BackgroundOsteoprotegerin (OPG) is a glycoprotein that has multifaceted role and is associated with several cancer malignancies like that of bladder carcinoma, gastric carcinoma, prostate cancer, multiple myeloma and breast cancer. Also OPG has been associated with several organ pathologies. The widespread expression of OPG suggests that OPG may have multiple biological activities that are yet to be explored.MethodsThe anchorage-independent sphere cultures of the adherent cells were instrumental in our study as it provided a deeper insight into the complexity of a 3D tumor. Cytokine profiling was performed for OPG’s detection in the microenvironment. ELISA and western blotting were performed to quantify the OPG secretion and measure the protein levels respectively. OPG expression was detected in human breast cancer tissue samples by IHC. To decipher OPG’s role in tumor aggressiveness both recombinant human OPG as well as OPG rich and depleted breast cancer cell conditioned media were tested. Western blotting and MTT assay were performed to detect changes in signaling pathways and proliferation that were induced in presence of OPG. Onset of aneuploidy, in presence of OPG, was measured by cell cycle analysis and western blotting. Finally, human Breast Cancer qBiomarker Copy Number PCR Array was used to detect how OPG remarkably induced gene copy numbers for oncogenic pathway regulators.ResultsSUM149PT and SUM1315M02 cells secrete high levels of the cytokine OPG compared to primary human mammary epithelial cells (HMEC). High expression of OPG was also detected in human breast cancer tissue samples compared to the uninvolved tissue from the same patient. OPG induced proliferation of control HMEC spheres and triggered the onset of aneuploidy in HMEC sphere cultures. OPG induced the expression of aneuploidy related kinases Aurora-A Kinase (IAK-1), Bub1 and BubR1 probably through the receptor activator of nuclear factor kappa-B ligand (RANKL) and syndecan-1 receptors via the Erk, AKT and GSK3(3 signaling pathway. Gene copy numbers for oncogenic pathway regulators such AKT1, Aurora-A Kinase (AURKA or IAK-1), epidermal growth factor receptor (EGFR) and MYC with a reduction in the copy numbers of cyclin dependent kinase inhibitor 2A (CDKN2A), PTEN and DNA topoisomerase 2 alpha (TOP2A) were induced in presence of OPG.ConclusionsThese results highlight the role of OPG in reprogramming normal mammary epithelial cells to a tumorigenic state and suggest promising avenues for treating inflammatory breast cancer as well as highly invasive breast cancer with new therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1837-1) contains supplementary material, which is available to authorized users.

Inflammatory and invasive breast cancers are aggressive and require better understanding for the development of new treatments and more accurate prognosis. Here, we detected high expression of PPARα in human primary inflammatory (SUM149PT) and highly invasive (SUM1315MO2) breast cancer cells, and tissue sections of human breast cancer. PPARα ligands are clinically used to treat dyslipidemia. Among lipid lowering drugs clofibrate, fenofibrate and WY14643, clofibrate showed high chemo-sensitivity towards breast cancer cells. Clofibrate treatment significantly induced PPARα DNA binding activity, and remarkably reduced cyclooxygenase-2/PGE2 and 5-lipoxygenase/LTB4 inflammatory pathways. Clofibrate treatment reduced the proliferation of breast cancer cells probably by inhibiting NF-κB and ERK1/2 activation, reducing cyclinD1, cyclinA, cyclinE, and inducing pro-apoptotic P21 levels. Surprisingly, the expression of lipogenic pathway genes including SREBP-1c (sterol regulatory element-binding protein-1c), HMG-CoA synthase, SPTLC1 (serine palmitoyltransferase long-chain), and Acyl-CoA oxidase (ACO) decreased with a concurrent increase in fatty acid oxidation genes such as CPT-1a (carnitine palmitoyltransferase 1a) and SREBP-2 (Sterol regulatory element-binding protein-2). Clofibrate treatment induced secretion of free fatty acids and effectively decreased the level of phosphorylated active form of fatty acid synthase (FASN), an enzyme catalyzing de novo synthesis of fatty acids. High level of coactivators steroid receptor coactivator-1 (SRC-1) and histone acetylase CBP-300 (CREB binding protein-300) were observed in the nuclear complexes of clofibrate treated breast cancer cells. These findings implicate that stimulating PPARα by safe, well-tolerated, and clinically approved clofibrate may provide a safer and more effective strategy to target the signaling, lipogenic, and inflammatory pathways in aggressive forms of breast cancer.

