Osteoprotegerin rich tumor microenvironment: implications in breast cancer.

The occurrence of single nucleotide polymorphisms (SNPs) in the OPG gene in association with breast cancer has been examined. Ney et al. 2013 [ 92 ] for the first time reported a significant association between the SNP rs3102735 (5′ near promoter region containing the minor allele C as well as for the homo- and heterozygous genotype with the minor allele C) of the OPG gene and the susceptibility of breast cancer in Caucasian populations. A 1.5-fold increased risk of breast cancer was associated with SNP rs3102735 [ 92 ]. Another study reported that the C allele of the OPG SNP rs2073618 and the T allele of the OPG SNP rs2073617 occurred more frequently in breast cancer patients [ 93 ]. The variant C allele of 950 T/C in the OPG promoter has been reported to play a major role as a genetic safe guard against progression in patients with prostate cancer [ 94 ]. The analysis of OPG gene variants C950T (promoter) and C1181G (exon 1) revealed that the presence of polymorphic 1181G/950T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease [ 95 ]. Apart from breast cancer, genetic variations in form of polymorphisms in OPG and RANKL have also been associated with bone fractures in premenopausal patients with systemic lupus erythematosis (SLE) [ 96 ]. OPG/A163G polymorphism has been suggested to contribute to the genetic regulation of bone mineral density or bone turnover markers in Slovak population and thus could increase or decrease osteoporosis risk [ 97 ]. 163A/G (rs3102735) and 950T/C (rs2073617) polymorphisms of OPG have been evaluated in patients with pre-eclampsia (60 cases of early-onset severe pre-eclampsia and 91 cases of late-onset pre-eclampsia) [ 98 ]. Kwan et al., 2014 used a meta-analysis of GWAS from FIVE studies comprising > 10 000 individuals from European and Asian origin, and identified two genome-wide significant loci (8q23-q24.1 and 17q11.2) and one locus on chromosome 14 associated with OPG levels with near genome-wide significance [ 99 ]. In conclusion, they discovered that variants > 100 kb upstream of the gene encoding OPG are associated with variation in circulating OPG levels and identified another new significant locus on chromosome 17q11.2 as well as a suggestive locus on chromosome 14q21.2 associated with the trait [ 99 ]. They estimated that over half of the heritability of age-adjusted OPG levels could be explained by all SNPs studied [ 99 ].

Metastasis is an intricate multistep process, which is closely associated with worse prognosis of patients with tumors, and spread of the tumor. For example, bone metastasis is considered lethal and is an active area of research for potential therapeutic developments targeting bone cancer metastases. Bone metastases occur in more than 70% of breast cancer patients and cause severe skeletal complications such as fractures, spinal cord compression, bone pain, and hypercalcemia [ 26 ]. Patients with estrogen receptor-positive (ER+) breast cancer constitute a major clinical population who are at risk for bone metastases [ 26 ]. More than 50% of primary breast cancer cells express OPG and RANK, while RANKL could be detected only in 14-60% [ 25 ]. OPG is one of the important players of the OPG/ receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) triad (Figure 2 ), and is a secreted member of the TNFR superfamily of proteins [ 27 ]. OPG functions as an en­dogenous antagonist receptor that prevents the biological effects of RANKL, both membranous and soluble, and thus acts as an inhibitor of bone remodeling and resorption. Unlike RANK and RANKL, OPG does not have a transmembrane domain or cytoplasmic domain [ 28 ]. OPG is thus a decoy receptor, which interferes with the osteoclastic RANKL/RANK signaling to prevent bone loss [ 29 ]. OPG can bind to various cell surface receptors such as Syndecan-1, membranous RANKL, and αVβ5 integrin in order to trigger different cell survival pathways particularly, ERK, PI-3K/Akt pathway which results in expression of various cell survival favorable genes. Simultaneously, stable β-catenin of the canonical Wnt/β-catenin pathway, which is turned on in the majority of cancer cells, translocate to nucleus and turns on OPG gene expression. The OPG gets secreted out of the cell in the microenvironment, and can exert its paracrine and autocrine effects. The released OPG can affect the nearby healthy cells thus driving them towards tumorigenesis. The released OPG can also exert autocrine effects on the cancer cells by binding to cell surface receptors. The binding of OPG turns on the vicious cell survival signaling in cancer cells thus favoring their growth, proliferation, survival, angiogenesis [ 6 ]. The four cysteine rich pseudo repeats form an elongated structure and binds to one of the grooves of the active RANKL therefore preventing RANKL/RANK interaction and hence osteoclastogenesis. OPG production is modulated by sev­eral cytokines, vitamins, estrogens and other molecules, thereby modulating osteoclastogenesis and bone resorp­tion. OPG production is induced by 1α, 25-dihydroxyvi­tamin D3, estrogens, pro-inflammatory cytokines such as interleukin-1 (IL-1) and TNF-α as well as transforming growth factor-β (TGF-β), whereas parathyroid hormone (PTH) and glucocorticoids inhibit OPG production. OPG expression is modulated by biphosphonates in osteoblasts [ 30 , 31 ]. Another study has also highlighted the possibility of bone derived OPG to increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process thus promoting the breast cancer mediated bone osteolysis [ 32 ]. Aggressive breast cancer malignancies metastasize to bone and are associated with dysregulation of the RANK/RANKL/OPG pathway and can increase the RANKL/OPG ratio, which would favor excessive osteolysis [ 33 ]. OPG blocks the maturation of bone resorbing osteoclast cells in the bone microenvironment thus preventing bone resorption. OPG does not have a transmembrane domain or cytoplasmic domain [ 11 ]. A high affinity anti-RANKL monoclonal antibody denosumab (AMG162; bone antiresorptive drug) targeting the osteoblast/cancer cell interphase has been developed to prevent bone loss in prostate and breast cancer bone metastases by slowing down bone turnover, hence prostate and breast cancer growth on the skeleton [ 29 ]. Clinical phase III study showed that even patients with breast cancer without bone metastases but with reduced bone density due to treatment with aromatase inhibitors benefit from treatment with denosumab [ 34 ]. The treatment significantly increased bone density after 1-2 years compared with the placebo treated control group [ 34 ]. AMGN-0007 is a recombinant OPG construct and suppressed bone resorption when administered to multiple myeloma (MM) and breast cancer patients during a phase I clinical trial [ 35 ]. Knocking down OPG expression in triple-negative breast cancer cells led to a significant reduction in metastasis in the chick embryo metastasis model. A reduction in metastasis was observed from both a primary tumor and by intravenous injection of tumor cells, suggesting a direct impact of OPG on metastasis [ 2 ]. The discovery that OPG is a potent inhibitor of osteoclast activity and maturation initiated research into the possibility of using this molecule as a therapeutic agent for the treatment of a variety of conditions associated with increased bone resorption, including tumor-induced bone disease [ 36 ].

