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. 2014 May;15(5):593-601.
doi: 10.4161/cbt.28165. Epub 2014 Feb 20.

Phospholipase D1 and Choline Kinase-α Are Interactive Targets in Breast Cancer

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Free PMC article

Phospholipase D1 and Choline Kinase-α Are Interactive Targets in Breast Cancer

Mayur Gadiya et al. Cancer Biol Ther. .
Free PMC article

Abstract

A consistent metabolic hallmark observed in multiple cancers is the increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which is closely related to malignant transformation, invasion, and metastasis. Enzymes in choline phospholipid metabolism present attractive targets to exploit for treatment, but require a clear understanding of the mechanisms underlying the altered choline phospholipid metabolism observed in cancer. Choline kinase-α (Chk-α) is an enzyme in the Kennedy pathway that phosphorylates free choline (Cho) to PC, and its upregulation in several cancers is a major contributor to increased PC levels. Similarly, increased expression and activity of phospholipase D1 (PLD1), which converts phosphatidylcholine (PtdCho) to phosphatidic acid (PA) and Cho, has been well documented in gastric, ovarian and breast cancer. Here we report a strong correlation between expression of Chk-α and PLD1 with breast cancer malignancy. Data from patient samples established an association between estrogen receptor (ER) status and Chk-α and PLD1 expression. In addition, these two enzymes were found to be interactive. Downregulation of Chk-α with siRNA increased PLD1 expression, and downregulation of PLD1 increased Chk-α expression. Simultaneous silencing of PLD1 and Chk-α in MDA-MB-231 cells increased apoptosis as detected by the TUNEL assay. These data provide new insights into choline phospholipid metabolism of breast cancer, and support multiple targeting of enzymes in choline phospholipid metabolism as a strategy for treatment.

Keywords: RNA interference; breast cancer; choline kinase-alpha; magnetic resonance spectroscopy; phospholipase D1.

Figures

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Figure 1. (A) Relative fold change in PLD1 mRNA and Chk-α mRNA in patient-derived tumor samples that are either ER+ (n = 11) or ER (n = 8). (B) Immunoblots representing the protein expression of Chk-α in nonmalignant MCF-12A cells, non-metastatic ER+ MCF-7 cells and highly metastatic ER MDA-MB-231 cells. (C) Immunoblots representing the protein expression of PLD1 in MCF-12A, MCF-7, and MDA-MB-231 cells. GAPDH was used as a loading control.
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Figure 2. Relative fold change of Chk-α and PLD1 mRNA in MDA-MB-231 cells (n = 7 per treatment group): untreated (1), treated with 25 nM PLD1-siRNA (4), treated with 100 nM PLD1-siRNA (5), treated with 75 nM Chk-α-siRNA (6), treated with 100 nM Chk-α-siRNA (7), and treated with 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (8). MDA-MB-231 cells were also treated with Dfect alone (2), and 100 nM control siRNA (3) (n = 5) as negative controls. Values represent mean + SE, #P < 0.09, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, compared with untreated control cells using ΔCt values.
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Figure 3. (A) Representative immunoblots of Chk-α and PLD1 in MDA-MB-231 cells: untreated (1), treated with 25 nM PLD1-siRNA (3), 100 nM PLD1-siRNA (4), 75 nM Chk-α-siRNA (5), 100 nM Chk-α-siRNA (6), and 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (7). Cells treated with 100 nM control siRNA (2) were used as negative controls. (B) Relative band intensity for Chk-α and PLD1 as compared with GAPDH was calculated after the treatment of MDA-MB-231 cells (n = 3–6 per group) with 25 nM PLD1-siRNA (4), 100 nM PLD1-siRNA (5), 75 nM Chk-α-siRNA (6), 100 nM Chk-α-siRNA (7), and 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (8). Cells treated with 100 nM control siRNA (3) were used for comparison. Values represent mean + SE, #P < 0.06, *P ≤ 0.05, **P ≤ 0.01 (one-tailed unpaired t test), compared with control siRNA treated cells (2).
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Figure 4. Representative images of TUNEL assay performed in MDA-MB-231 cells (n = 5 per group) after transfection with: 25 nM PLD1-siRNA (4), 100 nM PLD1-siRNA (5), 75 nM Chk-α-siRNA (6), 100 nM Chk-α-siRNA (7), and 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (8). MDA-MB-231 cells were treated with DNase I (1) as a positive control while untreated (2) and 100 nM control siRNA (3) treated samples served as negative controls. Panels from left to right represent a bright field view of the cells taken with a 40× objective, propidium iodide staining (red), incorporation of fluorescein-12-dUTP (green), and a merged image of propidium iodide and fluorescein-12-UTP incorporation. Propidium iodide staining represents the total number of cells in the panel while the intensity of fluorescein is indicative of the number of double strand breaks in DNA. Multiple images with the same acquisition parameters were taken from each sample to represent the entire sample.
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Figure 5. Images obtained from the TUNEL assay were quantified using MATLAB software as described in Materials and Methods and intensity/pixel values were calculated. The intensity/pixel were obtained for samples (n = 5) following transfection with 25 nM PLD1-siRNA (4), 100 nM PLD1-siRNA (5), 75 nM Chk-α-siRNA (6), 100 nM Chk-α-siRNA (7), and 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (8) in MDA-MB-231 cells. MDA-MB-231 cells were treated with DNase I (1) as a positive control while untreated (2) and 100 nM control siRNA (3) treated samples were considered as negative controls. Values represent mean + SE, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
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Figure 6. Percent cell viability/proliferation (%) as determined by MTS assay in MDA-MB-231 cells (n = 4 per group). MTS assays were performed 3 d after 48 h of treatment with Dfect (1), 100 nM control siRNA (2), 25 nM PLD1-siRNA (3), 100 nM PLD1-siRNA (4), 75 nM Chk-α-siRNA (5), 100 nM Chk-α-siRNA (6), and 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (7). Values represent mean + SE, #P < 0.075, *P ≤ 0.05, **P ≤ 0.01, compared with control siRNA treated cells unless otherwise indicated.
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Figure 7. (A) Representative high-resolution 1H MR spectra showing the GPC, PC, Cho region of MDA-MB-231 cell extracts: untreated (1), treated with 25 nM PLD1-siRNA (2), treated with 100 nM PLD1-siRNA (3), treated with 25 nM PLD1-siRNA + 75nM Chk-α-siRNA (4), treated with 75 nM Chk-α-siRNA (5), and treated with100 nM Chk-α-siRNA (6). (B) Histogram showing quantified levels of PC (white box), GPC (gray box), and tCho (black box), in MDA-MB-231 cells (n = 4 per group): untreated (1), treated with Dfect (2), treated with 100 nM control siRNA (3), treated with 25 nM PLD1-siRNA (4), treated with 100 nM PLD1-siRNA (5), treated with 75 nM Chk-α-siRNA (6), treated with 100 nM Chk-α-siRNA (7), and treated with 25 nM PLD1-siRNA + 75 nM Chk-α-siRNA (8). Values represent mean + SE, *P ≤ 0.05, compared with untreated control cells unless otherwise indicated.

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