Bone sialoprotein 1;  BNSP;  BSPI;  Early T-lymphocyte activation 1;  ETA-1;  Nephropontin;  OPN;  Osteopontin;  Osteopontin-C;  Osteopontin-D;  Osteopontin/immunoglobulin alpha 1 heavy chain constant region fusion protein;  PSEC0156;  SPP-1;  Secreted phosphoprotein 1;  Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1);  SPP1;  SPP1/CALPHA1 fusion;  Urinary stone protein;  Uropontin

The protein product of gene Spp1 is known by many names; however, osteopontin (OPN) is the most widely used and preferred. Osteopontin was first identified in osteoblasts and is primarily known for its role in bone mineralization, hence derives the name “osteo” for bone and pontin from “pons”- bridge in Latin. Orthologous gene analysis of Spp1 gene indicates that it is found in 39 eukaryotic genera. In humans, OPN is expressed in many organs and plays an important role in many cellular, physiological, immunological, and pathological processes; thus, OPN is a multifunctional protein. It belongs to one of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of secreted phosphoproteins; other proteins in this family include integrin-binding sialoprotein (IBSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). OPN was first described by Donald Senger, of Massachusetts Institute of Technology (Senger et al. 1980), and then by many other groups (Prince et al. 1987; Oldberg et al. 1986). The protein was first characterized for glycosylation by Franzén and Heinegård (Franzen and Heinegard 1985). However, the role of OPN in cancer was first proposed by Smith and Denhardt in 1987 (Smith and Denhardt 1987). This chapter will give a brief overview of OPN and its significance in cancer.

Nascent OPN is 300–314 amino acid long and approximately 33 kDa protein; however, after glycosylation the molecular weight ranges from 44–75 kDa. The protein is comprised of negatively charged, hydrophilic amino acids such as Asp and Glu. OPN contains a signal peptide for translocating the translated protein into the endoplasmic reticulum for posttranslational modification. The protein undergoes phosphorylation, sulfation, and glycosylation. The type and amount of posttranslational modification depend on the cell type and thus perform different function (Sodek et al. 2000). Therefore, the molecular weight varies from species to species and cell types to cell types. The protein FAM20C phosphorylates OPN at multiple sites within the S-x-E/pS motif in the extracellular medium. N- and O-glycosylation are carried out at by glycosyltransferases at Asp and Ser/Thr, respectively. OPN is then secreted out of the cell where it is known as sOPN; however, not all OPN is secreted; the intracellular OPN (iOPN) is found in cytoplasm and nucleus (Inoue and Shinohara 2011). (This chapter addresses sOPN as OPN.)

The multiple integrin-binding domains (α_vβ₁, α_vβ₃, α_vβ₅), CD44-binding domain, aspartate domain, and cell-attachment sequence Arg-Gly-Asp (RGD) are all peculiarities of osteopontin. In addition it also contains heparin- and calcium-binding regions as well as matrix metallopeptidase and thrombin cleavage sites (Fig. 2). A brief overview of OPN interacting with different proteins can be visualized in Fig. 3. The cleavage of OPN gives rise to two different isoforms, OPN-R or OPN-L. However, there are splice variants that lead to isoform OPN-a, OPN-b (lacking exon 5), and OPN-c (lacking exon 4). The function of these isoforms is a matter of great interest among researchers, due to the fact that they are associated with cancer (Anborgh et al. 2011).

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OPN is classified as intrinsically disordered protein (IDPs) (Kurzbach et al. 2013), meaning that the tertiary structure of this protein is not well defined and forms various 3D structures (Fig. 4). Since it has many interacting domains, the disordered structure may facilitate its interaction with various receptors. Its multitude of binding domains and posttranslational modification make OPN a multifunctional protein.

