Abstract
Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system (“AFFL”). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.
Highlights
S 1.1 Oxysterols and breast cancer U Breast cancer (BCa) is the most commonly diagnosed cancer among woman [1] and elevated LDL-cholesterol is a predictor of poor outcomes [2]
D OHC activates the liver X receptor (LXR) leading to enhanced expression of transcription factors E that promote the epithelial to mesenchymal transition [9, 11], and modifies immune γδ-T cells and T polymorphonuclear-neutrophil function to promote Estrogen Receptor (ER)-negative BCa metastasis [12]
Little is known regarding the levels of AC oxysterols in ER-positive and ER-negative BCa tissue
Summary
S 1.1 Oxysterols and breast cancer U Breast cancer (BCa) is the most commonly diagnosed cancer among woman [1] and elevated LDL-cholesterol is a predictor of poor outcomes [2]. Oxysterols can A be formed by either enzymatic or non-enzymatic oxidation of cholesterol. The M enzymatically formed 27-hydroxycholesterol (27-OHC) is of particular interest; In BCa, 27-OHC induces cell proliferation and tumour growth via its ability to bind and activate the Estrogen Receptor (ER) [9, 10] being at least part of the molecular evidence that links elevated cholesterol with BCa [9]. A related P sidechain oxysterol, 25-OHC, has been found elevated in the circulation of BCa patients under E going treatment for metastatic disease [13]. The other enzymatically formed sidechain oxysterols (scOHC) can play roles, as e.g. LXR activators. Little is known regarding the levels of AC oxysterols in ER-positive and ER-negative BCa tissue. Methods for studying oxysterols in breast cancer tissues are needed
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