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Nat Rev Cancer. Author manuscript; available in PMC Sep 1, 2008.
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PMCID: PMC2484121

Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs): Multifunctional proteins in cancer


Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein — small integrin-binding ligand N-linked glycoproteins (SIBLINGs) — are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets.


The progression of malignant cells requires complex interactions with the host tissues. Tumour survival and metastasis necessitate overcoming immune surveillance, extracellular matrix barriers and limiting nutrients. Effectively stopping cancer progression appears to require the use of a panel of therapeutic modalities able to interfere with the multiple stages of cancer cell invasion and dissemination. An alternative to targeting specific stages in progression (for example, angiogenesis and metastasis) would be to target select molecules that have key roles in multiple stages of cancer development.

Small integrin-binding ligand N-linked glycoproteins (SIBLINGs1), a family of five integrin-binding glycophosphoproteins comprising osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE), are an emerging group of molecular tools that cancer cells use to facilitate their expansion. SIBLINGs are soluble, secreted proteins that can act as modulators of cell adhesion as well as autocrine and paracrine factors by their interaction with cell surface receptors such as integrins. OPN is the only SIBLING for which there is unequivocal evidence of its role in many steps of cancer development and progression, but accumulating data also implicate other family members, notably BSP and DSPP26. The involvement of SIBLINGs in many of the crucial steps for malignant progression makes them potentially valuable candidates for effective anticancer therapies. In this Review, we describe the major characteristics of SIBLINGs, including their proposed roles in normal tissue and the major activities they display during malignant progression. Finally, we discuss their potential as therapeutic targets and prognostic markers.

Discovery and characteristics of SIBLINGs

SIBLINGs are currently a family of five identically orientated tandem genes within a 375,000 bp region on chromosome 4 (FIG. 1a). The genes (DMP1, DSPP, integrin-binding sialoprotein (IBSP, which encodes BSP), MEPE and secreted phosphoprotein 1 (SPP1, which encodes OPN)) are probably a result of an early gene duplication and divergence1. The term SIBLING refers to the gene family’s unifying genetic and biochemical characteristics in general, not to functional activity. SIBLINGs are defined as small, soluble RGD motif containing, integrin-binding ligands to distinguish them from large extracellular matrix proteins such as fibronectin (FN1), collagen and thrombospondin (THBS1). Comparison of any one SIBLING to itself at the amino acid sequence level throughout evolution (predominantly in birds and mammals) shows that they are poorly conserved. Each SIBLING member appears to be able to drift substantially as long as it remains hydrophilic and flexible, and retains a number of motifs and member-related short amino acid sequences. For example, using standard sequence comparison basic local alignment search tool (BLAST) programs7, human and mouse OPN are identical at only 63% of the amino acid positions and the comparison with chicken drops to 30% identity, although all retain, for example, at least one RGD and N-linked oligosaccharide motif each. Therefore, the SIBLING family was defined structurally by the conserved motifs within their exons, including an abundance of acidic amino acids, the RGD motif, similar post-translational modification motifs (for example, casein kinase phosphorylation and various glycosylation events) and more recently the recognition that at least one site of controlled proteolysis appears to be important in all members (FIG. 1b). The SIBLINGs that have had their three-dimensional structure solved by NMR analysis (BSP and OPN) are extended and flexible in solution, a property shared by a number of proteins that have multiple binding partners and that are involved in bridging macromolecular components (for example, certain ribosomal proteins). Consistent with that observation, SIBLINGs can bind to a number of different protein families, including integrins (through both RGD and non-RGD motifs) and other cell-surface proteins, members of the matrix metalloproteinase (MMP) family and complement factor H (CFH). These interactions enable cell surface localization and sequestration of MMPs and CFH by at least OPN, BSP and DMP1, which in turn enables their biological activities (extracellular matrix degradation and evasion of complement-mediated lysis for example).

Figure 1Figure 1
Chromosomal localization and exon–intron similarities of human SIBLInG genes

Four acidic members (BSP8, DMP1 (REF. 9), DSPP10 and OPN11) were discovered as abundant proteins trapped within the mineralized matrices of bone and dentin. In the early years of study, each of these proteins was thought to be both skeletal tissue-specific and to have a role in directly nucleating hydroxyapatite crystals within mesenchymal tissues through their phosphate groups and/or polyacidic amino acid domains9,12. From the 1990s, various combinations of SIBLING proteins were discovered to be significantly upregulated in a number of epithelial tumours that are known to frequently exhibit pathological microcalcifications and to have strong propensities to metastasize to bone13. More recently it has been shown that all five of the SIBLINGs are also expressed in the epithelial cells of metabolically active normal ducts of the salivary gland and kidney14,15, but not in metabolically passive normal ducts16. SIBLINGs are secreted proteins that can be localized through interactions with receptors either on the cell’s own surface, enabling autocrine activities, or by diffusing short distances to nearby cells where they may function in paracrine signalling. SIBLINGs propagate biological signals by initiating integrin signalling and by binding and sequestering other proteins to the cell surface.

Roles of SIBLINGs in normal tissues

SIBLINGs bind to cell surface integrins and sometimes CD44 in normal tissues and function as signal transducers to promote cell adhesion, motility and survival (FIG. 2) through activating kinase cascades and transcription factors. The biological activities of SIBLINGs are also modulated by proteolytic processing, which can reveal cryptic binding sites and can remove or separate functional domains, thereby modulating cell adhesion and migration (FIG. 1b).

