HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

Abstract

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using 13C MRS to monitor [1,2-13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by 31P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using 1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.

Choline kinase alpha—Putting the ChoK-hold on tumor metabolism

“…Subsequent gene expression profiling in 17AAG-treated breast cancer cells, however, found no change in ChoK expression and attributed the HSP-90-inhibitor effects to choline transporter and phospholipase induction [76]. HDAC inhibitors Vorinostat, LAQ-824, and belinostat provide another example running contrary to this conventional wisdom, in that they induce differentiation and apoptosis in cancer cells but cause a rise in PC and ChoK levels [77–80]. …”

Choline metabolism-based molecular diagnosis of cancer: an update

“…In addition to Chk-α, PC-PLC, PLD1 and PLD2, an elevation of choline uptake by choline transporters, followed by phosphorylation by Chk-α can also increase endogenous PC [57]. Increased expression of the high-affinity choline transporter CHT1 with a Km of ∼2 μM, also called solute carrier family 5 member 7 (SLC5A7) [58], has been observed in breast cancer cells [15].…”

Metabolic Imaging of Pancreatic Ductal Adenocarcinoma Detects Altered Choline Metabolism

“…Chk catalyzes the phosphorylation of Cho using ATP, as a phosphate donor, to produce PC. An elevation of choline uptake by choline transporters, followed by phosphorylation by choline kinase can also increase endogenous PC (11). Increased expression of the high-affinity choline transporter CHT1 with a K m of ~ 2 µM, also called solute carrier family 5 member 7 (SLC5A7) (12), has been previously observed in breast cancer cells (10).…”