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The regulation of OPN is influenced by many factors and is not very well understood. It is observed that OPN is upregulated by many activators such as EGF, hepatocyte growth factor, platelet-derived growth factor, parathyroid hormone, nitric oxide, TNF-α, IL-1β, angiotensin II, TGF-β, hyperglycemia, and hypoxia (Mazzali et al. 2002). It is not exactly clear how OPN is regulated; however, the Spp1 gene promoter elements may help to understand. The promoter driving Spp1 gene contains TATA-like, CAAT-like motifs along with RE-1 element. The RE-1 element consists of RE-1a (−55 to −86) which harbors binding sites for transcription factors Sp1, glucocorticoid receptor, and the E-box-binding factors; and RE-1b (−22 to −45) contains octamer motif-binding protein (OCT-1/OCT-2). It was also observed that myc binds to RE-1a and OCT-1 binds RE-1b and these elements work synergistically (Wang et al. 2000). However, the promoter also shows presence of Ras-activated enhancer sequence, vitamin D responsive element, and interferon-inducible element (Mazzali et al. 2002). In addition to these regulators, miRNAs have also been identified to target OPN expression. Our lab and others have shown that hsa-miR-299-5p, hsa-miR-335-5p, and hsa-miR-146a-5p target human OPN expression (http://mirtarbase.mbc.nctu.edu.tw/index.php).

OPN is known primarily for its role in bone mineralization and modeling and secondarily as a cytokine. However, this protein is now deemed as multifunctional protein. The activation of Spp1 gene by various factors, splicing of pre-mRNA that lead to various translated isoforms, as well as the different binding sites on OPN indicate the gene to protein events are intricately regulated. This could result in a multilevel regulatory capacity of this protein (Anborgh et al. 2011). OPN is found in many animal species such as mouse, primates, cat, and dogs and serves a similar role within these organisms. The role of OPN in promoting cancer through nonclassical Wnt and Hedgehog (Hh) signaling pathway has been shown (Shevde and Samant 2014; Mitra et al. 2012). OPN modulates Wnt signaling by influencing β-catenin to transcribe oncogenes downstream with the help of transcription factor T-cell factor 4 (TCF4). It also regulates many signaling pathways required for tumor progression (Mi et al. 2009). Similarly, OPN activates the nonclassical Hh signaling, mediated by transcription factor GLI1 (Das et al. 2013). These pathways are important in the development of embryos and in maintaining tissue/organ architecture and wound healing; however, dysregulation of these pathways through OPN is responsible for cancer (Shevde and Samant 2014). OPN is expressed in breast, colorectal, endometrial, head and neck, liver, lung, ovarian, pancreatic, prostate, renal, skin, testis, and urothelial cancers, as well as in carcinoid, glioma, lymphoma, and melanoma (Uhlen et al. 2015). There is convincing evidence that OPN plays a crucial role in modulating cancer by virtue of its role in inflammation, cell proliferation, metastasis, and anticancer drug resistance. The significance of OPN is such that it is used as a marker for cancer in addition to other markers. The overexpression of OPN is an indication of cancer progression and decreased levels of OPN are indicative of a better prognosis; thus, it has clinicopathological implications and can therefore be used for diagnosis along with other cancer markers (Weber 2011) (Fig. 5).

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Role in cancer: OPN is produced and secreted in normal cells of different organs in varying amounts. However, it has been observed that the expression of OPN levels increases in the cancerous cells (Table 1). Spp1 gene is predominantly observed to have missense mutation than any other mutations (nonsense, nonstop, indel) (Fig. 6). These mutations may alter OPN and however it may or may not be functionally relevant to cancer. However, any alteration in the promoter region of Spp1 gene could have a direct impact on OPN-mediated functions. A comparison of promoter regions in both breast cancer and normal specimens shows polymorphism at −443 position. This position is important for melanoma metastases and is a proposed binding site for transcription of c-Myb. The polymorphism at −443 with cytosine (homozygous allele) showed up-regulation of Spp1 compared to a heterozygous allele containing thymine/cytosine or homozygous thymine. The mutations at −443 position might favor the c-Myb activity and enhance the Spp1 transcription. Similarly, the polymorphism in the Spp1 promoter at −155_156GG position is linked to increased risk of glioma.

