Signature of sphingolipid-related genes in cancer

Current anti-cancer strategies are mainly focussing on the validation of the following targets and signaling pathways: growth factor signaling, cell cycle regulators, cancer stem cells, apoptosis regulators, extracellular matrix components, and mediators of angiogenesis and metastasis, as well as survival advantages. Intriguingly, some lines of evidence strongly suggest that sphingolipids are the active players in several of these pathways. Thus, sphingosine 1-phosphate has emerged as a potent trigger of proliferation, cytoskeleton organization and migration, adherence- and tight junction assembly, and morphogenesis. Besides the tumor cells, sphingosine 1-phosphate acts on endothelial cells as a strong pro-angiogenic factor. This peculiar lipid mediator functions extracellular through binding to the cell type-specific repertoire of G-protein-coupled receptors. In turn, ceramide acts as functional antipole of sphingosine 1-phosphate; diseased-driven aberrant levels of intracellular ceramide may lead to dysregulation of apoptotic cell death as well as development of a multidrug resistance phenotype in cancer cells. These are just two examples among the plethora of sphingolipid metabolites logically emphasizing why the complete understanding of formation and degradation of sphingolipid mediators via the interconnected network of cellular enzymes in health versus diseased conditions associated with increased tumorigenic potential is of high priority. The primary goal of this study is to further characterize cellular checkpoints, which redirect the physiologically balanced sphingolipid system to the pathological situations leading to development and progression of solid tumors as well as acquisition of therapy resistance. For a systematic analysis, we propose to use a combination of (i) in silico data-driven approach based on the analysis of an available genome-wide microarray data sets and dissecting altered sphingolipid-associated gene signatures, and (ii) a knowledge-driven approach based on the real-time PCR gene expression profiling using pre-designed multigene signature (panel of 45 genes; composition is selected on the basis of data mining of sphingolipid biology and creation of gene interactive network). Gene expression profiling will be performed with well-characterized clinical specimens from three types of solid tumors. Profiling will be further done with the established cell lines of respective tumor types and chemotherapy resistance models with sensitive parental and drug-selected sublines. Created gene expression data sets will be further analyzed to find regularities in the expression patterns within defined types of solid tumors to identify multigene signatures with prognostic value. To get associations with other disease-relevant mechanisms (e.g. proliferation-, invasion-, metastasis-, tumor stroma- and angiogenesis-associated) a subset of tumor samples with evidence for a significant correlation between gene(s) expression from sphingolipid-related multigene signature and clinical parameter(s) will be analyzed further by Affymetrix microarray. To estimate functional relevance of the selected candidate genes, in vitro cell-based experiments will be performed to cover a range of cellular responses. Disease-associated action of the sphingolipid-modifying enzymes will be aligned with the cellularissue levels of bioactive sphingolipid mediators (Sphingolipidomics). Conceptualization of obtained data might be used not only for basic research, but also presumably for therapeutic target proposal and building of prognostic and/or predictive sphingolipid-related, tumor-associated multigene expression signatures.

The perception that not only proteins but also lipids play critical roles in many aspects of cell regulation uncovers fundamentally novel mechanisms which can be targeted upon dysregulation in various diseases. Among those are sphingolipids. The scientific project P 23228-B13 conducted by Assoc.-Prof. Dr. Diana Mechtcheriakova and supported by the Austrian Science Fund (FWF) is pioneering in this field in respect of an innovative methodology and integrative approach to explore the role of the complex sphingolipid system in the pathobiology of cancer. The main finding is that the sphingolipid-associated events are among the pathological mechanisms underlying the epithelial to mesenchymal transition (EMT) program associated with metastasis in non-small cell lung cancer. This novel finding, that has significant importance for cancer research and may yield novel therapeutic strategies, was achieved in collaboration with national and international top experts from diverse disciplines. Furthermore, the project yielded an innovative analysis algorithm, named by the research team as MuSiCO/from Multigene Signature to Patient-Orientated Clinical Outcome. MuSiCO was developed with the intention of bridging basic and translational research for the benefit of the patient with a special focus given to lung adenocarcinoma, serous ovarian cancer, and brain malignancies. It consolidates patient-specific gene expression data, based on gene signatures specifically developed by the research team, with previously accumulated knowledge of omics research and state-of-the-art statistical modeling for survival prediction. Understanding the necessity for implementation of more integrative, systems biology-based approaches for analysis was decisive for the success of the project. This in turn was triggered by understanding the complexity of the sphingolipid machinery. Sphingolipids belong to a complex class of natural lipid molecules synthesized by a cell and released to plasma. Food is an additional natural source of basic sphingolipids, which can be taken up and undergo further modifications by specific cellular enzymes resulting in sphingolipid metabolites that possess new properties. On the one hand, sphingolipids maintain their basic functions as components of the cellular membrane; on the other hand, ground-breaking discoveries of the last decades have identified sphingolipid mediators among the active players that regulate a variety of vital cellular and biological processes including cell growth, survival, differentiation, and migration. By way of example, the balance between sphingosine 1-phosphate, or S1P, and ceramide, known as sphingolipid rheostat, is considered as one of the critical factors for a cell to make a decision to survive/proliferate or to go to apoptosis and die. Under physiological conditions, the proper function of diverse sphingolipid molecules is guaranteed by a tightly coordinated way of action of their synthesizing, degrading and modifying enzymes, as well as of their specific cell surface receptors and transporters altogether building up the entire sphingolipid machinery. Under pathophysiological, disease circumstances, such complexity may render multiple breakpoints. The herein newly developed algorithm is able to map those breakpoints on the gene and lipid levels. The first part of the study has been published in 2016 in Oncotarget, one of the top and most prestigious scientific journals in the field of Oncology. Further part of the study has been published in 2014 in PLoS ONE, the top journal in the category Interdisciplinary Sciences. Additional publication(s) are in preparation.