Abstract
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.
Highlights
Breast cancer is the most common cancer and the second leading cause of death from malignancy in women in the United States
Strong immunofluorescence staining for fatty acid synthase (FASN) and ACC1 was observed in the breast cancer cells compared to the control human mammary epithelial cells (HMEC) cells [19]
We recently reported that the breast cancer microenvironment is rich in OPG, which can influence healthy HMEC cells and drive them towards tumorigenesis [3]
Summary
OPG (osteoprotegerin) is a secreted member of the TNFR (tumor necrosis factor receptor) superfamily of proteins. It is a secretory protein and, as evident from the name, it blocks the maturation of bone resorbing osteoclast cells in the bone microenvironment preventing bone resorption [2]. Our previous study [3], for the first time, identified that OPG is secreted and expressed at very high levels from SUM1315MO2 (invasive breast cancer cell line), SUM149PT and SUM190PT cells (inflammatory breast cancer cell lines). OPG could reprogram the normal HMEC cells by inducing cell survival, proliferation, aneuploidy and upregulating CD24 expression. OPG induced upregulation in gene copy numbers for oncogenic pathway regulators such Aurora A, EGFR, AKT/PI3K, and MYC in HMEC spheres [3]
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