The tumor microenvironment (TME) is the cellular environment in which the tumor exists and provides its niche for growth, progression, survival and evolution. The normal microenvironment provides crucial signaling to maintain appropriate tissue architecture, inhibit cell growth and suppress the malignant phenotype, and acts as a barrier to tumorigenesis [ 5 ]. Incorrect signals from TME can destabilize tissue homeostasis, initiate, promote, and push normal cells to malignant phenotype [ 5 ]. The TME is highly complex and dynamic, and includes blood vessels, immune cells, inflammatory cells, and components of the extracellular-matrix (ECM). TME is also rich in various cytokines, chemokines, ECM proteins, growth and angiogenic factors involved in autocrine but also paracrine cell signaling. The field of TME has expanded our understanding of cancer as more than a single factor driven disease. Rather, cancer biology involves complex reciprocal interaction and co-evolution between cancer cells and host stromal cells, interplay of soluble growth factors and chemokines as the key mediators involved in oncogenic signaling pathways of tumors. The list of anti-cancer therapies and clinical trials based on the role of the microenvironment in various types of cancers has grown long and it includes endostatin, Bevacizumab (Avastin), MK-2416, Anastroazole, Bay 43-9006, DX2400, Celecoxib, and PG545 etc. [ 5 ]. The majority of these are multikinase inhibitors blocking tumor cell growth pathways such as BRAF, Bcr-Abl, c-Kit, vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, PDGFR and colony-stimulating factor-1 receptor [ 5 ]. Proteomic landscape of the breast cancer TME includes diverse factors involved in tumor growth, proliferation, metastasis, vascularity, evading cell death pathways and host immune system. In our previous study we demonstrated that inflammatory and invasive breast cancer TME is rich in OPG, chemokines such as urokinase-type plasminogen activator receptor (uPAR), Oncostatin M (OSM), IL-6 and GRO-α [ 6 ]. Here, we focus only on osteoprotegerin (OPG) as one of the factors present in the TME of inflammatory and invasive breast cancer cell lines, and discuss how it multitasks various functions to drive tumorigenesis.

At present, one in eight women in the United States will develop breast cancer [ 1 ]. Recent advances in breast cancer detection and treatment have decreased the mortality rate of breast cancer [ 1 ] but the success of treatment relies largely on detection of the disease at early stages [ 1 ]. A lack of knowledge regarding the molecular mechanisms underlying breast tumor progression to invasive and then metastatic disease limits the ability to treat advanced disease. The identification of factors that promote metastasis is essential for the development of new breast cancer therapies and a further reduction in breast cancer mortality [ 2 ]. Inflammatory breast cancer (IBC) is a highly aggressive, angioinvasive, and a highly metastatic form of breast cancer. IBC is associated with a high incidence of early nodal and systemic spread. Systemic chemotherapy, adjuvant therapy, surgery and radiation have not improved disease prognosis or overall survival of IBC patients [ 3 , 4 ].

