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DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130 - PubMed

  • ️Tue Jan 01 2019

DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130

Francesca Nardi et al. Sci Rep. 2019.

Abstract

Acyl-CoA:diacylglycerol acyltransferase I (DGAT1) is a key enzyme in lipogenesis which is increased in metabolically active cells to meet nutrient requirements. DGAT1 has been recognized as an anti-obesity target; however, its role in the tumor microenvironment remains unclear. We postulated that, in prostate cancer (PCa) cells, augmented lipogenesis and growth are due to increased DGAT1 expression leading to microtubule-organizing center (MTOC) amplification. Thus, therapeutic targeting of DGAT1 potentially has tumor suppressive activity. We tested whether blocking DGAT1 in PCa cells altered MTOC and lipid signaling. Western blot and immunofluorescence were performed for MTOC and triglyceride mediators. Treatment with a DGAT1 inhibitor was evaluated. We found a stepwise increase in DGAT1 protein levels when comparing normal prostate epithelial cells to PCa cells, LNCaP and PC-3. Lipid droplets, MTOCs, and microtubule-regulating proteins were reduced in tumor cells treated with a DGAT1 inhibitor. Depletion of the non-centrosomal MTOC protein GM130 reduced PCa cell proliferation and migration. Inhibition of DGAT1 reduced tumor growth both in vitro and in vivo, and a negative feedback loop was discovered between DGAT1, PEDF, and GM130. These data identify DGAT1 as a promising new target for suppressing PCa growth by regulating GM130, MTOC number and disrupting microtubule integrity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

TAG-related proteins are dysregulated and suppression of DGAT1 decrease lipid content in prostate cancer cells. (A) ATGL (56 kD), DGAT1 (55 kD), and PEDF (50 kD) levels were analyzed by western blot in NHPrE, LNCaP and PC-3 cells. (B) ATGL, (C) DGAT1, and (D) PEDF densities were normalized by GAPDH in NHPrE, LNCaP and PC-3 cells. (E) LNCaP and PC-3 cells were treated with 1 µM DGAT1 inhibitor for 24 h, and, after fixation, LDs were analyzed with Oil-Red-O which stains neutral lipids in red. Size bar: 10 µm. (F) The total number of LDs per single cell in LNCaP (grey) and PC-3 cells (black) was counted (n = 50). For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2
Figure 2

DGAT1 inhibitor reduces cell proliferation and migration in prostate cancer cells. LNCaP and PC-3 cells were treated with 1 µM DGAT1 inhibitor for 24 h. (A) The proliferation rate was evaluated by the percentage of cells positive for PCNA staining/total cells (n = 50). The migration rate was evaluated by the percentage of (B) LNCaP cells and (C) PC-3 cells able to move into the scratched area at the times of 12 and 36 h. (D) Cell invasion was analyzed at the time of 24 h. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 3
Figure 3

DGAT1 inhibition decreases ncMTOC-related proteins in prostate cancer cells. (A) LNCaP and PC-3 cells were treated with 1 µM DGAT1 inhibitor for 24 h, and, after fixation, the cells were stained with mouse anti-pericentrin antibody (green) and DAPI (blue) to visualize the MTOCs and nucleus, respectively. Size bar: 10 µm. (B) The total number of pericentrin-positive MTOCs was counted per single cell (n = 50). (C) γ-tubulin (48 kD), ninein (243 kD), GM130 (140 kD), and CETN1 (20 kD) levels were analyzed by western blot in PC-3 control (CTR) and treated with 1 µM DGAT1 inhibitor or 10 nM PEDF. (D) γ-tubulin, (E) ninein, (F) GM130, and (G) centrin 1 (CETN1) densities were normalized by GAPDH. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4
Figure 4

DGAT1 inhibition regulates critical MT-associated proteins and disrupts the MT network in aggressive prostate cancer cells. (A) CLASP2 (141 kD) and α-tubulin (55 kD) levels were analyzed by western blot in PC-3 control (CTR) and treated with 1 µM DGAT1 inhibitor or 10 nM PEDF. (B) CLASP2 and (C) α-tubulin densities were normalized by GAPDH. (D) PC-3 cells were treated with 1 µM DGAT1 inhibitor for 24 h, and, after fixation, the cells were stained with rabbit anti-α-tubulin antibody (red) and DAPI (blue) to visualize the MTs and nucleus, respectively. Single color (red) images of α-tubulin were processed to enhance the network of MTs using Filament Sensor software (images in black and white). Size bar: 10 µm. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. **P < 0.01, ***P < 0.001.

