Global metabolic profile identifies choline kinase alpha as a key regulator of glutathione-dependent antioxidant cell defense in ovarian carcinoma.

Epithelial Ovarian Cancer (EOC) "cholinic phenotype", characterized by increased intracellular phosphocholine content sustained by over-expression/activity of choline kinase-alpha (ChoKα/CHKA), is a metabolic cellular reprogramming involved in chemoresistance with still unknown mechanisms.By stable CHKA silencing and global metabolic profiling here we demonstrate that CHKA knockdown hampers growth capability of EOC cell lines both in vitro and in xenotransplant in vivo models. It also affected antioxidant cellular defenses, decreasing glutathione and cysteine content while increasing intracellular levels of reactive oxygen species, overall sensitizing EOC cells to current chemotherapeutic regimens. Natural recovering of ChoKα expression after its transient silencing rescued the wild-type phenotype, restoring intracellular glutathione content and drug resistance. Rescue and phenocopy of siCHKA-related effects were also obtained by artificial modulation of glutathione levels. The direct relationship among CHKA expression, glutathione intracellular content and drug sensitivity was overall demonstrated in six different EOC cell lines but notably, siCHKA did not affect growth capability, glutathione metabolism and/or drug sensitivity of non-tumoral immortalized ovarian cells. The "cholinic phenotype", by recapitulating EOC addiction to glutathione content for the maintenance of the antioxidant defense, can be therefore considered a unique feature of cancer cells and a suitable target to improve chemotherapeutics efficacy.

pool targeting CHKA (NM_001277), together with their relative negative controls siRNA (QIAGEN), applying the same transfection protocols and siRNA concentration.

Quantitative Real Time PCR (qRT-PCR)
Total RNA was extracted from EOC cell lines using the RNA Spin Mini Isolation Kit (GE Healthcare Europe GmbH) following the manufacturer's protocol. cDNA was generated from 2μg of RNA using High-Capacity cDNA Archive Kit (Applied Biosystems) and qRT-PCR was carried out in triplicate using the 7900 HT FAST Real-Time PCR System (Applied Biosystems). Probes used for amplification were from Applied Biosystems (CHKA HS00608045_m1; xCT/SLC7A11 HS00204928_m1 and GAPDH HS03929097_g1). The ΔΔCT method was used to determine the quantity of the target sequences. In all qRT-PCR experiments GAPDH was amplified as endogenous control and I64-hTERT cells were used as calibrator. Analyses were performed using SDS software 2.2.2 (Applied Biosystems).

Drug treatment assays
For cytotoxicity assays, stably silenced or transient silenced cells 72 hours after transfection, were seeded at 7.5x10 4 cells/well in 96-well flat-bottom plates and exposed for 7h to cisplatin (DDP) (TEVA Italia s.r.l) at serial dilution from 1x10 -4 to 1x10 -7 M, or doxorubicin (doxorubicin hydrochloride Pfizer, Italia s.r.l) at serial dilution from 1x10 -4 to 1x10 -7 M. Effects of drugs treatment were assessed 48h later using a CellTiter-GLO luminescent cell viability assay performed according to the manufacturer's instructions (Promega, Madison, WI). Luminescence was measured using an Ultra multiplate reader (Tecan Group, Mannedorf/Zurich, Switzerland).

Cell migration assays
Migration assays were performed using 24-Well Fluorimetric Cell Migration Assay (Merck Millipore, MA, USA). Cells were seeded in the upper chamber at 5×10 5 cells/ml in 0.3 ml serumfree culture media. Media supplemented with 10% FCS was placed as a chemoattractant in the bottom well in a volume of 0.5 ml. After incubation for 24 h at 37°C in an atmosphere containing 5% CO2, assay was performed according to the manufacturer's instructions (Merck Millipore, MA, USA). Migration was measured using a microplate reader (Tecan Group, Mannedorf/Zurich, Switzerland) with excitation/emission wavelengths of 485/535nm.

Cell invasion assays
Invasion capability was assayed using Fluoro Blok system in a 24-well companion plate (from Becton Dickinson) coated with Matrigel. 5×10 4 cells/well were seeded onto the apical surface of the inserts in 0.75 ml of serum-free colture media. The basal chamber of the wells was filled with 650 μL of media supplemented with FCS as chemoattractant. Cells were incubated in the Fluoro-Blok multiwell insert system for 24 hours at 37°C in a humidified atmosphere of 5% CO 2 and labeled with 4g/ml calcein AM (Molecular Probes, Life Technologies) in Hanks' balanced salt solution (Life Technologies) for 1 hour at 37°C and 5% CO 2 . Fluorescence was measured using a microplate reader (Tecan Group, Mannedorf/Zurich, Switzerland) with excitation/emission wavelengths of 485/535nm.

Metabolomic profiling
Metabolomic profiling analysis was performed by Metabolon as previously described (17) analyzing 8 replicates of parental, ΔLuc-sh-RNA and sh-CHKA transduced INTOV11 and SKOV3 cells collected 24 and 72 hours post seeding. Extracts from all samples were split into equal parts for analysis on the gas chromatography/mass spectrometry (GC/MS) and Liquid chromatography/mass spectrometry (LC/MS/MS) platforms. Sample Accessioning. Each sample received was accessioned into the Metabolon LIMS system and was assigned by the LIMS a unique identifier that was associated with the original source identifier only. This identifier was used to track all sample handling, tasks, results etc. The samples (and all derived aliquots) were tracked by the LIMS system. All portions of any sample were automatically assigned their own unique identifiers by the LIMS when a new task is created; the relationship of these samples is also tracked.
All samples were maintained at -80 ºC until processed. Sample Preparation. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. A recovery standard was added prior to the first step in the extraction process for QC purposes. Sample preparation was conducted using aqueous methanol extraction process to remove the protein fraction while allowing maximum recovery of small molecules. The resulting extract was divided The sample extract was dried then reconstituted in acidic or basic LC-compatible solvents, each of which contained 8 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion optimized conditions and the other using basic negative ion optimized conditions in two independent injections using separate dedicated columns. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5mM Ammonium Bicarbonate. The MS analysis alternated between MS and data-dependent MS 2 scans using dynamic exclusion. Raw data files are archived and extracted as described below. Gas chromatography/Mass Spectroscopy (GC/MS). The samples destined for GC/MS analysis were re-dried under vacuum desiccation for a minimum of 24 hours prior to being derivatized under dried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). The GC column was 5% phenyl and the temperature ramp was from 40° to 300° C in a 16 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning singlequadrupole mass spectrometer using electron impact ionization. The instrument was tuned and calibrated for mass resolution and mass accuracy on a daily basis. The information output from the raw data files was automatically extracted as discussed below. biochemicals which were detected in all samples from one or more groups but not in samples from other groups were assumed to be near the lower limit of detection in the groups in which they were