Figure 2
SIBLINGs mediate cell–matrix interactions and cellular signalling

For example, OPN interacts with a variety of integrins, including αvβ3, αvβ5, αvβ1, α4β1, α8β1 and α9β1, as well as CD44 splice variants. Integrin-mediated cell adhesion and migration are stimulated in assays in which full-length OPN is immobilized on tissue culture dishes. Thrombin cleavage of OPN separates the integrin- and CD44-binding domains, which in some cases promotes adhesion over migration. For example, the thrombin-generated amino-terminal OPN fragment binds to αvβ3 and αvβ5 integrins (through RGD17) or to α9β1 and α4β1 integrins (through the cryptic SVVYGLR sequence18) and promotes cell adhesion. The carboxy-terminal fragment binds to CD44 variant 6 (CD44v6) — and sometimes to CD44v3 by a heparin bridge — and promotes the formation of foci, invasion and tumorigenesis19. under specific conditions, OPN is also a substrate for MMP3 and MMP7, and the resulting OPN fragments facilitate adhesion and migration in vitro through activation of β1-containing integrins20. OPN has also been shown to be a substrate for liver transglutaminase and plasma transglutaminase factor IIIa, resulting in protein crosslinking21 and enhanced cell adhesion, spreading, focal contact formation and migration22. Through interactions with cell-surface receptors, OPN and its proteolytic fragments modulate cell adhesion and migration.

Through their action on the transcription factor nuclear factor κB (NFκB), SIBLINGs can also affect cellular proliferation, differentiation and apoptosis in normal tissues. BSP increases survival and decreases apoptosis of bone marrow-derived monocytes and macrophages through enhanced NFκB signalling23. BSP can also induce NFκB -dependent bone resorption by inducing osteoclastogenesis and osteoclast survival23. OPN activation of NFκB promotes survival of activated T cells through phosphorylation of the kinase IKKβ (also known as inhibitor of NFκB kinase β (IKBKB)) and inhibition of the transcription factor FOXO3 (REF. 24). A role for OPN-induced activation of NFκB in the survival of dopaminergic neurons25, endothelial cells26 and dendritic cells27 has also been reported.

The above-mentioned pathways (migration, adhesion and apoptosis evasion) are crucial in the development and progression of cancer as nascent neoplasms must successfully navigate these pathways to survive. Therefore it seems possible that the effects of SIBLINGs in cancer biology are due in part to modulation of these pathways.

SIBLINGs and tumour progression

Tumour progression involves a sequential series of events that confer a survival advantage to transformed cells. These events begin with neoplastic transformation and continue through the subversion of proliferation blockades, growth restriction, physical barriers and host defense systems. Cancer cell survival requires proliferation, interaction with the extracellular matrix to create space to grow, pathways for nutrient access and escape of the cells to a new environment. Successful progression also involves cellular responses and evasion of immune surveillance.

Cancer cell adhesion and proliferation

Cancer cells bind to SIBLINGs and their various proteolytic fragments through a variety of integrin receptors by both RGD-dependent and RGD-independent interactions. OPN, and perhaps DMP1, can also interact with specific splice variants of CD44 that are expressed by cancer cells. Interestingly, OPN binding to CD44v6 results in the propagation of cytosolic signals that enhance integrin activation and thus migration (an example of inside-out signaling) in colon HT29 cells28. Tumour cells were stimulated to spread following the interaction of CD44 and OPN apparently through β1 integrins, which have well-characterized roles in enabling cell adhesion29,30. OPN exhibiting reduced serine/threonine phosphorylation by casein kinase induces the adhesion of human breast cancer cells almost sixfold more than hyperphosphorylated OPN31, highlighting the possible modifying roles of the many post-translational events on SIBLING functions.

The adhesive properties of the SIBLINGs have also been investigated in the context of bone targeting and recognition by metastasizing cancer cells, and these molecules have been implicated in enhancing the affinity of metastatic cancer cells for bone (discussed in more detail below). Breast cancer cells expressing active αvβ3 integrin adhere to BSP-enriched mineralized bone as well as to recombinant BSP during in vitro adhesion and invasion assays32. Thus, exogenously added BSP peptides strongly inhibited breast cancer cell adhesion to extracellular bone matrix at micromolar concentrations33. Furthermore, multiple myeloma cells adhere to OPN, indicating that the increased stromal expression of OPN that is associated with this disease might be one of the factors enhancing the retention of these cells in the bone marrow34. Although the RGD domain of DMP1 has been shown to mediate the adhesion and spreading of dental pulp cells in vitro35, no data about DMP1-mediated cancer cell adhesion are available.

SIBLINGs may also affect cancer cell proliferation. BSP accelerates the proliferation of breast cancer cells in vitro6. Furthermore, IBSP-transfected breast cancer cells show increased primary tumour growth following injection into the mammary fat pad of nude mice36. OPN stimulates human prostate cancer cell line proliferation when transferred to a mouse xenograft model system37. OPN-induced proliferative responses mainly occur through activation of the epidermal growth factor receptor (EGFR)38 and integrin-mediated intracellular Ca2+ signalling39. The intracellular signalling pathways operant in OPN modulation of cell proliferation and migration have been well characterized (for a review see REF. 18). The binding of OPN to CD44 promotes cell migration through kinase cascades involving phospholipase Cγ, protein kinase C, phosphatidylinositiol 3-kinase (PI3K) and Akt, a serine/threonine kinase that regulates cell cycle progression, growth factor-mediated survival and cell migration (FIG. 2a). The binding of αvβ3 by OPN is associated with SRC kinase-mediated complex formation between αvβ3 and EGFR, which activates the mitogen-activated protein kinase (MAPK) pathway and results in the promotion of tumour growth. The potential effects of SIBLINGs other than BSP and OPN on cell proliferation have not been investigated.

Invasion and extracellular matrix degradation

High cancer cell motility combined with increased expression of proteases that degrade the extracellular matrix is generally predictive of invasive capability40,41. OPN and BSP are expressed at high levels by numerous cancers and might contribute to their invasive potential. Functional studies using over-expression of OPN in two prostate cancer cell lines reveal that OPN increases invasion and enhances the ability of cancer cells to intravasate into blood vessels in a mouse neoplastic model37. OPN also enhances in vitro migration of various types of cancer cells including melanoma42, breast43, and multiple myeloma44. Similarly, transfection of a breast cancer cell line with IBSP cDNA stimulated migration and invasion in vitro36.