Figure 5
Figure 5

Loss of GM130 is one mechanism responsible for growth suppression induced by DGAT1 inhibitor in aggressive prostate cancer cells. (A) GM130 (140 kD), DGAT1 (55kD), and PEDF (50 kD) levels were analyzed by western blot in PC-3 cells transfected with siRNA targeting GM130 or with negative control siRNA. (B) GM130, (C) DGAT1, and (D) PEDF densities were normalized by GAPDH. (E) The proliferation rate of PC-3 cells transfected with siRNA targeting GM130 was evaluated by the percentage of cells positive for PCNA staining/total cells and compared to the negative control (n = 50). (F) Cell migration of PC-3 cells transfected with siRNA trageting GM130 was measured at the times of 24 and 48 h and compared to the negative control. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. **P < 0.01, ***P < 0.001.

Figure 6
Figure 6

PEDF acts as an inhibitor of DGAT1 in aggressive prostate cancer cells. (A) ATGL (56 kD), perilipin 2 (PLIN2) (51 kD), DGAT1 (55 kD), and PEDF (50 kD) levels were analyzed by western blot in PC-3 control (CTR) and treated with 1 µM DGAT1 inhibitor or 10 nM PEDF. (B) ATGL, (C) PLIN2, (D) DGAT1, and (E) PEDF densities were normalized by GAPDH. (F) PC-3 cells were treated with 10 nM PEDF for 48 h, and, after fixation, the cells were stained with rabbit anti-DGAT1 antibody (red), BODIPY (green) and DAPI (blue) to localize DGAT1 protein, LDs and nucleus, respectively. Size bar: 10 µm. (G) PC-3 cells were treated with 10 nM PEDF for 48 h, and, after fixation, the cells were stained with rabbit anti-PEDF antibody (red), BODIPY (green) and DAPI (blue) to localize PEDF protein, LDs and nucleus, respectively. Size bar: 10 µm. For the western blot analysis, each protein has been detected by sequential staining and stripping using the same membrane. Data are presented as mean ± SEM. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7
Figure 7

Inhibition of DGAT1 suppresses tumor growth in vivo. Grafts containing PC-3 cells with or without DGAT1 inhibitor were implanted under the kidney capsule of SCID mice, and, after 2 weeks, the kidneys were harvested. (A) Tumor volume was calculated in the treated and untreated tumors. (B) H&E stain was performed in kidney sections of treated or untreated tumors using 20x and 40x objectives (I: invasion; N: necrosis). Size bars: 20 μm (C) ATGL-positive LD density in treated vs untreated tumor tissues was analyzed (n = 50). (D) PC-3 cell proliferation in vivo was analyzed using BrdU staining (n = 50). (E) Immunohistochemical stains were performed for BrdU and GM130 to analyze cell proliferation and intracellular GM130 protein, respectively. Size bars: 20 μm. Data are presented as mean ± SEM. Student’s unpaired t test. ****P < 0.0001.

Figure 8
Figure 8

Model of DGAT1 inhibition of lipid-MTOC axis in prostate cancer. This is a model of lipid-MTOC crosstalk in prostate cancer. At baseline, PCa cells are lipid-rich with MTOC amplification and a complex and dense MT network. At the site of the Golgi apparatus, GM130 and CLASP2-coated MTs are strongly expressed. PC-3 cells express high levels of DGAT1 and low levels of PEDF protein. After treatment with a DGAT1 inhibitor, the LD density is reduced as well as the number of MTOCs. The expression levels of proteins regulating ncMTOCs (GM130 and CLASP2) and MTOCs in general (γ-tubulin and ninein) were significantly decreased. Structural changes in the MT network post-treatment revealed fragmentation and loss of many linear MTs. Blockade of the lipid-MTOC axis via DGAT1 inhibitor resulted in suppression of prostate cancer growth and invasion both in vitro and in vivo.

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