Osteopontin (Spp1), Table 1

The levels of OPN expressed in normal and cancer organs modified from the data CAB002212 at the protein atlas (http://www.proteinatlas.org/)

Organs expressing OPN	Level of OPN expressed
	Normal	Cancer
Brain	Not detected	High, medium
Breast	Low	High, medium
Cervix	Not detected	High, medium, low
Colon/rectum	Medium	High, medium, low
Endometrium	Low	High, medium, low
Head and neck	Low	Medium, low
Kidney	High	High, medium, low
Liver	Not detected	High, medium, low
Lungs	Medium	Medium, low
Lymph node	Low	Medium, low
Ovary	Not available	High, medium, low
Pancreas	Medium	High, medium, low
Prostate	Not detected	High, medium, low
Skin	Not detected	High, medium, low
Stomach	Low	High, medium
Testis	Not detected	High, medium, low
Urinary bladder	Low	High, medium, low

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Cell adhesion: OPN promotes cell adhesion by interacting with integrins present on the cell surface through the RGD-dependent mechanism (Liaw et al. 1994). However, OPN is also known to help adhesion without the RGD by not interacting with alpha v integrin (Katagiri et al. 1996). Another interesting aspect is that the balance of glycosylation and phosphorylation determines the function of OPN in cell adhesion. The O-glycosylation of OPN determines the level of phosphorylation and modulates cell adhesion. O-glycosylation suppresses phosphorylation which reduces cell adhesion (Kariya et al. 2014). The thrombin cleavage sites on OPN are important for cell adhesion as the deletion of cleavage sites or inhibition of thrombin resulted in decreased cell adhesion (Beausoleil et al. 2011; Schulze et al. 2008). This has implication on establishment of metastatic cell at other organ or sites in the body.

Epithelial-mesenchymal transition (EMT): The transition of epithelial to mesenchymal phenotype or vice versa is required for developmental processes, wound healing, and the maintenance of structural integrity (Kalluri and Weinberg 2009). However, EMT is the characteristic of cancer development. The dysfunction in these transition events leads to cancer progression and is commonly observed in the bladder, brain, breast, cervical, colon/rectum, liver, lungs, ovarian, prostate, pancreas, renal, and skin (Heerboth et al. 2015). OPN regulation of EMT was demonstrated in a mouse xenograft model (Bhattacharya et al. 2012). It was also noted that the up-regulation of OPN promotes EMT in breast, colorectal, gastric, liver and ovarian cancer. OPN mediates EMT downstream (downstream of what?) with transcription factors such as TWIST, ZEB, and SNAIL (Kothari et al. 2016). In cancer, EMT is the interplay of HF-1 α, PI3K/AKT, and NF-κB pathways mediated or initiated by OPN.

Metastasis: As OPN facilitates EMT, this eventually leads to metastasis; the general prerequisite for metastasis is through the mesenchymal phenotype. It starts with increased motility and invasiveness and the migrated cells establish (cell adhesion) and form a tumor (angiogenesis) on the site, by virtue of OPN (Larue and Bellacosa 2005). The isoforms of OPN are an important part of this process. Metastasis increases in the cells expressing OPN and the inhibition of OPN reverses it. In cases of non-small lung cancer, metastasis from the lungs was reduced when OPN was neutralized with a monoclonal antibody (Shojaei et al. 2012). Hepatic metastasis in colorectal cancer was associated with OPN due to decreased homotypic adhesion and increased heterotypic adhesion (Kyprianou et al. 2012). Similarly, OPN has been established as a key player in the metastasis from breast cancer and lungs and bone metastasis is established (Ibrahim et al. 2000).

Role in immunity: OPN is a secretory cytokine and plays an active role in inflammation at the systemic or local cancer microenvironment. External injection of OPN recruits macrophages and T-cells at the site demonstrating its chemotactic function. OPN also increases the cell adhesion of these cells, and if the gene Spp1 is silenced or OPN is neutralized with antibodies, the cells adhesion is reduced. The recruitment of leucocytes creates a pro-inflammatory environment around and supports Th1 and suppresses Th2 immune response (Mazzali et al. 2002). OPN stimulates IL-12 production and suppression of IL-10 as a pro-inflammatory cytokine; however, it is also known to be anti-inflammatory. Under stress or inflammation, the enzyme nitric oxide synthase (iNOS) produces nitric oxide (NO) in the cells, and this NO induces OPN to inhibit iNOS. A feedback inhibition of iNOS is regulated by OPN and thus acts as anti-inflammatory (Denhardt et al. 2001). The pro- and anti-inflammatory feature of OPN makes it ideal for cancer progression. A balanced inflammatory microenvironment is important for cancer, as limited inflammation restricts tumor growth, while excess inflammation causes tumor regression (Coussens and Werb 2002). It is interesting to know that OPN produced by tumors or a host acts as an immunosuppressor in the metastatic microenvironment probably due to the regulation of inflammation and favorable cancer growth (Sangaletti et al. 2014). In the tumor microenvironment, OPN offers this strategy of self-survival and proliferation for the tumor mass (or cancer cell population), in addition to favoring angiogenesis, resisting apoptosis.

