Altered Lipid Raft Composition Correlates with Oncogenic Potential

Abstract

IntroductionMutant gain‐of‐function (GOF) p53 acts as an epigenetic regulator to promote oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that cholesterol accumulation alters lipid raft signaling, contributing to oncogenesis. To address the relationship between cholesterol and oncogenic properties, we measured cholesterol levels in a panel of cell lines and correlate these levels with both GOF p53 expression and oncogenic properties. To directly tie lipid raft composition to oncogenic properties, we utilized the SW13 cell line with two epigenetically distinct cell subtypes with differing oncogenic properties. The SW13‐ subtype has an epithelial morphology and is highly proliferative, while the SW13+ subtype has a mesenchymal‐like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13‐ cells appear to adopt a SW13+ phenotype. Therefore, we treated with both HDACi to control the subtype conversion and shRNA to knock‐down GOF p53 expression. This experimental approach allows the composition of raft fractions to be correlated to the oncogenic properties of each SW13 subtype, with and without GOF p53 expression.MethodsProliferation rate and MMP expression were quantitated by EdU assays and in situ zymography respectively. Lipid raft fractions were isolated using the detergent‐resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression, and analyzed by mass spectrometry. Cholesterol assays were used to quantify cholesterol levels after treatment, and western blots were performed to assess levels of p53 and raft markers.ResultsCell lines expressing GOF p53 have 2 ‐ 4 fold increase in normalized levels of cholesterol when compared to a p53‐null cell line (p=0.02). Within the SW13 cell line subtypes, each subtype appears to have different oncogenic properties. First, p53 knock‐down alters cell behavior by decreasing MMP activity in SW13+ (p = 0.05) but not SW13‐ cells. Second, an increased rate of proliferation was measured for SW13‐ (p = 0.01) but not for SW13+ cells. We observed differential expression of genes coding for lipid raft components including GPI anchors, cholesterol biosynthesis, and sphingolipids; additionally, multiple raft‐related proteins are also differentially expressed. Suggestive of a functional difference in rafts, our data suggest a shift from planar rafts in the SW13‐ to caveolar rafts in the SW13+ line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered when GOF p53 is knocked down.ConclusionsOur data support the hypothesis that cholesterol levels and lipid raft signaling are significantly altered in cell lines expressing GOF p53 mutations. These changes correlate with distinct oncogenic properties and may correlate to oncogenic potential. Ongoing studies are aimed at delineating the link between GOF mutant p53 and raft function.