Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase α1 (ChoKα1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoKα1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.

. Time-course immunoblot analysis of cell cycle inhibitors in MCF-7 cells. MCF-7 cells were treated with 1 µM of EB-3D up to 24 h.  Figure 2A. Each band has been normalized to β-actin and represented as a fold change with respect to the untreated control (t0). Data are presented as mean ± SEM of at least three independent experiments. Differences between treatment and its control were analyzed using one-way ANOVA with Bonferroni correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure S5. Additional analysis of senescence-like phenotype in normal and cancer cell lines. (A) βgalactosidase (X-gal)-staining in MCF-7 breast cancer cells. MCF-7 were seeded onto a chamber slide, treated with 1 µM of EB-3D or 2.5 µM of Compound C (AMPK inhibitor) for 72 h or alternatively pretreated with Compound C for 2 h, and then exposed to EB-3D for 72 h. After treatment cells, were stained according to the manufacturer's protocol. Original magnification 10×. (B) GPCho/PCho molar ratio quantified from 1 H-NMR spectra of water-soluble extracts from MDAMB-231 cells treated with DMSO or EB-3D 1 µM for the indicated time points, retrieved from Figure 1A. Metabolite levels are normalized to each time point control. Differences between treatment and its control were analyzed using one-way ANOVA with Bonferroni correction. **** p < 0.0001. (C) Representative 1 H-NMR spectra from the 3.20-3.25 ppm region of water-soluble extracts from MDA-MB-231 cells after 48 h of treatment with DMSO (red line) and 1 µM of EB-3D (blue line). (D) Flow cytometry analysis of cellular senescence using C12-FDG probe in normal mammary MCF-10A cell line and in normal human fibroblast treated with 1 µM of EB-3D for 72 h. Figure S6. Flow cytometry analysis of cell death. MDA-MB-231 cells were treated with 1 µM of EB-3D or an anticancer drug cocktail (composed by Cis-Pt 20µM, 5-FU 1µM, Doxo 0.5µM) or with the simultaneous addition of EB-3D and the drug cocktail for 72 h. Bars represent the mean ± SEM of three independent experiments. Statistical significance was determined using ANOVA with Newman-Keuls correction. Asterisks indicate a significant difference between treated and control (DMSO). * p < 0.05, *** p < 0.001. Figure S7. ChoKα protein sequence alignment generated by EMBOSS Needle Global alignment tools between human and mouse protein sequences (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Residues highlighted in yellow (Tyr333, Tyr345, Phe361, Trp420, Trp423, Ile433, Phe435, Tyr440) shown to be important for interaction with EB-3D or its stabilization are perfectly conserved. Also, Asp306 catalytic base for ATP hydrolysis is conserved. Vertical bar (|) indicates fully conserved residue. Colon (:) indicates conservation between groups of strongly similar properties. Period (.) indicates conservation between groups of weakly similar properties.

Cell Viability Assay and Drug Combination Sensitivity Assay
Individual wells of 96-well tissue-culture microtiter plates were inoculated with 100 µL of complete medium containing MDA-MB-231 or MDA-MB-468 (3.5 × 10 3 cells) or MCF-7 (5 × 10 3 cells). The plates were incubated at 37 °C in a humidified 5% CO2 incubator for 18 h prior to the experiments to ensure exponential growth. After medium removal, 100 µL of fresh medium containing serial dilutions of single drugs or drug combinations (at a fixed molar ratio) was added to each well and incubated at 37 °C for 48/72 h. The percentage of DMSO in the medium in no case exceeded 0.5%. Cell viability was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) test. Briefly, MTT was added to each well at a final concentration of 0.5 mg/mL and incubated for 3 h at 37 °C. The quantity of formazan (presumably directly proportional to the number of viable cells) was measured by recording changes. Viable cells with active metabolism converted MTT into a purple-colored formazan product, which was next solubilized with acidified isopropanol to measure the changes in absorbance at 570 nm using a plate reading spectrophotometer (Victor, Perkin Elmer). The GI50 was defined as the compound concentration required to inhibit cell proliferation by 50% in comparison with cells treated with the maximum amount of DMSO (since EB-3D is solubilized in DMSO) and considered as 100% viability. Each drug concentration/combination was performed in triplicate. To determine the synergistic, additive, or antagonistic effects of the drug combinations, CalcuSyn software (version 2.0, Biosoft) based on the method of the combination index (CI) described by Chou was used (22), where synergism is defined as CI < 1, additivity as CI = 1, and antagonism as CI > 1.
In additional experiments, the accurate determination of the cell proliferation rate was determined by trypan blue exclusion assay. 7.5×10 4 cells were seeded in six-well plates and incubated at 37 °C in a humidified 5% CO2 incubator for 18 h prior to the experiments to ensure exponential growth. Cells were then treated with EB-3D or DMSO (time 0) and then collected by trypsinization after 72 h. Alternatively, after 72 h of EB-3D treatment, cells were washed extensively and then treated with EB-3D (EB-3D continuous) or DMSO (EB-3D WASHOUT) for a further 72 h. Collected cells were resuspended in 0.4% trypan blue solution (Thermo Fisher Scientific) and counted on a hemocytometer. Only trypan blue negative cells were considered viable cells.

Magnetic Resonance Spectroscopy ( 1 H-MRS)
MDA-MB-321 breast cancer cells were seeded and cultured for 24 h in complete growth medium and then treated with EB-3D or DMSO for the indicated time points. Water-soluble extracts were obtained using the dual-phase extraction method. Briefly, cells were resuspended with ice-cold methanol and vigorously vortexed. Samples were incubated on ice for 15 min, mixed with chloroform (1:1), vortexed vigorously, and kept on ice for 10 min. Finally, water was added and shaken well. Samples were stored at 4 °C overnight for phase separation and later centrifuged at 15,000× g at 4 °C for 30 min. The upper water/methanol phase containing water-soluble cellular metabolites (Cho, PCho, and GPCho) was treated with 50 mg of chelex beads (Sigma-Aldrich, Milan, Italy) and then removed by filtration. Following methanol evaporation, the remaining water phase was lyophilized. Water-soluble extracts were resuspended in deuterated water containing 7.006 × 10 −8 moles of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, Sigma-Aldrich) as an internal standard for NMR spectral analysis.
NMR spectra were analyzed using Bruker Topspin software (Bruker Biospin). Signal integrals of N(CH3)3 of Cho at about 3.202 ppm, PCho at about 3.224 ppm, and GPCho at about 3.232 ppm in water-soluble extracts were determined, normalized to cell number and cell volume, and compared with the standards. To determine concentrations of water-soluble extracts, peak integrations (Imet) from 1 H spectra for PCho, GPC, and Cho were compared with that of the internal standard TSP (ITSP).