Invasive cells have the capacity to degrade the extracellular matrix through at least two pathways of controlled proteolysis: the urokinase-type plasminogen activator (uPA, also known as PLAU) pathway and the MMP pathway. Several studies using recombinant OPN show that this SIBLING significantly increases in vitro invasiveness. For example, OPN increases the invasiveness of pancreatic cancer cells45 and non-small cell lung carcinoma cells46. Treatment of breast cancer cells with OPN results in higher invasiveness through the basement membrane analogue, Matrigel, and increases both PLAU mRNA expression and urokinase activity47. In a metastatic murine mammary cancer cell lines model, the binding of OPN to integrin receptors induces MMP2 and uPA expression through integrin-linked kinase (ILK)-dependent AP1 activity48. It has recently been shown that OPN induces αvβ3 integrin-mediated AP1 activity and uPA secretion by activating SRC–EGFR–ERK (extracellular signal-regulated kinase) signalling pathways and further demonstrates a functional molecular link between OPN-induced integrin- and SRC-dependent EGFR phosphorylation and ERK- and AP1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells49. Thus, both increased cell motility and induction of uPA expression are possible mechanisms of increased invasiveness of breast epithelial cells in response to OPN.

Potential mechanisms for SIBLING-enhanced matrix degradation have been described. BSP and OPN induce the activation of MMP2 in GCT23 giant cell tumour cells50. OPN binding to αvβ3 is associated with PI3Kmediated NFκB activation and nuclear factor-inducing kinase (NIK, also known as mitogen-activated protein kinase kinase kinase 14 (MAP3K14)) activation of AP1 and NFκB, which stimulate uPA-dependent MMP9 activation51. Thus, at least two SIBLINGs can induce MMP expression. Interestingly, NIK-dependent MMP9 activation has been recently implicated in melanoma growth and metastasis to lung52.

BSP, DMP1 and OPN bind to and modulate the activity of MMP2, MMP9 and MMP3, respectively53. SIBLING-mediated MMP activation includes both making the proMMPs enzymatically active to some degree and reactivating MMPs that are inhibited by tissue inhibitors of MMP (TIMPs)53. The expression of these three SIBLINGs and their cognate MMPs was correlated in a number of different cancer types54. BSP promoted invasion of several osteotropic cancer cell lines in vitro by apparently localizing MMP2 to the cell surface through αvβ3 (REF. 55). DMP1 enhanced the invasion potential of a colon cancer cell line by bridging MMP9 to integrins and, perhaps, CD44 (REF. 56). Another mechanism might involve enzymatic processing of SIBLINGs that alters invasion and migration properties. For example, increased hepatocellular carcinoma cell invasion was attributed to OPN peptides cleaved by MMP9 and thrombin57. Interestingly, transglutaminase crosslinking of OPN forms proteolysis-resistant inactive OPN polymers that reduced breast cancer cell invasion and migration in vitro58.


Metastasis is a complex process characterized by multiple stages: malignant cells proliferate and spread from the primary tumour mass, invade adjacent capillary or lymphatic vessels, resist immunological attacks and eventually gain access to secondary sites where they proliferate to form a new tumour (for a review see REF. 59). Throughout this multi-step progression, cancer cells interact with extracellular matrix proteins, endothelial cells, platelets, stromal cells and other organ-specific structures. Multifunctional extracellular matrix proteins such as the SIBLINGs are expected to have key roles in metastasis as they affect adhesion, migration and matrix degradation (FIG. 3).

Figure 3
The role of SIBLING proteins at different steps of the metastatic cascade

Compelling evidence from cancer cell transfection experiments and mouse xenograft models demonstrate that high-level OPN expression can confer a metastatic phenotype on cells that originally formed benign tumours60. Since this initial observation, numerous studies conducted with human biological tissue from various cancer types consistently report that tumours that are likely to progress to more advanced stages present with de novo or increased expression of SIBLINGs (TABLE 1). In particular, the correlations observed between high levels of OPN expression in tumour cells and their subsequent metastatic dissemination were supported by gain- and loss-of-function studies demonstrating the pro-metastatic role of OPN (for a review see REF. 61). Thus, the introduction of an OPN expression vector into non-metastatic rat mammary epithelial cells resulted in lung metastasis development in 55% of the inoculated animals that produced primary tumours60 and the antisense inhibition of OPN inhibited osteolytic metastases of human breast cancer cells62. Hostproduced OPN also appeared to be of importance for metastasis development, as melanoma cells that do not express OPN showed reduced lung and bone metastases when injected into OPN-deficient mice compared with wild-type mice63.

Table 1
SIBLING expression in normal and malignant human tissues as well as correlation with disease progression

High expression of SIBLINGs is associated with osteotropic cancers including breast64, prostate65 and lung66 as well as multiple myeloma67. A recent microarray and functional genomic study in an experimental mouse model demonstrated a functional association between OPN, interleukin 11 and either chemokine receptor 4 (CXCR4) or connective tissue growth factor (CTGF) in breast cancer cells that favours bone metastasis68. Overexpression of BSP also enhanced experimental bone metastasis69. The affinity of BSP-expressing cancer cells for bone is emphasized in a study where the transfection of IBSP cDNA into a brain-metastasizing breast cancer cell line subclone was sufficient to induce bone metastases, although no bone lesions were observed with the control line70.

The expression of SIBLINGs by breast and prostate carcinoma prompted the hypothesis that osteotropic cancer cells can become ‘bone-like’ or express osteomimetic properties that favour ‘seeding’ in the skeleton by improving their adhesion, proliferation and/or survival in bone. It was shown that during the malignant transformation of prostate epithelium, a switch of gene expression that confers an osteoblastic phenotype (including expression of SIBLINGs) may occur71. Indeed, the expression of certain crucial transcription factors that are known to regulate the expression of bone-related genes, such as RUNX2 and MSX, is altered in prostate cancer cells in a way that favours the acquisition of an osteoblast-like profile by these cells (see below for details). The expression of ‘bone’ proteins by cancer cells does not necessarily target cell metastasis to bone, rather it is more likely that the expression of transcription factors regulating SIBLING genes such as RUNX2 produces a mesenchymal phenotype that finds in bone a fertile soil for survival. More recently, Notch signalling and ERK activation have been shown to be important for the osteomimetic properties of prostate cancer bone metastatic cell lines72. In good agreement with the osteomimicry theory, a parallel between the gene expression profiles of human breast cancer cells with a high propensity to metastasize to bone and differentiating osteoblast cells was revealed73. Interestingly, the osteomimicry gene expression profile observed in osteotropic breast cancer cells is comprised not only of SIBLINGs but also of other proteins that are associated with the acquisition of an osteoblastic phenotype, including core-binding factor β (CBFβ, a RUNX co-transcription factor) and the osteoblastic cell–cell adhesion protein cadherin 11 (CDH11)73.

