Abstract 451: HIF-1 alpha silencing in MDA-MB-231 human breast cancer cells alters choline phospholipid metabolism

Abstract

Abstract Hypoxia-inducible factor-1 (HIF-1) over-expression has been associated with an increased patient mortality rate in many cancer types including breast cancer. Suppression of HIF-1 gene expression has been shown to inhibit tumor growth. Here we have studied the effect of HIF-1 silencing on the metabolism of MDA-MB-231 cells using a magnetic resonance (MR)- compatible cell perfusion assay. Previous studies have identified choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), and the resulting increase of PC and total choline (tCho) as markers of an aggressive phenotype. We found that HIF-1 silencing resulted in significantly reduced Chk expression together with reduced tCho and PC, compared to parental cells. The sequence for shRNA against HIF-1α was cloned into a lentivirus vector with a green fluorescent protein (GFP) reporter construct (pRRL-pGK-GFP). Viral supernatant preparation and transduction of MDA-MB-231 breast cancer cells was performed following standard protocol. Transduced cells were validated for HIF-1α knock-down by western blots and by quantitative real-time polymerase chain reaction (q RT-PCR). Cell perfusion studies were performed using an MR-compatible perfusion assay to determine intracellular levels of metabolites using 1H and 31P MR spectroscopy (MRS). Experiments were performed in triplicates. The Mann Whitney-U test was used to determine statistical significance (p <0.05). HIF-1α protein expression increased following treatment with 200μM of the hypoxia mimetic CoCl2 in parental MDA-MB-231 cells but not in cells transduced with HIF-1α shRNA. Reduced expression of Chk in HIF-1α silenced cells was observed following CoCl2 treatment, while its expression was induced in parental cells. Quantitative data from 1H and 31P MRS showed significantly reduced levels of tCho (p <0.05) and PC (p < 0.01) in HIF-1α silenced cells compared to parental MDA-MB-231 cells, confirming that silencing of HIF-1α reduced levels of choline containing metabolites by reducing Chk expression. We previously observed that Chk is up regulated under hypoxia and have established a HIF-1 binding site on the Chk promoter. The reduction of choline metabolites in HIF-1α silenced cells further confirms the role of HIF-1α in the regulation of Chk. The reduction of Chk, total choline and PC levels in HIF-1α silenced cells are also typical of a less aggressive metabolic phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 451.