sections, mature active osteoblasts appear as cobble-stone-shaped cells placed in a single file on the bone surface they have just produced.
Osteomimicry: How to Become a Bone-Like Cell
The concept of osteomimicry started to grow with the evidence that clinical prostate cancer samples expressed bone matrix proteins, such as OCN, BSP and osteopontin (OPN), at significantly higher levels compared to the normal tissue. In 1992, Curatolo et al. proposed OCN and bone ALP as potential markers in the follow-up of prostate cancer patients with bone metastases . One of the first works that clearly defined the phenomenon of osteomimicry dated 1999, by Koeneman and co-authors, who came to the following conclusion: “We propose the hypothesis that osseous metastatic prostate cancer cells must be osteomimetic in order to metastasize, grow and survive in the skeleton” . In this paper, the authors showed an autocrine and paracrine role for the transcription factors Runx2 and MSX in prostate cancer cells, which in turn increased the expression of the bone matrix proteins OPN, OCN and BSP, thus mimicking osteoblast features.
Consistently, a later work by Knerr et al., who investigated the crosstalk between osteoblasts and prostate cancer cells, found that osteoblast-released factors influenced tumour cells’ behaviour by slowing down their proliferation, changing their adhesion properties and inducing an osteomimicry profile, characterised by an increased expression of ALP, BMP2, Col1α1, OPN and OCN . Consistent with a reciprocal influence between tumour cells and osteoblasts, conditioned medium from osteoblasts induced mineralisation of the prostate cancer cell line LNCaP-19 and the expression of the osteogenic profile as well as of the genes involved in the metastatic progression.
Over the years, the landscape of osteomimicry has become more complex, also following the advances in the osteoblast biology. Moreover, similar osteomimicry pathways have been observed also in breast cancer cells, as shown by Bellahcène and colleagues, who demonstrated an osteoblast-like gene signature in human osteotropic breast cancer cells, characterised by a significant overexpression of pro-osteoblastic genes . On this basis, in the following paragraphs we will illustrate the principal players contributing to the osteomimicry profile of prostate/breast cancer cells.
Expression of this transcription factor by tumour cells is a crucial determinant in the acquisition of osteomimicry properties by prostate cancer cells. Yeun and colleagues also demonstrated an activation of Runx2 driven by the transcription factor TWIST, which in turn enhanced osteomimicry of prostate cancer cells . Runx2 expression is significantly increased in breast cancer cells, compared to normal breast tissue, in association with the osteoblast-related genes BSP and OPN, likely regulating their expression. Conversely, the overexpression of a dominant-negative mutant form of Runx2 in breast cancer cells inhibited their ability to induce bone metastases, likely by reducing RANKL and TNFα secretion. The role of Runx2 as a master gene regulating the expression of several osteomimicry genes has also been highlighted by Tan and colleagues  (Fig. 3a). In this work, they demonstrated the expression by breast cancer samples of a set of bone-related genes (BRGs), which partially match with the previous osteomimicry gene signature identified by Bellahcène et al.  and Kang et al. . These BRGs are highly co-expressed in bone metastases when compared to breast cancer metastases in other organs and their expression is activated by the transcription factor Runx2 which, as suggested by the authors, acts as a “master mediator during the transformation of epithelial breast cancer cells into osteomimetic cells”  (Fig. 3a). The expression of BRGs’ profiling in turn increased multidrug resistance in tumour and bone microenvironment.
Based on this finding, we can assume that the expression of Runx2 by tumour cells is an important detail in the picture of osteomimicry.
The central role of Wnt/β-catenin pathway in osteoblast differentiation has been clearly elucidated. What has been recently demonstrated is also an involvement of Wnt in the regulation of osteoclasts by osteoblasts, thus further strengthening the role of this pathway in bone homeostasis. Coherently with the osteogenic role of this signalling, its effect on osteoclasts is mainly inhibitory. In fact, it has been demonstrated that Wnt3a produced by osteoblasts impaired osteoclastogenesis in co-cultures with bone marrow cells . Wnt16 is another recently discovered negative regulator of osteoclastogenesis produced by osteoblasts, which inhibits RANKL expression .
Due to the complex role of Wnt in bone homeostasis, it is conceivable to hypothesise an involvement of this pathway in bone-related malignancies, especially with regard to the mechanisms of prostate cancer-induced osteoblastic metastases. It is known, since many years, that both prostate and breast cancer cells express Wnts, such as Wnt2, Wnt5a and Wnt7b, while Hall and colleagues demonstrated not only an autocrine effect of Wnts in stimulating prostate cancer cell proliferation, but also a paracrine role in osteoblasts, increasing their activity (Fig. 3b) .