The crosstalk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. In our previous study, we discovered Osteoprotegerin (OPG) as one of the cytokines heavily secreted by breast cancer cells. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to normal human mammary epithelial HMEC cells. OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer. Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assays highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of the COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells. Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and fatty acid synthase (FASN). FASN is a multifunctional enzyme involved in lipid biosynthesis. Immunofluorescence staining revealed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be crosstalk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Interestingly, knocking down OPG by CRISPR/Cas9 gene editing in breast cancer cells decreased FASN expression at the protein level. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 and COX-2 inhibitor celecoxib individually decreased the number of lipid bodies/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3/7 and caspase-9 activation. But a more efficient and effective decrease in lipid bodies/cell and survival kinase signaling was observed upon combining the drug treatments for the aggressive cancer cells. Collectively, the novel biological crosstalk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic target to treat highly invasive breast cancer.

Osteoprotegerin (OPG) is a soluble decoy receptor for tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). It belongs to the tumor necrosis factor receptor superfamily (TNFRSF). OPG was initially discovered to contribute to homeostasis of bone turnover due to its capability of binding to receptor activator of nuclear factor-kappaB (NF-kB). However, apart from bone turnover, OPG plays important and diverse role(s) in many biological functions. Besides having anti-osteoclastic activity, OPG is thought to exert a protective anti-apoptotic action in OPG-expressing tumors by overcoming the physiologic mechanism of tumor surveillance exerted by TRAIL. Along with inhibiting TRAIL induced apoptosis, it can induce proliferation by binding to various cell surface receptors and thus turning on the canonical cell survival and proliferative pathways. OPG also induces angiogenesis, one of the hallmarks of cancer, thus facilitating tumor growth. Recently, the understanding of OPG and its different roles has been augmented substantially. This review is aimed at providing a very informative overview as to how OPG affects cancer progression especially breast cancer.

The endoplasmic reticulum (ER) regulates protein folding, post-translational modifications, lipid synthesis, and calcium signaling to attenuate the accumulation of misfolded proteins causing ER stress and maintains cellular homeostasis. The tumor microenvironment is rich in soluble cytokines, chemokines, growth, and angiogenic factors and can drive the ER’s abnormal functioning in healthy cells. Cancer cells adapt well to the tumor microenvironment induced ER stress. We identified that the inflammatory breast cancer (IBC) cells abundantly express osteoprotegerin (OPG) and their tumor microenvironment is rich in OPG protein. OPG also called osteoclast differentiation factor/osteoclastogenesis inhibitory factor (OCIF) is a soluble decoy receptor for receptor activator of nuclear factor-kappa B ligand (RANKL). Employing mass spectrometry analysis, we identified a set of ER chaperones associated with OPG in IBC cell lysates (SUM149PT, SUM1315MO2) compared to healthy human mammary epithelial cells (HMEC). Proximity ligation assay (PLA) and immunoprecipitation assay validated the interaction between OPG and ER chaperone and master regulator of unfolded protein response (UPR) GRP78/BiP (glucose-regulated protein/Binding immunoglobulin protein). We detected remarkably high gene expression of CCAAT enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), protein disulfide-isomerase (PDI), PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) in SUM149PT and SUM190PT cells when compared to HMEC. Similarly, tissue sections of human IBC expressed high levels of ER stress proteins. We evaluated cell death and apoptosis upon Salubrinal and phenylbutyrate treatment in healthy and IBC cells by caspase-3 activity and cleaved poly (ADP-ribose) polymerase (PARP) protein assay. IBC (SUM149PT and SUM190PT) cells were chemosensitive to Salubrinal treatment, possibly via inhibition in OPG secretion, upregulating ATF4, and CHOP, thus ultimately driving caspase-3 mediated IBC cell death. Salubrinal treatment upregulated PDI, which connects ER stress to oxidative stress. We observed increased ROS production and reduced cell proliferation of Salubrinal treated IBC cells. Treatment with antioxidants could rescue IBC cells from ROS and aborted cell proliferation. Our findings implicate that manipulating ER stress with Salubrinal may provide a safer and tailored strategy to target the growth of inflammatory and aggressive forms of breast cancer.