Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy

Abstract

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20-50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods.

Keywords: breast cancer; choline kinase; choline metabolism; magnetic resonance spectroscopy; ovarian cancer; phosphatidylcholine phospholipase C; targeted therapy.

Figure 1

Detection of the ¹H-MRS total choline (tCho) metabolic profile in epithelial ovarian cancer (EOC) cells and in MRS/MRSI clinical examinations. The phosphatidylcholine cycle and its links with tyrosine kinase receptors’ stimulation, post-receptor signaling pathways, and cancer cell biological features are sketched in the central scheme. (A–C) High resolution 9.4-T ¹H-MRS tCho profile of aqueous extracts of (A) ovarian surface epithelial cells (OSE); (B) SKOV3 cell line and its in vivo-passaged cell variant SKOV3.ip, characterized by higher in vivo tumorigenicity and twofold higher HER2 protein expression level (in the insert, Western blot analysis of HER2, 185 kDa); (C) SKOV3.ip cells exposed for 24 h to the competitive PC-PLC inhibitor D609, compared with control untreated cells. (D) Absolute activity rates (nmol/10⁶ cells × h, mean ± SD) of ChoK and PC-PLC measured in aqueous extracts of different EOC cell lines, compared with those of non-tumoral immortalized hTERT cells (number of independent experiments, n ≥ 3 for all cell lines). (E,F) Examples of in vivo detection of (E) ¹H-MRS tCho peak (3.2 ppm) in a single voxel and (F) tCho map obtained by MRSI in EOC patient examined at 1.5 T. For further details, see Ref. (, , –51). The central scheme was adapted from Figure 1 of Ref. (43). Abbreviations: Enzymes: ChoK, choline kinase (EC 2.7.1.32); CT, cytidylyltransferase (EC 2.7.7.15); LPL, lysophospholipase (EC 3.1.1.5); PCT, phosphocholine transferase (EC 2.7.8.2); PD, glycerophosphocholine phosphodiesterase (EC 3.1.4.2); PLA1, phospholipase A1 (EC 3.1.1.32); PLA2, phospholipase A2 (EC 3.1.1.4); PLC, phosphatidylcholine-specific phospholipase C (EC 3.1.4.3); PLD, phospholipase D (EC 3.1.4.4). Metabolites: AA, arachidonic acid; CDP-Cho, cytidine diphosphate-choline; Cho, free choline; DAG, diacylglycerol; GroP, sn-glycerol-3-phosphate; GPCho, glycerophosphocholine; Ino, myo-inositol; LPA, lysophosphatidate; LPtdCho, lysophosphatidylcholine; PA, phosphatidate; PCho, phosphocholine; PtdCho, phosphatidylcholine; tCho, total choline-containing metabolites (GPCho + PCho + Cho). Transporters: CHT, choline high-affinity transporters; CTL, choline transporter-like proteins; OCT, organic cation transporters.

Figure 2

Subcellular localization of PC-PLC in human BC cell lines. (A) PC-PLC activity rates (nmol/mg protein × h, mean ± SD, n ≥ 3) measured in MCF-7, SKBr3, and MDA-MB-231 BC cell lines, possessing different levels of HER2 and EGFR expression, compared with that of the human mammary epithelial cell line of fibrocystic origin MCF-10. (B) Confocal laser scanning microscopy (CLSM) examinations (3D reconstruction images) of the same cell lines as in (A). Cells were either fixed and permeabilized (upper panels, scale bars 20 μm) or maintained unfixed (bottom panels, scale bars 5 μm) and stained for PC-PLC detection with rabbit anti-PC-PLC antibodies (pseudo-color gray); nuclei were stained with DAPI (blue). (C) Colocalization of PC-PLC and HER2 on plasma membrane of SKBr3 cells. CLSM detection of PC-PLC and HER2 was performed on fixed and permeabilized (upper panels) or unfixed cells (bottom panels, 3D reconstruction images) using rabbit polyclonal anti-PC-PLC antibodies (green) and anti-HER2 W6/100 monoclonal antibody (red). Colocalization areas are represented in yellow. Scale bars, 8 μm. (D) HER2 downmodulation in SKBr3 cells exposed for different time intervals to the competitive PC-PLC inhibitor D609. CLSM analyses of unfixed SKBr3 cells cultured in complete medium in absence (t = 0) or presence of D609 (6 and 24 h). After washing, cells were stained with anti-PC-PLC (green) and anti-HER2 antibodies (red). For further details, see Ref. (28, 34).