The known biological activities of OPN and BSP support their role in promoting metastases to the bone, and to other organs. It is likely that future explorations will identify SIBLINGs as essential regulators of the metastatic phenotype. This phenotype is presumably influenced by stromal and inflammatory cells that are closely associated with primary and metastatic tumours. Identifying the specific roles of SIBLINGs in cancer–stroma interactions and signalling cascades involving growth factor–growth factor receptor and cell–matrix interactions could result in the development of additional novel and refined strategies for the prevention and treatment of metastases. Tumour cell survival (at both primary and distant sites) requires successful counter-responses to immune surveillance.

Inflammation and complement evasion

The interplay between inflammation and cancer is currently an area of intense research74. Inflammation is part of the innate immune system and can provide an immediate, although non-specific, response. SIBLINGs can have roles in immune cell migration into sites of matrix turnover and degradation as well as in infection and inflammation. One paradigm for SIBLING function and metabolism within the immune system is that OPN is secreted by activated macrophages, leukocytes and activated T lymphocytes7577 and is also a chemotactic cytokine for macrophages78, dendritic cells27 and neutrophils79. It is possible that OPN expression by tumours actually promotes inflammation-induced cancer growth and progression through, for example, the promotion of macrophage and neutrophil infiltration. Tumour cells that secrete OPN might be propagating chronic inflammation, which can accelerate transformation and tumour progression. OPN might also enhance tumour survival by downregulating macrophage nitric oxide synthase expression and downstream production of nitric oxide80. Cytokines can also alter SIBLING levels. Studies have reported that both OPN and DSPP are upregulated by the macrophage inflammatory protein MIP3α (also known as CCL20), a chemokine involved in modulating cell-mediated immunity81, which is known to promote pancreatic cancer cell migration82. The overall action of OPN on the immune system is to regulate the function of macrophage and macrophage-derived cells (that is, osteoclasts). The expression and potential biological activities of the other SIBLINGs in immune cells have not been studied; however, given their adhesive properties and other shared biochemical properties with OPN, it is possible that they have similar as yet undiscovered modulatory roles in inflammation.

Another component of the innate immune response is the complement system, which is composed of about 26 proteins that combine with antibodies and/or cell surface molecules as part of the humoral response. The complement cascade has a role in inflammation, immune adherence, opsonization, viral neutralization, cell lysis and localization of antigen83. Nearly all cells are subject to constant low levels of complement attack, but only cells that do not express the correct cell surface proteins, thereby inactivating the early steps of the cascade, are killed. CFH is a major negative regulatory factor that quenches complement- mediated lysis. Cells that become transformed may escape the complement system during their transit through the patient’s circulation by upregulating genes that help to control this aspect of immune surveillance. As such, the expression of SIBLINGs (specifically OPN, DMP1 and BSP) by tumour cells might provide such a selective advantage for survival by mediating the binding of CFH to the cell surface through integrins and/or CD44. The activated CFH then inhibits the formation of the membrane attack complex and subsequent cell lysis (FIG. 2d). In vitro experiments have demonstrated that these three SIBLINGs can protect murine and human cancer cell lines from attack by complement8486.


Angiogenesis promotes tumour growth as well as metastatic spread through a complex interplay of positive and negative mediators of extracellular matrix degradation and endothelial cell and vasculature recruitment. There is evidence that αvβ3 is a key angiogenesisassociated receptor that is significantly upregulated on the surface of activated endothelial cells87. OPN and BSP have been shown to act as pro-angiogenic factors and, based on their RGD motifs, it is likely that the other SIBLINGs may also interact with αvβ3 integrin and influence the behaviour of endothelial cells. OPN contributes to the genesis of new capillaries infiltrating the cancer lesion in several in vivo models88,89. BSP also promotes angiogenesis in the chicken chorioallantoic membrane assay through binding αvβ3 (REF. 90).

The integrin αvβ3 mediates the migration of activated endothelial cells during vessel formation. SIBLINGs, as ligands for αvβ3 through the RGD sequence, may stimulate endothelial cell migration. It is also possible that SIBLING modulation of protease activity (uPA or MMP) generates bioactive fragments of extracellular matrix components responsible for angiogenesis. Experimental evidence suggests that antagonizing the ligation of SIBLINGs to integrins is a promising approach for the inhibition of angiogenesis and associated tumour growth. For example, blocking the interaction between OPN and αvβ3 inhibits angiogenesis and stops lung cancer growth in mice88. The αvβ3 integrin was shown to be important for OPN-mediated NFκB induction and survival, as adding a neutralizing anti-β3 integrin antibody blocked NFκB activity and induced endothelial cell death when cells were plated on OPN26. A recent study demonstrates that OPN triggers vascular endothelial growth factor-dependent breast tumour growth and angiogenesis by autocrine and paracrine mechanisms91. Thus, it is possible that, through their interaction with αvβ3, the SIBLINGs may also cooperate with molecules that have important biological functions during angiogenesis and tumoural growth processes, including MMPs, growth factors and their receptors.


