Abstract 652: Magnetic resonance spectroscopy detects silencing of the novel anticancer target GDPD5 in human breast cancer cells
Authors: Mailin Döpkens, Tiffany R. Blackwell, Farhad Vesuna, Venu Raman, Balaji Krishnamachary, Zaver M. Bhujwalla, Dieter Leibfritz, Kristine Glunde
Journal: Cancer Research (2010)

Abstract Altered choline phospholipid metabolism in breast cancers provides multiple targets for anticancer therapy. Malignant transformation of breast cancer cells results in a switch from high glycerophosphocholine (GPC) and low phosphocholine (PC) to low GPC and high PC. Glycerophosphocholine phosphodiesterase (E.C. 3.1.4.2; GPC-PDE) catalyzes the degradation of GPC to Cho and glycerol-3-phosphate. The GPC-PDE gene(s) responsible for the low GPC concentration in breast cancer cells have not yet been identified. Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is a GPC-PDE that is rapidly inhibited by NaCl and urea (NaCl/urea) in renal cells, and may be a candidate gene for GPC-PDE in breast cancer cells. We chemically inhibited GPC-PDE with NaCl/urea in nonmalignant MCF-12A, and malignant MCF-7 and MDA-MB-231 breast epithelial cell lines. We stably downregulated GDPD5 using short hairpin RNA against GDPD5 (GDPD5-shRNA) delivered by lentiviral transduction in MCF-7 breast cancer cells. Fully relaxed high-resolution 1H magnetic resonance spectroscopy (MRS) of cell extracts was performed on Bruker Avance 500 MR Spectrometer to quantify metabolites. Cell viability/proliferation was measured by WST-1 proliferation assay. Quantitative RT-PCR (qRT-PCR) detected significantly higher GDPD5 mRNA levels compared to the mRNA levels of GDPD1, 2, 3, and 4 in the respective cell line for MCF-12A, MCF-7, and MDA-MB-231 cells. GDPD5 levels were significantly higher in MDA-MB-231 compared to MCF-7 and MCF-12A cells. MRS metabolite quantification demonstrated that exposure of MCF-12A, MCF-7, and MDA-MB-231 cells to NaCl/urea (n=3), as well as transduction with GDPD5-shRNA in MCF-7 cells (n=2), significantly increased GPC and decreased PC, resulting in a decreased [PC]/[GPC] ratio. An increased [PC]/[GPC] ratio is associated with increased malignancy in breast cancer cell lines. We observed a switch from low GPC and high PC to high GPC and low PC following NaCl/urea treatment and following GDPD5-shRNA transduction. GDPD5 inhibition by NaCl/urea significantly decreased cell proliferation/viability in MCF-12A, MCF-7, and MDA-MB-231 cells. Inhibiting or down-regulating GDPD5 altered the choline phospholipid metabolite profile of breast cancer cells toward a less malignant metabolic profile. GDPD5 is at least partially responsible for the decreased GPC levels in breast cancer cells, as indicated by high GDPD5 mRNA and low GPC metabolite levels in MDA-MB-231 cells. Decreased proliferation detected upon GDPD5 inhibition with NaCl/urea further corroborated the importance of GPDP5 in breast cancer. These results indicate that GDPD5 may provide a future target for anticancer therapy. MRS could be used to monitor the GPC increase following downregulation of GDPD5 by RNA interference in such future therapies. This work was supported by NIH R01 CA134695 (to K.G.). 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 652.