Functional Characteristics of Tumor-Associated Protein Spot14 and Interacting Proteins in Mouse Mammary Epithelial and Breast Cancer Cell Lines

Abstract

Abstract : Thyroid Hormone Responsive Protein Spot14 (S14) is known to be necessary for high rate de novo fatty acid synthesis in tissues that synthesize lipid; however, S14 is also correlated with poor prognosis of breast cancer. The molecular mechanism of S14 remains illusive, but two paradigms exist: one that implicates S14 in transcriptional events and the other in metabolic processes. In vivo, S14 null mammary epithelial cells (MECs) at lactation day 4 showed changes in gene expression for various glycolysis, pentose phosphate shunt, and de novo fatty acid synthesis mRNAs; but unexpectedly, loss of Spot14 increased these mRNAs thereby contradicting previous reports from the literature. Despite literature evidence for direct involvement in regulating gene expression, S14 over-expression studies showed only minor mRNA changes in normal mouse CiT3 cells. S14 may not directly influence mammary epithelial gene expression. In contrast, Nile Red and Bodipy staining of cytoplasmic lipid droplets showed a S14 dependent increase, and metabonomic profiling in short term S14 overexpression revealed significantly increased CH2 acyl lipids. Despite this shift toward lipogenic metabolism, no changes in the abundance of glycolytic or lipogenic enzymes were observed, suggesting that S14 may operate with enzymes. Mass Spectrometry identified a handful of glycolytic enzymes and motor proteins that putatively associate with S14. In CiT3 mammary epithelial cells, S14 seems to function at the protein level along the glycolysis pathway to somehow tip metabolism towards fatty acid synthesis.