The identification of Wnts as the key players in bone metastases took advantage from the employment of typical inhibitors of this pathway, such as DKK-1, which was able to significantly increase in vivo the osteolytic potential of the C4-2B prostate cell line, which usually gives mixed (osteoblastic/osteolytic) metastases, thus switching towards an osteolytic phenotype. Consistently, a significantly higher expression of the Wnt/nuclear β-catenin pathway has been observed in bone metastasis samples of prostate cancer patients . Finally, Wnt molecules are significantly more expressed in prostate cancer cell lines that are osteotropic, such as PC3, compared to DU145 and LNCaP arising from brain and lymph node metastases, respectively . Taken together, all these results highlight the role of Wnts in prostate cancer tropism to the bone.
As already discussed, RANKL is a key regulator of tumour invasion and bone colonisation by fuelling, along with other factors, the vicious cycle. Indeed, it has been documented that the autocrine production of RANKL by prostate cancer cells, as well as RANKL arising from osteoblasts, induced bone and visceral metastases by a mechanism involving AP4 and c-Myc/Max transcription factors  (Fig. 4). RANKL signalling also reduced androgen receptor expression in prostate cancer cells, thus inducing androgen resistance, and drove their ability to colonise bone and recruit bystander cells in the bone metastasis  (Fig. 4a).
As stated above, OPG is a well-known molecule involved in osteoclast differentiation . Although OPG expression is not exclusive of the bone, its key role in the RANKL/RANK-mediated regulation of osteoclastogenesis makes it a pillar in bone metabolism. In fact, this decoy receptor produced by osteoblasts binds to RANKL thus avoiding its interaction with RANK expressed by osteoclast precursor, eventually inhibiting osteoclastogenesis and bone resorption . The involvement of OPG in prostate cancer is known since many years. Therefore, serum OPG levels have been associated with disease progression and response to androgen ablation in patients with prostate cancer.
Although the final effect of OPG in the bone is an inhibition of osteoclast resorption, which would not fit with the pro-bone colonising effect of osteomimicry, OPG is also a decoy receptor for Tumour necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) that inhibits TRAIL-induced apoptosis. This has been clearly demonstrated by Holen et al. , who also found that the antiapoptotic effect of OPG was reversed by co-administration of 100-fold molar excess of RANKL, thus pointing out the importance that cancer-derived OPG may have in the survival of hormone-resistant prostate cancer cells (Fig. 4b). Consistently, Knerr et al.  demonstrated that osteoblast-derived factors released in conditioned media stimulate prostate cancer cell expression of osteomimicry genes, including OPG.
A similar antiapoptotic effect of OPG was also confirmed in breast cancer by Neville-Webbe and colleagues , showing that bone marrow stromal cells isolated from breast cancer patients release OPG in the conditioned medium, which in turn protects breast cancer cells from TRAIL-induced apoptosis. Consistently, a significant upregulation of OPG in an osteotropic MDA-MB-231 subclone was observed compared to the parental and multiorgan-metastasising cell line, thus indicating a positive correlation between OPG expression and breast cancer cells homing and colonising the bone. Induction of proliferation, angiogenesis and aneuploidy in human mammary epithelial cells by OPG was also observed, along with an upregulation of the cell adhesion protein, CD24. Finally, Higgs and colleagues  proposed the development of OPG variants retaining RANKL binding and lacking TRAIL binding, thus allowing TRAIL-mediated apoptosis, which in turn inhibits osteolytic lesions and promotes TRAIL-induced apoptosis of tumour cells.
Taken together, these data demonstrate that OPG, produced by tumour cells, gives them an advantage in terms of survival and the ability to metastasise the bone.
Bone Matrix Proteins
Several noncollagenous proteins contribute to the organic bone matrix. However, their expression is not exclusive of this tissue. Among them, BSP, OPN and matrix extracellular phosphoglycoprotein (MEPE), along with dentin matrix protein (DMP)1 and dentin sialophosphoprotein (DSPP), belong to the Small Integrin-Binding Ligand N-linked Glycoproteins (SIBLINGs), a family of proteins mainly involved in the regulation of cell adhesion, motility and survival .
OPN is a secreted adhesive glycoprotein with a functional RGD cell-binding domain that interacts with several integrins, such as αvβ1, αvβ3, αvβ5, α4β1, α8β1, α9β1 and with the cell–cell interaction, adhesion and migration receptor, CD44 . Different functional forms can be taken by OPN, according to the different post-translational changes (i.e. glycosylation, phosphorylation and sulphation). Moreover, OPN is subjected to cleavage by thrombin, thus changing its function and ability to interact with integrins.