All SIBLINGs are expressed by bones and teeth, and it was originally thought that they acted to directly regulate hydroxyapatite crystal formation92. Outside of the skeletal system, pathological dystrophic calcification associated with the upregulation of OPN and/or BSP has been observed. Notable among these are atherosclerotic vascular plaques, renal osteodystrophy and kidney stones93. Because of their earlier association with mineralization in bone, the expression of BSP and OPN has been studied in cancers such as breast and thyroid carcinomas, in which ectopic calcification occurs64,94,95. Although calcifications are usually associated with benign lesions, certain patterns of calcification — such as tight clusters with irregular shapes — may indicate the presence of a premalignant tumour. Tumours from the primary sites of bone-seeking cancers frequently contain foci of dystrophic calcifications in the form of hydroxyapatite microcalcifications. The cause(s) of these ectopic calcifications are ill-defined and the exact role of the SIBLINGs in the formation of such calcifications is not known. Although it was initially thought that mineral deposition might occur because of the increased local concentration of SIBLINGs (which have well-described characteristics of nucleators of mineralization) it has also been reported that OPN actually blocks ectopic calcification96. It is also possible that SIBLING association with ectopic calcification primarily controls and diminishes the immune and inflammatory response that is provoked by inappropriate crystal deposition.

Nevertheless, SIBLING-expressing tumours are more readily detectable clinically at early stages, on the basis of associated abnormal mammographic calcifications. Most of the breast calcifications detected at mammography are benign. Radiologists must be able to identify typically benign breast calcifications that do not require biopsy to prevent unnecessary procedures and to reduce patient anxiety. It will be interesting to determine whether the high expression of SIBLINGs that is associated with the detection of suspicious microcalcifications will help to identify lesions that are likely to evolve towards malignancy. The expression of SIBLINGs in premalignant lesions has not yet been investigated and is an interesting field of investigation to fully understand the role of these proteins during cancer progression.

Regulation of SIBLING genes in cancer cells The promoter regions of SPP1, IBSP, DMP1 and DSPP have been cloned in different species and they exhibit a number of consensus regulatory sequences, such as potential binding sites for AP1 and NFκB transcription factors. Regulation of SIBLING genes has been best studied in the context of osteoblastic and odontoblastic cell differentiation (BOX 1). RUNX2, a member of the RUNX transcription factor family, is a transcription factor that is crucial for the regulation of genes that support bone formation97 and as such it is involved in the control of OPN98, BSP99, DMP1 (REF. 100) and DSPP101 expression. All the RUNX proteins are intimately associated with tumour progression, invasion and metastasis102. Notably, RUNX2 is aberrantly expressed at high levels in breast and prostate tumours and cells that metastasize to the bone103. Interestingly, RUNX2 also transactivates SPP1 (REF. 104) and IBSP105 in breast cancer cells, suggesting that the expression of SIBLINGs might be subject to the same regulation both in normal osteoblasts and in cancer cells. Human myeloma cells with active RUNX2 protein produce OPN that is involved in the pathophysiology of multiple myeloma-induced angiogenesis106. In colorectal cancer, gene-profiling studies identified a positive correlation between metastatic colon tumours and increased OPN expression107. More recently, RUNX2 and ETS1 were identified as crucial transcriptional regulators of OPN expression in a murine colorectal cancer cell line and their suppression using antisense oligonucleotides resulted in significant downregulation of OPN108. Several additional signalling pathways and transcription factors that are associated with cancer progression regulate OPN expression in models of breast cancer, melanoma and leukaemia (for reviews see REFs 4,61). These include AP1, MYC, OCT1 (also known as POU2F1), upstream stimulating factor (USF), v-Src, transforming growth factor β (TGFβ)BMP–SMAD–HOX (homeobox), WNT–β catenin–adenomatous polyposis coli (APC)– glycogen synthase kinase 3 (GSK3)–transcription factor 4 (TCF4), Ras–Ras response factor (RRF) and p53. The global picture of OPN gene transcriptional regulation in cancer cells is that of an intricate regulatory network. Studies of the proximal promoter regions of other SIBLING genes are needed to identify regulatory elements that could be responsible for their overexpression in cancer.

Box 1 Expression and distribution of SIBLINGs in normal tissues

With the exception of osteopontin (OPN), which was independently discovered in several tissues, the distribution of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family in normal tissues was originally believed to be limited to bones and teeth92. In these calcified tissues, the presumed function of the family was a role in the biomineralization of matrix and a number of published in vitro studies do support such a role (for a review see REF. 159). With one exception, the dentin of dentin sialophosphoprotein (Dspp)-null mice160, all of the SIBLING gene knockout mouse models have little if any significant change in basic matrix mineralization. Early reports indicated that OPN is also a component of human breast milk161, and is expressed in chronic inflammatory cells162 and kidney163, as well as some other epithelia164. Terasawa et al.165 reported the expression of dentin matrix protein 1 (DMP1) in several mouse soft tissues including liver, muscle, brain, pancreas and kidney. Matrix extracellular phosphoglycoprotein (MEPE) was originally discovered in tumours causing osteomalacia and was shown at that time to be expressed (mRNA only) predominantly in bone and brain with “very low levels of expression” in lung, kidney and placenta122. Recent studies have demonstrated that all five members of the SIBLING family are expressed in metabolically active ductal epithelial cells of the salivary gland and kidney14,15 and all but MEPE were expressed in eccrine sweat ducts16. MEPE expression appears to be limited to ductal cells that actively transport phosphate16. The role of the SIBLINGs in normal soft tissues is now a subject of intense investigation. SIBLINGs expression can also be transcriptionally regulated (see table). BSP, bone sialoprotein; DLX5, distalless homeobox 5; FRE, fibroblast growth factor response element; HDAC3, histone deacetylase 3; HOX, homeobox; PPARγ, peroxisome proliferator-activated receptor γ.

Consistent with its role in tumour initiation and progression, SPP1 expression is also repressed by tumour suppressors such as BRCA1 and phosphatase and tensin homologue (PTEN), and metastasis suppressors such as breast cancer metastasis suppressor 1 (BRMS1). BRCA1 expression inhibits SPP1 promoter transactivation and hence suppresses OPN expression109. A BRCA1 mutation in human primary breast cancer lesions is associated with OPN overexpression, suggesting that it may confer increased tissue-specific cancer risk, in part, by disruption of the suppression of OPN transcription by BRCA1 (REF. 109). The tumour suppressor PTEN antagonizes PI3K, which is responsible for promoting cell growth, survival and tumorigenesis, when over-stimulated in cancer cells. OPN was shown to act downstream of the PI3K pathway in melanoma and glioma cancer cells. A link has been found between OPN expression at both the mRNA and protein level that involves PI3K activation of OPN and may help explain how PTEN loss contributes to the development of these malignancies110,111. By contrast, BRMS1 inhibits OPN expression through the inactivation of NFκB and subsequent binding to the SPP1 promoter. Thus, downregulation of OPN might be one of the mechanisms of metastasis suppression by BRMS1 (REF. 112).

