Lipid Droplet Biosynthesis Impairment through DGAT2 Inhibition Sensitizes MCF7 Breast Cancer Cells to Radiation

Abstract

Breast cancer is the most frequent cancer in women worldwide and late diagnosis often adversely affects the prognosis of the disease. Radiotherapy is commonly used to treat breast cancer, reducing the risk of recurrence after surgery. However, the eradication of radioresistant cancer cells, including cancer stem cells, remains the main challenge of radiotherapy. Recently, lipid droplets (LDs) have been proposed as functional markers of cancer stem cells, also being involved in increased cell tumorigenicity. LD biogenesis is a multistep process requiring various enzymes, including Diacylglycerol acyltransferase 2 (DGAT2). In this context, we evaluated the effect of PF-06424439, a selective DGAT2 inhibitor, on MCF7 breast cancer cells exposed to X-rays. Our results demonstrated that 72 h of PF-06424439 treatment reduced LD content and inhibited cell migration, without affecting cell proliferation. Interestingly, PF-06424439 pre-treatment followed by radiation was able to enhance radiosensitivity of MCF7 cells. In addition, the combined treatment negatively interfered with lipid metabolism-related genes, as well as with EMT gene expression, and modulated the expression of typical markers associated with the CSC-like phenotype. These findings suggest that PF-06424439 pre-treatment coupled to X-ray exposure might potentiate breast cancer cell radiosensitivity and potentially improve the radiotherapy effectiveness.

Keywords: DGAT2; breast cancer; cancer stem cells; lipid droplet; lipid metabolism; radiotherapy.

Figure 1

Effects of PF-06424439 on proliferation, cell cycle and cell death of MCF7 cells. (A) Cell viability of MCF7 cells treated with different concentrations (1, 10, 50, 100, and 200 µM) of PF-06424439 compared with control (CTL) for 24, 48, 72, 96 h, as assessed by Presto blue assay and measured at 590 nm by using a spectrophotometer. RFU: Relative Fluorescence Units. (B) IC₅₀ was calculated in response to 72 h of exposure to different concentrations of PF-06424439. (C) Cell viability of MCF7 incubated with 10 μM of PF-06424439 for 72 h. (D) Cell cycle distribution was analyzed in MCF7 cells treated with 10 μM of PF-06424439 for 72 h compared with untreated cells. (E) Quantitative analysis of cells in G1, S and G2/M phases was determined by Flow Cytometry after PI staining as reported in the “Materials and Methods” section. (F) The fold change of dead MCF7 cells incubated with 10 μM of PF-06424439 for 72 h was evaluated by Flow Cytometry using PI staining. All data are presented as mean ± SD of three independent experiments for each assay (* p  ≤ 0.05, ** p  ≤ 0.01).

Figure 2

The combined effect of PF-06424439 pre-treatment and radiation enhanced radiosensitivity of MCF7 cells. (A) Clonogenic assays were performed to verify the ability of untreated (control, CTL) or PF-06424439 pre-treated MCF7 cells irradiated with different doses of X-rays (2, 4, 6 Gy) to form colonies. (B) Representative images of flasks with crystal violet-stained MCF7 colonies after 18 days from 72 h PF-06424439 pre-treatment and radiation (2, 4, 6 Gy). (C) γ-H2AX immunostaining was carried out to detect DSBs produced after 30 min from 6 Gy irradiation of MCF7 cells untreated or 72 h pre-treated with PF-06424439. Hoechst 33342 dye was used to counterstain nuclei and 100 cells per sample were imaged at the confocal microscope with a 63× oil-immersion objective. (D) The average number of γ H2Ax foci red staining per cell nucleus was shown as bar plot and quantified using Image J 1.52p software. (E–G) LD540 green staining of cytoplasmic LDs in MCF7 cells untreated or pre-treated with PF-06424439 for 72 h and irradiated with 6 Gy, after 30 min and 72 h from irradiation. 100 cells per sample were acquired with 63× oil-immersion objective. (F–H) Image J analysis was performed to quantify LD amount at 30 min and 72 h after irradiation. For all confocal images the scale bar is 20 μm. All experiments were performed in triplicate and expressed as mean ± SD (* p  ≤ 0.05, ** p  ≤ 0.01).

Figure 3

MCF7 pre-treatment with 10 µM PF-06424439 followed by radiation modulates lipid gene metabolism and CSC-related gene expression. (A) RT-qPCR results showing the relative mRNA expression of HMGCR, FASN, and DGAT2 as well as LD-associated genes, PLIN1, TIP47, PLIN5 after 30 min and 72 h from irradiation (6 Gy) in MCF7 cells untreated and pre-treated with 10 µM PF-06424439. (B) The relative mRNA expression of CSC markers, CD44, and CD166, evaluated by RT q-PCR at 30 min and 72 h after irradiation. Data are represented as means ± SD from three different experiments performed in triplicate (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 4

PF-06424439 pre-treatment reduces epithelial to mesenchymal transition on MCF7 cells. (A) Migration assay was performed to determine the effects of PF-06424439 on the MCF7 invasive capacity within 36 h from the scratch. (B) Migration was significantly decreased in cells pre-treated with PF-06424439; the bar plots show the percentage of wound closure rate from 24 to 36 h, quantified by ImageJ Fiji software. (C) Western blotting analysis of Vimentin and E-cadherin protein expression in MCF7 cells analyzed after 72 h of treated 10 µM PF-06424439 treatment. (D,E) Protein levels were quantified by ImageJ software and the results were normalized to the control. HSC70 was used as a loading control. (F) RT q-PCR for EMT markers (E-cadherin, Vimentin, and Snail) of MCF7 cells untreated or pre-treated with PF-06424439 and exposed to 6 Gy, 30 min and 72 h after irradiation. Data were collected from three independent experiments and are shown as means ± SD (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).