Omics approach towards understanding the function of bioactive lipids in osteosarcoma (2017)

Osteosarcoma is the most common form of primary bone cancer in humans and well as dogs. The treatment options for OS include a multi-agent induction chemotherapy and radical excision of the tumor followed by adjuvant chemotherapy. Despite the aggressive treatment course, the survival rates are poor. In patients with localized disease, 5-year survival rates are approximately 65%. However, for metastatic disease at diagnosis or recurrence, the 5-year survival rates have plateaued to only 20%. Although progress has been made towards improving treatment options, the early detection and subsequent control of metastasis have been challenging in OS. This work is aimed at discovering the various proteomic and lipidomic biomarkers in metastatic and non-metastatic osteosarcoma as well as development of lipid mediators as potential therapeutics.&#13;\nWe take a high-throughput approach towards identifying specific membrane proteins and lipids that are differentially expressed in normal osteoblasts, non-metastatic and metastatic osteosarcoma. As described in chapter 2, a high throughput peptide fingerprinting enabled us to identify various membrane proteins that are differentially expressed in metastatic versus non-metastatic osteosarcoma. Membrane proteins are the largest class of drug targets and this study identifies proteins that can targets for drugs without having to overcome the delivery of drugs across the plasma membrane. However, the functional study of membrane proteins is complicated by the fact that they exist in an amphipathic environment and lose their structure in aqueous buffers. Thus we stabilize the membrane proteome of osteosarcoma cells in nanodiscs (chapter 3). Nanodiscs are lipid bilayers surrounded by membrane scaffold protein and provide the amphipathic environment for membrane proteins. In this study we showed that lipid composition affects the protein composition incorporated. &#13;\nSince lipid composition affects protein incorporation and lipid-protein interactions are important for cell signaling processes, we investigated the global cellular lipidome of osteosarcoma and osteoblast cells through a high throughput lipidomics approach (chapter 4). We identified that several lipid classes and lipid species are differentially regulated in normal cells versus non-metastatic cells vs metastatic cells. These include both structural lipids as well as signaling lipids. Since signaling lipids play an important role in various physiological functions, we chose to further explore the role of a specific class of signaling lipids, known as endocannabinoid epoxides, in osteosarcoma (chapter 5). Endocannabinoid epoxides are metabolites of omega-3 fatty acids that arise from a cross-talk of phospholipase D and cytochrome P450 epoxygenase enzymes. They have previously been shown to have anti-inflammatory and anti-angiogenic properties. In this study, we show that they also possess anti-tumorigenic activity. Furthermore, we synthesize novel analogs of specific endocannabinoid epoxides that show reduced susceptibility to biological enzymes, making them better therapeutic candidates.&#13;\nTherefore, we have performed a thorough proteomic and lipidomic characterization of osteosarcoma that can serve as biomarkers as well as initiate the development of novel therapeutics. The lipid mediators synthesized in chapter 5 show promise in cellular studies and can be taken forward into mice models for osteosarcoma and bone cancer pain. These will likely show similar effects in other cancers and studies can be expanded to carcinomas and other cancers as well.