Despite the wealth of literature on OPG, there are many questions still unresolved, including the exact role of OPG in bone metastasis of breast cancer and the therapeutic potential of targeting OPG. Besides roles in cancer, OPG plays role(s) in bone metabolism, endometriosis, periodontal disease, thyroid disease and coronary heart disease [ 11 ]. Though OPG can modulate breast tumor growth and progression, future studies required to fully determine the mechanism of effect and overall outcomes of the different types of interactions required to determine whether strategies to block OPG signaling would be effective in blocking the development of primary breast tumors [ 43 ]. At this point OPG can no longer be considered solely in the bone microenvironment in breast cancer and caution must be exercised in the development of systemic treatment strategies aimed at increasing OPG levels. Localized delivery of OPG to the bone may be more appropriate to inhibit osteolysis linked metastatic breast cancer [ 43 ]. Conditionally replicating adenoviruses (CRAds) have been used as anticancer agents designed to infect and lyse tumor cells. In a murine model of osteolytic bone metastases of breast cancer, the CRAd armed with shortened OPG (sOPG)-Fc reduced tumor burden in the bone and inhibited osteoclast formation more effectively than an unarmed CRAd suggesting the role of OPG [ 17 ]. Breast cancer development in BRCA1/2 mutation carriers is a consequence of autonomous and nonautonomous cell factors, which serve as excellent targets for cancer prevention. BRCA-mutation carriers were reported to have lower mean values of free serum OPG in particular in BRCA1-mutation carriers compared with controls [ 100 ]. OPG may become a new biomarker and a target of treatment for patients with colorectal cancer since many studies have revealed the clinicopathologic significance of OPG expression by using clinical tissue samples from patients [ 78 ]. The effects of sustained expression of OPG using a recombinant adeno-associated viral (rAAV) vector in a mouse model of osteolytic breast cancer has clearly indicated the potential of rAAV-OPG therapy for reducing morbidity and mortality in breast cancer patients with osteolytic bone damage [ 101 ]. With all this development in the understanding of OPG, there might be more therapeutic avenues to manipulate OPG to predict and manage aggressive forms of breast cancer.

OPG was first identified by sequence homology to the tumor necrosis factor receptor (TNFR) family during a rat intestine cDNA sequencing project and was named based on its function (Latin: os bone, protegere to protect) [ 7 ]. OPG was independently discovered and alternatively named osteoclastogenesis inhibitory factor (OCIF) [ 8 ], TR1 [ 9 ], and follicular DC-derived receptor-1 (FDCR-1) [ 10 ], which are found to be identical to OPG. The human OPG gene is located at chromosome 8q23-24 and is composed of 401 amino acids [ 11 ]. The OPG protein comprises 401 amino acids of which 21 form a signal peptide [ 12 ] (Figure 1 ). Human and murine OPG consist of four cysteine rich pseudo repeats located in the N-terminal, two death domains, and a heparin-binding site located in the C-terminus and a 21 amino acid signal peptide [ 11 ]. Signal peptide is cleaved to generate a mature form of 380 amino acids (Figure 1 ). At the N terminus, there are four domains (D1-D4), which have cysteine-rich TNF receptor homologous motifs [ 12 ]. These motifs are required and are sufficient for binding to its major target, the receptor activator of nuclear factor (NFΚB) ligand (RANKL), and for inhibiting osteoclastic differentiation and activation [ 12 ]. At the C terminus, there are tandem death-domain homologous regions (D5 and D6) followed by a heparin-binding site (D7) [ 12 ] (Figure 1 ). More specifically at position 400, there is a cysteine required for homodimerization of the molecule (Figure 1 ). OPG is a secreted protein with no transmembrane or cytoplasmic domain, and is produced as a monomer (55-62 kDa) that undergoes homodimerization [ 11 , 12 ]. OPG is secreted as a disulfide-linked homodimeric glycoprotein with four or five potential glycosylation sites, generating a mature form of OPG of 110-120 kDa [ 12 ]. The dimeric form of the protein exhibits a greater affinity for RANKL and a higher heparin-binding capacity than the monomeric form. The heparin binding site (D7) of OPG facilitates its binding to cell membrane-associated heparan sulfates. Heparan sulfates are expressed on the cell surface as heparan sulfate proteoglycans (HSPGs), and are involved in cell signaling, controlling cell behavior, actin cytoskeleton regulation, cell adhesion, and cell migration [ 13 ]. For biological use, D7, D5, and D6 are removed, and the remaining amino acids 22-194 OPG peptide is fused to the Fc domain of human IgG1 (OPG-Fc), which maintains the potent dimeric nature of full-length OPG and exhibits a significantly increased circulating half-life [ 12 , 14 ]. OPG is highly expressed in the adult lung, heart, kidney, liver, thymus, lymph nodes and bone marrow [ 11 ]. OPG is syn­thesized by several cells including stromal cells, osteo­blasts, vascular smooth muscle cells, B lymphocytes, and articular chondrocytes [ 11 ].