Drug resistance: The anticancer drugs are effective in targeting cancer cells; however, the chemotherapy regime faces difficulties due the drug resistance. The mutations in cancer may increase the transporter activity or degrade the drug to reduce the intracellular concentration of drug. Alternate mechanism involves mutation in target protein or epigenetic modification, alteration in DNA repair, or cell cycle processes (Florea and Büsselberg 2013). OPN is one of the proteins that is responsible for drug resistance as an effect of the aforementioned processes. OPN leads to up-regulation of the ABCB1 and ABCG2 transporters that causes drug resistance. Conversely, silencing of Spp1 gene leads to sensitivity to actinomycin-D, cyclophosphamide, doxorubicin, paclitaxel, and rapamycin (Shevde and Samant 2014). Knockout of Spp1 in MDA-MB-231 shows that sensitivity to doxorubicin and radiobiologicals is increased. Similarly, overproduction of OPN increases resistance to oxaliplatin in colorectal cancer (Ng et al. 2015) and cisplatin in oral squamous cell carcinoma (Luo et al. 2015). Elevated levels of OPN in cancer patients are also linked with poor prognosis. Thus, it is very clear that drug resistance and OPN are positively correlated.

Direct or indirect inhibitors of OPN have potential for therapeutics. Aptamers are DNA, RNA, or peptide that form complex three-dimensional structure to occupy an active site or bind to target protein resulting in its inactivation. These molecules hold promise for cancer therapeutics and the list against the cancer targets are increasing day by day (Sun et al. 2014). OPN-R3 aptamers that directly inhibit OPN have been developed, and its administration has reversed breast cancer growth and reduced metastasis (Mi et al. 2008; Talbot et al. 2011). Similar results were observed when Spp1 gene transcripts were silenced using siRNA and shRNA (Shevde and Samant 2014). Also, an antibody against OPN was able to reduce the metastasis significantly in non-small lung cancer (Shojaei et al. 2012). Currently there are very few known inhibitors of OPN. However, agelastatin A alkaloid is 1.5–16 times more potent than cisplatin and is found in the sponge Agelas dendromorpha. Agelastatin A has been beneficial in bladder, breast, colon, and skin cancer. This alkaloid stalls the cancerous cells in the G2 phase of the cell cycle by downregulating β-catenin while upregulating the cellular Tcf-4 that inhibits OPN (Mason et al. 2008). Similarly, another indirect way of targeting OPN is by argatroban, a thrombin inhibitor. The activity of OPN is enhanced by the cleavage of OPN at thrombin sites by thrombin; however, this can be hindered by argatroban (Schulze et al. 2008). These therapeutics need thorough investigation of the short- and long-term consequences in normal physiology, as abnormal morphogenesis and lactation deficiencies have been reported in mammary glands due the inhibition of OPN (Nemir et al. 2000).

Besides the normal function of OPN in bone modeling, it is clear that it is one of the important players in cancer. The role of OPN is important for understanding the complexity and diversity of cancer. Currently, the snapshots of OPN’s operation and its ability to modulate inflammation, cell proliferation, apoptosis, metastasis, and drug resistance are known. However, the utilization of this knowledge to target this influential molecule has not been achieved yet. The challenges for the development of drugs targeting OPN are due to IDP nature and unclear implication of impairing OPN function. On the other hand, OPN can be used as a significant cancer diagnostic marker (along with other markers) to distinguish OPN associated with normal and cancer in the clinical setup.