SIBLINGs as prognostic indicators in cancer

The first SIBLING found to be overexpressed in cancer was OPN113,114. Since then, large numbers of studies have established that increased expression of OPN is a consistent feature for most known human malignancies (TABLE 1). Increased expression of BSP was originally observed in human breast cancer64 and its putative role in the acquisition of an osteotropic phenotype by metastatic cancer cells soon led to the extension of this observation to other bone-seeking cancers such as prostate65, lung66, thyroid115 and cervical carcinoma116, as well as multiple myeloma67 (TABLE 1). Recently, BSP was detected in pancreas117, skin118 and oral119 carcinomas, a group of neoplastic lesions that are not particularly osteotropic when they metastasize. These observations suggest that, as is the case for OPN, BSP expression in cancer cells is not restricted to cancer cells metastasizing to bone but is a common feature of the malignant phenotype. The expression profile in cancer of the three other SIBLINGs — DMP1, DSPP and MEPE — has not been evaluated in detail to date, but data indicate that DMP1 and DSPP are upregulated in several human malignancies54. Immunohistochemical studies demonstrated increased expression of DSPP in human prostate2 and oral119 cancers, and high levels of DMP1 were observed in human lung120 and breast121 cancers. It is expected — based on the detection of DMP1 and DSPP in a variety of human malignancies compared with their normal corresponding tissues on a mRNA–cDNA array54 — that future studies will verify high expression of these proteins as a consistent feature of most human cancers (TABLE 1). By contrast, MEPE expression seems to be much more restricted than that of the other SIBLINGs, so far being found to be expressed in normal cells associated with phosphate transport16. Interestingly, only tumours that result in oncogenic hypophosphataemic osteomalacia appear to express MEPE122. Screening of MEPE expression at the mRNA level in a collection of human normal and cancer tissues revealed minimal expression in all tissues analysed54. This observation suggests that MEPE, unlike other SIBLINGs, does not intervene during cancer progression. The reasons for this are still unclear. Enhanced expression of SIBLINGs is not only associated with several tumour types, but their levels of expression are also often directly correlated to specific stages of clinical progression. Gene expression analyses have identified SPP1 to be among the most strongly upregulated genes in human colon cancer123. In this study, OPN expression was shown to be an independent prognostic marker for poor overall survival. These observations were supported by a recent study that found that colon cancer patients with tumours expressing high levels of OPN have significantly reduced survival124. In another study, OPN was shown to be a predictor of outcome that was independent of clinical characteristics (such as age, lesion size and histological type) in ovarian carcinoma. The prognosis for survival within 36 months was <5% for patients with increased OPN and 75% for those with no OPN increase125. Clear cell renal carcinoma patients with OPNpositive tumours also exhibited worse prognosis than patients who had tumours lacking OPN126. The prognosis for patients with increased OPN in cervical cancers is also extremely poor, as none survived within 24 months, compared with the 67% survival rate observed within the same time period for patients with no OPN increase127. Other cancers with a positive correlation between OPN expression and poor prognosis are breast128, prostate129, head and neck130,131, thyroid132, non-small cell lung133 and hepatocellular134 carcinomas (TABLE 1).

Increased BSP expression in primary breast135 and prostate65 carcinoma is also associated with tumour progression. In non-small cell lung carcinoma, BSP expression is associated with bone metastasis development and could be useful in identifying high-risk patients who could benefit from novel modalities of surveillance and preventive treatment136. Expression of other SIBLINGs might also correlate with tumour progression. DSPP expression correlates with aggressiveness in human prostate cancers2 and in oral cancer119. unique among the SIBLINGs, DMP1 expression was inversely associated with progression in human breast cancer121. The positive prognostic value of DMP1 for breast cancer patients has only been reported in one study and awaits confirmation. Small interfering RNA (siRNA)-mediated repression of DMP1 enhances migration of human breast cancer cells in vitro121. Thus, it can be speculated that the expression of DMP1 alters cancer cell motility and hence reduces local invasion and metastatic spreading. This effect could be achieved through a competition of DMP1 with BSP and/or OPN for their binding to the cell membrane integrin receptors and the subsequent activation of their corresponding MMP partner. Such putative mechanisms urge investigations of whether modulation of SIBLING expression might differentially affect cancer cell behaviour.

SIBLINGs can also be detected in the blood, and it is therefore not surprising that several studies have established a correlation between blood levels of OPN and BSP and the presence of a malignant tumour. However, the high affinity interaction of CFH with several SIBLINGs, including OPN and BSP84, masks all known antibodybinding sites and therefore interferes with accurate direct measurement of these proteins in the serum137. Interestingly, this masking implies that the biological activities of OPN and BSP might be limited to autocrine and/or paracrine effects as the abundant (0.5 mg/ml in blood) complement protein will quickly bind and inactivate them as they diffuse away from their sites of secretion and action. OPN, however, is interesting because 5–10% of the total amount of this SIBLING in the blood escapes the masking by CFH by mechanisms that are currently unknown. Through careful preparation of plasma, this fraction of OPN has been successfully used in many studies, as mentioned below, but when serum is analysed this fraction is masked by CFH. One confounding factor for the use of blood OPN levels is that inflammation also increases OPN levels138. In patients with bone metastases, increased plasma (not serum) levels of OPN have been suggested to be the result of both cancer cell secretion and accelerated bone turnover34. Similar to increased expression in tumour cells themselves, increased OPN plasma levels appears to be a marker for metastatic progression and poor survival in patients with breast139, prostate140, renal cell141, lung142, gastric143, head and neck144, hepatocellular145, cervical127 and pancreatic cancers146. Serum BSP (after disruption of the SIBLING–CFH complex) is significantly increased in patients with colon, breast, prostate and lung cancer137. BSP blood level also predicts bone metastasis development in patients with breast cancer147; predicts tumour burden, neoplastic bone involvement and prognosis in multiple myeloma148; and is an independent prognostic factor for human prostate cancer-related death149. No data on the blood levels of DMP1, DSPP or MEPE in human malignancies are available. Although detection of increased blood levels of OPN and BSP bear obvious diagnostic and prognostic value, such tests are not yet available to clinicians.

