Brendan Coutu and Dr. John Prince, Department of Chemistry and Biochemistry
Introduction
Cancer not only affects individuals and families but has the greatest economic effect worldwide than any other premature cause of death.1 Diverse aspects of cancer biology are under investigation to better understand the mechanism of action of different cancer types, that biomarkers can be identified for early diagnosis, and, most importantly, that a lasting cure can be discovered.32 In the development of cancer, cells undergo a number of phenotypic changes, known as the hallmarks of cancer.2 The hallmarks of cancer are described as sustained proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis, and resisting cell death. Each of these hallmarks are caused by genomic mutations that hijack the normal function of a cell. Although the proteins involved in each of these processes have been established, their action are modulated by a relatively unknown lipidome.
Kidney cancer is one of many types of cancer which has a high occurrence of metastasizing.3 To better understand the effect that this cellular state has on the lipidome, a model of kidney cancer was investigated. Upon treatment of MDCK cells with hepatocyte growth factor (HGF) the oncogenic pathway, c-Myc, is constitutively turned on.4 This effectively models kidney cancer as a high concentration of HGF induces uncontrollable cell proliferation.
A series of lipidomic analyses were applied to this cancer model to better understand the effect cancer has on the cellular lipidome. Through a time dependent study of the effect of HGF on lipid species, it can be ascertained that lipids play a vital role in the progression of kidney cancer.
Materials/Methods
Eighteen plates of MDCK cells were grown to confluency. These plates were divided into six different samples. The first set was not induced with HGF (acting as a control). After the remaining samples were induced with 1 ml of 12 mM HGF they were harvested respectively after 1.5, 3, 6, 12, and 24 hours. Upon harvesting, 0.1 ml of 1 mM coenzyme Q10 was added which acted as an internal standard for mass and intensity recalibration. Cells were lysed via triteration to ensure that no lipid oxidation occurred which can be associated with sonication. Lipids were extracted from the cells via a Bligh and Dyer lipid extraction.5 An intrasource separation was applied to the samples via LiOH.6
After samples were randomized, each plate of each sample was analyzed three times in an LTQ Orbitrap using an ESI head. Default parameters were used except a windows method of MS/MS data acquisition was utilized and the samples were analyzed twice in negative ion mode (without and then with LiOH) and then in positive ion mode (with a two minute period of spectral averaging upon polarity switching allowed).
Through Lipidomic Search the m/z peaks were identified as lipids. The significance of a lipid was determined based on a number of parameters including the coefficient of variance of the relative abundance (intensity) of a lipid between replicate runs and the increase or decrease in relative abundance of a lipid species across time points.
The lipid identifications of the top sixty lipid species were confirmed via MS/MS fragmentation in a subsequent analysis in which MDCK cells were treated with HGF for six hours. All sample preparation was as previously described.
Results
Through the lipidomic analysis of kidney cancer a number of significant lipids were identified that showed consistent expression across replicate sample analysis and increased or decreased expression after induction of HGF. The upregulation and downregulation of certain lipid groups could be correlated linearly with the amount of time a sample was treated with HGF. The effect of HGF on these lipids has not previously been observed (see Figure 1).
Discussion
This study gives further insights into the roles that lipids play in the progression of cancer. An increase in lipids made up of saturated fatty acids reveals an increase in de novo biosynthesis of lipids. Enzymes such as fatty acid synthase (FASN) are known to be up regulated in cancer cells. FASN synthesizes saturated fatty acids from Acetyl-CoA. Mammalian cells do not have the enzymes to synthesize poly-unsaturated fatty acids, instead these lipids are supplied by the mammal’s diet. An increase in saturated fatty acids can explain the increased rigidity in cellular membranes of cancer cells. The membranes of cancer cells are known to be less permissive to organic chemotherapy drugs than expected. As saturated lipids are packed tightly together they become less permeable to dissimilar molecules. As the roles of lipids become better understood in cancer key players in lipid biosynthesis pathways may become well-established targets for cancer therapeutics.
References
- McCarthy, N. Prostate cancer: Studying the classics.
- Hanahan, D. H. & Weinberg, Robert A. The Hallmarks of Cancer.
- Li, Y. et al. Functional and molecular interactions between the HGF/c-Met pathway and c-Myc in large-cell medulloblastoma
- Hilvo, M. et al. Novel theranostic opportunities offered by characterization of altered membrane lipid metabolism in breast cancer progression.
- Bligh, E. G., Dyer, W.J. A Rapid Method of Total Lipid Extraction and Purification.
- “Han, X. & Gross, R. W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.