The specific role of OPN in prostate cancer progression was highlighted by Thalmann et al. , who identified this protein as a paracrine and autocrine mediator of prostate cancer growth and progression, stimulating the anchorage-independent growth of tumour cells, likely by binding to the CD44 receptor. Carlinfante et al.  investigated the expression of OPN in skeletal metastases and matched primary tumours from breast and prostate cancer patients. They found a moderate to strong OPN expression in 42% of all breast tumours and in 56% of all prostate tumours, while significantly more breast cancer bone metastases showed high OPN expression compared to prostate tumour bone metastases . These results were strengthened by Shevde and colleagues , while in 2003 Kang et al. demonstrated that MDA-MB-231 cells carrying a triple overexpression of CXCR4, IL-11 and OPN exhibited a dramatic increase both in the stimulation of osteoclast adhesion and in the incidence of bone metastases . Finally, recent reports show that the upregulation of OPN due to aberrant Hedgehog signalling induced osteoclast bone resorption through upregulation of cathepsin K and MMP9, thus suggesting a direct effect of OPN in the enhancement of osteolytic lesions .
BSP is another glycoprotein highly expressed in the bone and produced by osteoblasts, osteocytes and hypertrophic chondrocytes . Similarly to OPN, several studies showed an involvement of BSP in malignancies, by increasing the growth of breast cancer cells in vitro and in vivo. With regard to the osteomimicry, Bellahcène and colleagues clearly demonstrated that BSP expression positively correlated with malignancies and bone metastasis development . Consistently, Waltregny and co-authors  found that prostate cancers expressed detectable levels of BSP, while no or low expression was observed in the adjacent normal glandular tissue and these levels positively correlated with tumour progression. Further studies conducted by Zhang and colleagues demonstrated that the overexpression of BSP stimulated the development of bone metastases . Moreover, BSP overexpression in a subclone of MDA-MB-231 that usually metastasises the brain (i.e. MDA4-231BR) induces osteotropism, thus switching metastasis predilection of these tumour cells from the brain to bone .
Consistent with all these findings, recent results show that local administration of siRNAs targeting OPN and BSP encapsulated in nanoparticles significantly reduced breast cancer-induced skeletal metastases in rat models .
Few reports have called into question also OCN as a determinant of osteomimicry. This protein, mainly produced by mature osteoblasts, is expressed by malignant prostate cancer cells and related bone metastases but not by normal prostate tissue . Huang and colleagues demonstrated that the promoter activity of OCN and BSP is significantly enhanced in an androgen-dependent prostate cancer cell line after treatment with conditioned medium from prostate cancer or bone marrow stromal cells, through a PKA-dependent mechanism .
Another bone matrix protein contributing to the osteomimicry properties of tumour cells is osteonectin (ON) [also known as Basement Membrane (BM-40) and secreted protein acidic and rich in cysteine (SPARC)]. This is a calcium-binding glycoprotein produced by osteoblasts, fibroblasts and endothelial cells involved in cell–matrix interactions, migration and angiogenesis. ON was found to be expressed at low levels in normal prostate tissue as well as in few primary prostate cancer samples, while high levels of ON were observed in most of the prostate cancer metastatic foci. Consistently, Jacob et al.  demonstrated the ability of ON to promote the migration and invasion of bone-metastasising prostate and breast cancer cells. Other groups highlighted the role of ON in prostate cancer-induced bone metastases, thus concluding that ON expression is increased in prostate cancer metastases and could therefore be an earlier marker of a less favourable outcome in prostate cancer patients.
β2-M is a major histocompatibility protein co-receptor which, besides its well-known role in immunity, has been involved in bone remodelling regulation. It induces bone mineral dissolution and calcium efflux by a mechanism at least in part mediated by IL-1β, as well as osteoblast proliferation. A role for this protein in osteomimicry of prostate cancer cells has also been demonstrated, through the stimulation of the expression of BMPs and RANKL, which in turn promote the EMT as well as bone metastasis development. β2-M seems to work by activating the PKA, eventually leading to an increased expression of some CREB target genes, such as OC and BSP. These results were confirmed by the same authors in 2011, demonstrating that β2-M overexpression in prostate, breast, lung and renal cancer cells significantly increases bone metastases and reduces survival in animal models . This is achieved by a mechanism requiring a complex between β2-M and hemochromatosis protein HFE, which in turn regulates intracellular iron homeostasis and HIF-1α .
MicroRNAs (miRNAs) and Osteomimicry
miRNAs are well-known regulators of tumour growth and bone metastases. With particular regard to osteomimicry, noteworthy to be mentioned is miR-218, which has been recently described as an inducer of osteogenesis by activating the Wnt signalling . Indeed, Hassan et al.  demonstrated a positive feedback loop between miR-218 and Wnt signalling, with the former acting by downregulating the inhibitors of the canonical Wnt pathway, such as DKK2, Sclerostin and Secreted Frizzled-Related Protein 2 (SFRP2). Interestingly, they found miR-218 to be highly expressed in metastatic breast cancer cells compared to normal mammary cells, which in turn also increased OPN and BSP expression as well as the release of CXCR4, thus supporting tumour growth in the bone.