SIBLINGs as therapeutic targets Because of the plausible roles of SIBLINGs in cancer as pro-oncogenic, pro-metastatic and pro-angiogenic molecules, these proteins also have the potential to be valuable targets for cancer therapy (FIG. 4).

Figure 4
SIBLINGs and their cell receptors are potential therapeutic targets for cancer therapy

Several studies have already demonstrated the value of OPN and BSP as therapeutic targets in preclinical animal models. Silencing these SIBLINGs using siRNA and short hairpin RNA (shRNA) technology, or interfering with their activities using specific antibodies, inhibits or reduces tumour progression and the development of metastases. Decreasing expression of OPN in human squamous oesophagus carcinoma using shRNA reduces tumour growth and lymph node metastases in vivo150. Similar experiments performed on murine colon cancer cells led to the inhibition of tumour growth and the formation of liver metastases151. Silencing of OPN or BSP using specific antisense oligonucleotides in a human breast cancer cell line resulted in a significant decrease of osteolytic bone metastases in nude rats62. Although the use of RNA interference for therapy is attractive, much work remains before such therapeutics become available to patients152.

Antibody-based anticancer therapies, such as antibodies directed against vascular endothelial growth factor, have recently met with clinical success and are now becoming available to cancer patients153. Antibodies directed against OPN were effective in inhibiting development of lung metastases in nude mice inoculated with human hepatocellular carcinoma cells. In this case, OPN, which has been identified as a major gene in the signature for hepatocellular carcinoma, acts as both a diagnostic marker and therapeutic target for metastatic dissemination154. Anti-BSP antibodies also have therapeutic potential particularly for the prevention and treatment of breast cancer bone metastases, as suggested by the significant reduction of osteolytic lesion size in a nude rat model of human breast cancer bone metastases155.

Both integrins and CD44 have well-established roles in tumour progression. Therefore, interfering with these receptor–ligand interactions by controlling receptor cell surface expression, blocking receptor–ligand binding or suppressing associated signal transduction are promising ways to block both tumour development and metastatic dissemination (FIG. 4). CD44 has been targeted by diverse therapeutic strategies, including cytotoxic and immunotherapeutic approaches156. Because of its involvement in many processes that accompany tumour development and metastatic dissemination of cancers, αvβ3 integrin has long been a candidate target for cancer therapy by specific antibodies, peptide inhibitors and non-peptide antagonists that mimic the binding domain of physiological ligands156,157. Proof of principle that such strategies block angiogenesis, as well as tumour growth and dissemination, has been obtained in several animal models and small molecule inhibitors of this receptor are under study as drug candidates.

Finally, the ectopic expression of BSP in osteotropic neoplasms recently inspired a gene therapy protocol in bladder cancer using a conditional-replicating adenovirus. A truncated IBSP promoter controlling the E1A/B lytic-regulating sequence was used to construct the adenovirus AD-BSP-E1A. This virus had lytic activity on human bladder cancer cell lines and significantly reduced the size of bladder tumours in an orthotopic mouse model, opening a promising new strategy for the treatment of aggressive yet sensitive bladder tumours158.

From the results so far, one can speculate that SIBLINGs (particularly OPN and BSP) are viable targets that seem likely to form the basis of new anticancer therapies in the future.

Conclusions and future directions

The aim of this Review is to present an overview of some of the data that implicate the SIBLING family at several steps of cancer development and progression. Although OPN is the family member for which there is the most abundant and convincing data of its role as a key player at most of the critical steps in the evolution of malignancies, studies have revealed that BSP holds the same multifunctional role in cancer biology. Such activities are expected to be associated with DSPP and DMP1 also.

Much remains to be learned about the involvement of SIBLINGs in cancer progression, and we hope this Review will inspire cancer researchers to look more closely at this family. Future investigations should validate the use of SIBLINGs as prognostic markers in large population studies, determine their value as surrogate markers for the prediction of metastases in cancer patients and explore their potential as predictive indicators of the patient response to a given therapy (chemotherapy and/or radiotherapy). Studies are also needed to test the potential value of SIBLINGs as markers for the difficult diagnosis of precancerous lesions such as those found in breast, prostate, colon and oral tumours. Refining our knowledge of the mechanisms involved in how SIBLINGs modulate MMPs (in the absence and/or presence of natural or synthetic MMP inhibitors) could lead to the development of potential biomimetics for use in interventions. It is also of interest to determine the biological relevance of the interactions between SIBLINGs and CFH. For therapeutic strategies, the potential synergy of the combined repression of two or more SIBLINGs should also be tested.

SIBLINGs have the biological plausibility to have active roles in tumour cell adhesion, proliferation, invasion, matrix degradation, immune functions (inflammation and complement evasion), angiogenesis and metastasis. Based on the data accumulated so far, it is tempting to speculate that one of the major roles for SIBLINGs and their proteolytic fragments is to orchestrate, in the near proximity of cancer cells, a dynamically changing microenvironment that supports the key local steps that need to be temporally and spatially coordinated for successful invasion. These include adhesion through interactions with specific integrins, matrix degradation through localization and perhaps activation of MMPs, and migration through activation of selective signalling pathways. Such multifunctional activities are possible thanks to the abilities of SIBLINGs to bind multiple proteins. It is also likely that SIBLINGs are crucial for tumour growth and metastasis because they may participate in angiogenesis. The possible collaboration and/or competition between SIBLINGs during these processes remain to be elucidated by future research. Although acquisition of new data is crucial, the results to date make a case for the future of SIBLINGs as prominent molecular tools for diagnostic, prognostic and therapeutic applications in cancer.

