Glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) expression correlates with malignant choline phospholipid metabolite profiles in human breast cancer

Abstract

Altered choline phospholipid metabolism is a hallmark of cancer, leading to malignant choline metabolite profiles consisting of low glycerophosphocholine (GPC) and high phosphocholine (PC) in human breast cancers. Glycerophosphocholine phosphodiesterase (GPC-PDE) catalyzes the degradation of GPC to free choline and glycerol-3-phosphate. The gene(s) encoding for the GPC-PDE(s) responsible for GPC degradation in breast cancers have not yet been identified. Here, we demonstrate for the first time that the GPC-PDE encoded by glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) is associated with breast cancer malignancy. Two human breast cancer cell lines (n = 8 and n = 10) and primary human breast tumor samples (n = 19) were studied with combined MRS and quantitative reverse transcription-polymerase chain reaction to investigate several isoforms of GDPD expression with respect to choline phospholipid metabolite levels. Of the five GDPDs tested, GDPD5 was found to be significantly overexpressed in highly malignant estrogen receptor negative (ER(-)) compared with weakly malignant estrogen receptor positive (ER(+)) human breast cancer cells (p = 0.027) and breast tumors from patients (p = 0.015). GDPD5 showed significantly positive correlations with PC (p < 0.001), total choline (tCho) (p = 0.007) and PC/GPC (p < 0.001) levels in human breast tumors. GDPD5 showed a trend towards a negative correlation with GPC levels (p = 0.130). Human breast cancers with malignant choline metabolite profiles consisting of low GPC and high PC levels highly co-expressed GDPD5, choline kinase alpha (CHKA) and phosphatidylcholine-specific phospholipase D1 (PLD1), whereas cancers containing high GPC and relatively low PC levels displayed low co-expression of GDPD5, CHKA and PLD1. GDPD5, CHKA and PLD1 were significantly overexpressed in highly malignant ER(-) tumors in our patient cohort. Our study identified GDPD5 as a GPC-PDE that probably participates in the regulation of choline phospholipid metabolism in breast cancer, which possibly occurs in cooperation with CHKA and PLD1.

Figure 1

A, representative ¹H MR spectra of the choline region from hydrophilic extracts of MCF-12A, MCF-7, and MDA-MB-231 cells. B, metabolite concentrations in [mM] quantified from ¹H MR spectra of MCF-12A (black bars), MCF-7 (gray bars), and MDA-MB-231 (white bars) cells. C, relative fold change in gene expression of GDPD1-5 and CHKA obtained from MCF-7 and MDA-MB-231 cells normalized to MCF-12A cells. D, representative GDPD5 immunoblots and relative fold change in GDPD5 protein levels normalized to MCF-12A cells. β-actin was used as loading control. Values are mean±standard error (SE). *P < 0.05, **P < 0.01.

Figure 2

A, Correlation of GDPD5 mRNA expression as a function of R = 2 ^Δ(Δct) and protein levels expressed as relative fold change normalized to MCF-12A versus relative choline phospholipid metabolite concentrations in human breast cancer cells normalized to MCF-12A. B, Correlation of GDPD5 versus CHKA mRNA and protein levels expressed as relative fold change normalized to MCF-12A. Values are mean±standard error (SE). The gene, protein, and metabolite levels were normalized to MCF12-A, and therefore the symbols for gene and protein expression data of MCF-12A superimpose.

Figure 3

A, representative ex vivo HR MAS spectra of the choline region from ER⁺/low PC/GPC and ER⁻/high PC/GPC tumors. B, metabolite concentrations [μmol/g] quantified from HR MAS spectra of ER⁺/low PC/GPC (gray bars) and ER⁻/high PC/GPC tumors (white bars). C, relative fold change in gene expression of GDPD1, 2, 3, and 5, CHKA and PLD1 obtained from ER⁻/high PC/GPC tumors and ER⁺/low PC/GPC tumors normalized to the sample with the lowest GDPD5 expression. Values are mean±standard error (SE). *P < 0.05, **P < 0.01.

Figure 4

A, correlation of GDPD5 expression versus choline phospholipid metabolite concentrations in human breast tumors. B, correlation of GDPD5 versus CHKA and GDPD5 versus PLD1 expression in human breast tumors.

Figure 5

A, high co-expression of GDPD5, CHKA, and PLD1 was associated with choline metabolite profiles consisting of low GPC and high PC levels in human ER⁻/high PC/GPC breast tumors, as determined by HR MAS MRS. B, low co-expression of GDPD5, CHKA, and PLD1 was accompanied by high GPC and relatively low PC levels in human ER⁺/low PC/GPC breast tumors. The Cho level remained the same in both tumor groups. GPC-PDE, glycerophosphocholine phosphodiesterase; PLC, phosphatidylcholine-specific phospholipase C; CTP, phosphocholine cytidylyltransferase; CDP, diacylglycerol cholinephosphotransferase; PLA2, phosphoplipase A2; Lyso-PLA1, lysophospholipase A1.