Do exosomes Contribute to Osteomimicry?
Exosomes and their involvement in tumour progression is a “hot topic”. Exosomes are small membrane-bound vesicles of 50–100 nm in size, produced by most cell types via endosomal mechanisms. They shuttle bioactive molecules such as RNA, DNA, lipids and proteins . The role of these particles in tumour initiation and progression, angiogenesis and in the establishment of the pre-metastatic niche and metastasis is well ascertained . Recent reports show the ability of tumour-derived exosomes to influence bone cell physiology, eventually promoting bone metastasis development. In particular, Itoh et al. showed that exosomes isolated from prostate cancer cells stimulate osteoblast differentiation , while Karlsson et al. found that prostate cancer cells inhibit osteoclast differentiation by interfering with the fusion of osteoclast precursors . Taken together, these results indicate the ability of tumour-derived exosomes to hijack the bone microenvironment, by redirecting physiologically important pathways to the tumour’s advantage. With regard to the specific impact of exosomes on tumour osteomimicry (e.g. the release of exosomes by resident cells to increase osteomimetic properties of tumour cells or, conversely, the use of exosomes by tumour cells like bone cells use them), this is still an uncovered topic.
Is There an Osteoclast Mimicry?
As described so far, osteomimicry usually refers to the propensity of tumour cells to acquire an osteoblast-like phenotype. Indeed, tumour cells that join the bone and metastasise also share some molecules typically, although not exclusively, expressed by osteoclasts, such as RANK, the activation of which increases tumour cell proliferation, as well as MMP9 and αVβ3 integrin. Although it has been fully ascertained that tumour cells are not able per se to resorb bone, recent reports indicate that tumour cells can fuse with preosteoclasts or bone marrow-derived cells. In particular, Andersen et al. showed that bone-resorbing osteoclasts from myeloma patients contain nuclei with translocated chromosomes of myeloma B cell clone origin. They conclude that tumour cells contribute significantly to the formation of bone-resorbing osteoclasts in multiple myeloma . Consistently, other results indicated that macrophages could fuse with tumour cells, leading to mature osteoclasts with increased resorption ability .
In agreement with an osteoclast-like phenotype, Tan and colleagues found, among the bone-related genes highly co-expressed in bone metastases compared to the primary tumours, thus representing an “osteomimicry gene signature”, also cathepsin K , which is highly and quite selectively expressed by osteoclasts and actively participates in the resorption of the organic bone matrix.
Understanding why tumour cells have a predilection to metastasise the bone is not an easy task, but noteworthy to be investigated, and this is particularly true for prostate and breast cancers. Indeed, the former present with a quite exclusive metastatic outcome in the bone, and therefore the prevention of its development could really “make the difference” for patients in terms of survival. With regard to breast cancer-induced bone metastases, although they represent an incurable disease, patients carrying bone-only malignancies have a good prognosis compared to patients with metastases in visceral organs. Moreover, other studies demonstrated that metastatic breast cancers confined to the skeleton are highly responsive to treatments and associated with a longer survival.
Although most studies are focused on breast and prostate carcinomas, due to the prolonged survival of patients affected by malignancies, it is now understood that there is an increase of the incidence of bone metastases also for other type of cancers. Therefore, it is important that future work addresses the molecular mechanisms underlying this new aspect of bone oncology, identifying differences and commonalities that could make any type of bone metastasis curable.
Among the phenomena that allow tumour cells to grow in the bone, osteomimicry plays a crucial role that is now known not to be an exclusive prerogative of breast and prostate cancer cells. As an example, in up to 10% of pulmonary carcinoid patients it has been observed an intratumoral ossification, with the expression of BMP-2 and osteocalcin by tumour cells. Therefore, although with low frequency, osteomimicry appears to feature malignancies also outside the bone.
Based on these data, there is an urgent need to deeply investigate the underlying molecular mechanisms that allow the acquisition of the osteomimicry profile by tumour cells in order to settle more efficacious therapies able to ameliorate the quality of life of bone metastatic patients and, most importantly, extend their life expectation.
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Conflict of interest
The authors declare that they have no conflict of interest.
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Rucci, N., Teti, A. Osteomimicry: How the Seed Grows in the Soil. Calcif Tissue Int 102, 131–140 (2018). https://doi.org/10.1007/s00223-017-0365-1
- Bone metastases
- Breast cancer
- Prostate cancer