At a glance

  • Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) are a family of glycophosphoproteins comprising osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE).
  • The genes encoding the SIBLINGs are located within a cluster on chromosome 4 and encode soluble, hydrophilic proteins sharing common functional motifs and domains, including an Arg–Gly–Asp (RGD) motif that binds αvβ3 integrin.
  • SIBLINGs were initially described as mineralized tissue-associated glycophosphoproteins and were thought to be functionally restricted to these tissues. Recent results show that they are more widely distributed and are expressed in nonmineralized normal tissue, such as metabolically active ductal epithelial cells.
  • Some SIBLINGs activate specific metalloproteinases (MMPs; BSP activates MMP2, OPN, MMP3 and DMP1, MMP9). These three SIBLINGs also bind complement factor H and prevent complement attack.
  • SIBLINGs are overexpressed in many cancers. OPN and, less so, BSP are by far the more widely studied to date and their levels of expression are correlated with tumour aggressiveness. SIBLINGs can be detected in the blood and their level of expression is associated with prognosis.
  • Among SIBLINGs, OPN is involved in almost all steps of tumour progression, including invasion, metastasis and angiogenesis.
  • In vitro and in vivo experimental models demonstrated that interference with SIBLINGs, such as small interfering RNA selective knockdown, has potential anticancer therapeutic value.
  • Identifying the specific roles of SIBLINGs in cancer–stroma interactions and signalling cascades involving growth factor–growth factor receptor and cell–matrix interactions could result in the development of additional and refined strategies for the prevention and treatment of metastases.


The work of A.B. and V.C. is supported by grants from the National Fund for Scientific Research (Belgium), the Centre Anti-Cancéreux of the University of Liège and the European Commission through METABRE Contract CEE LSHC-CT-2004-503049. The work of K.O. is supported by National Institute of Dental and Craniofacial Research/National Institutes of Health (NIH) Grant K23 017791- 01A1, Medical College of Georgia Research Institute Grant STP 00105W005, and the Wendy Will Case Cancer Fund. The research of L.W.F. is supported by the Intramural Research Program of the NIH, Department of Health and Human Services. The work of N.S.F. is supported by grants from the National Cancer Institute (R21CA87311 and R01 CA113865) and from the Department of Defense Congressionally Directed Medical Research Program (W81XWH-04-1-0844 and BC010478).


Integrins are a large family of heterodimeric cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. They promote stable interactions between cells and their environment and mediate intracellular signalling
The main, calcareous part of a tooth, beneath the enamel and surrounding the pulp chamber and root canals
RGD motif
A tripeptide, Arg–Gly–Asp (RGD), found in numerous proteins that support cell adhesion. A subset of the integrins recognize the RGD motif within their ligands, the binding of which mediates both cell–substratum and cell–cell interactions
Hydroxyapatite crystals
The principal inorganic constituent of bone matrix and teeth, imparting rigidity to these structures, and consisting of hydrated calcium phosphate, Ca5(PO4)3OH
Metabolically active normal duct
Epithelia, such as that of the kidney nephrons, that alter the tonicity of the fluid they process in the course of normal physiology. The kidney nephrons process isotonic urine into the voided hypotonic urine
Type 0 introns
Introns that disrupt an open reading frame between codon junctions and therefore permit any splicing combination to other type 0 exons without causing frameshifts
Metabolically passive normal duct
Epithelia, such as that of the lacrimal gland ducts, that do not alter the tonicity of the fluid they process in the course of normal physiology. Tears from the lacrimal acini are isotonic and are secreted unchanged through the duct system
A family of cell surface signal transducing glycoproteins involved in cell–cell interactions, cell adhesion and migration. CD44s bind hyaluronan, a high-molecular mass polysaccharide found in the extracellular matrix, and a variety of extracellular as well as cell surface ligands. CD44 exists in multiple spliced forms and shows a high variability in glycosylation
A family of enzymes that catalyse the crosslinking of proteins at a glutamine in one chain with lysine in another chain. Although the family members have different structures, they share an active site (Tyr–Gly–Gln–Cys–Trp) and strict Ca2+ dependence
A cell that breaks down mineralized bone and is responsible for bone resorption
Dental pulp cells
Cells that comprise the soft tissue forming the inner structure of a tooth and containing nerves and blood vessels as well as possibly dentin stem cells
Describes tumours that metastasize preferentially to the skeleton
Bone lesions
Lytic lesions are areas of the bone marked by destruction, whereas sclerotic lesions are areas of the bone marked by thickening or hardening. A mixed lytic and sclerotic lesion exhibits facets of both resorption (destruction) and thickening (formation)
The process whereby opsonins (antibodies or complement proteins) make an invading cell or microorganism more susceptible to phagocytosis by binding to its surface
Chicken chorioallantoic membrane assay
A biological assay using the well-vascularized chorioallantoic membrane of the chicken egg to evaluate the biological activity of pro- and anti-angiogenic factors
Renal osteodystrophy
A bone disease characterized by softening and fibrous degeneration of bone and the formation of cysts in bone tissue, caused by chronic renal failure
Eccrine sweat ducts
These ducts transport sweat to the surface of the skin and are involved in evaporative cooling
Oncogenic hypophosphataemic osteomalacia
Osteomalacia (softening of the bones) resulting from renal phosphate wasting and low serum 1,25-dihydroxy vitamin D secondary to the presence of a tumour of which complete resection results in rapid resolution of the symptoms and signs

Contributor Information

Akeila Bellahcène, Metastasis Research Laboratory, University of Liège, Belgium.

Vincent Castronovo, Metastasis Research Laboratory, University of Liège, Belgium.

Kalu U. E. Ogbureke, Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, GA, USA.

Larry W. Fisher, Craniofacial and Skeletal Diseases Branch, DIR, NIDCR, NIH, Bethesda, MD, USA.

Neal S. Fedarko, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.


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