Lysophosphatidic acid suppresses apoptosis of high-grade serous ovarian cancer cells by inducing autophagy activity and promotes cell-cycle progression via EGFR-PI3K/Aurora-AThr288-geminin dual signaling pathways

Abstract

Lysophosphatidic acid (LPA) and geminin are overexpressed in ovarian cancer, and increasing evidence supports their contribution to ovarian tumor development. Here, we reveal that geminin depletion induces autophagy suppression and enhances reactive oxygen species (ROS) production and apoptosis of high-grade serous ovarian cancer (HGSOC) cells. Bioinformatics analysis and pharmacological inhibition studies confirm that LPA activates geminin expression in the early S phase in HGSOC cells via the LPAR_1/3/MMPs/EGFR/PI3K/mTOR pathway. Furthermore, LPA phosphorylates Aurora-A kinase on Thr288 through EGFR transactivation, and this event potentiates additional geminin stabilization. In turn, overexpressed and stabilized geminin regulates DNA replication, cell-cycle progression, and cell proliferation of HGSOC cells. Our data provide potential targets for enhancing the clinical benefit of HGSOC precision medicine.

Keywords: Aurora-AThr288; EGFR transactivation; LPA; apoptosis; autophagy; geminin.

FIGURE 1

Depletion of geminin suppresses autophagy activity, enhances ROS production, and induces the apoptosis of HGSOC cells. (A) Immunoblotting analysis of geminin protein levels in A2780 cells as indicated. shNC, A2780 cells transfected with shNC; sh-GMNN, geminin-knockout A2780 cells transfected with shRNA. Quantification of three independent experiments was performed, normalized to tubulin, and expressed as a ratio of NC, mean ± SEM, Student’s unpaired t-test, ***p < 0.001. (B) IB analysis of apoptosis-related proteins’ expression levels in A2780 cells as indicated. Mean ± SEM, n = 3, Student’s unpaired t-test, **p < 0.01, ***p < 0.001, ns—non-significant. (C) Apoptosis analysis of A2780 cells under geminin depletion. The percentage of apoptotic cells was analyzed with flow cytometry. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001. (D) Representative phase-contrast images of bioreactor expanded A2780 cells at indicated days of cultivation. Scale bar 20 μm. (E) Graphical illustration of the average aggregate size measured at the indicated day of cultivation: Day 8, shNC n = 116, sh-GMNN n = 108; Day 16, shNC n = 130, sh-GMNN n = 161. Images were analyzed using Nikon NIS Elements D software, mean ± SD, Student’s unpaired t-test, ***p < 0.001. (F) The protein–protein associations about geminin protein. Data from STRING. (G) ROS analysis of A2780 cells under geminin depletion. The percentage of apoptosis cells was analyzed with flow cytometry. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001. (H) IB analysis of LC3B protein levels in A2780 cells as indicated. Mean ± SEM, n = 3, Student’s unpaired t-test, ***p < 0.001.

FIGURE 2

LPA enhances geminin expression via a LPAR_1/3/MMPs/EGFR/PI3K/mTOR signalling pathway in HGSOC cells. (A) The mRNA expression levels of geminin in A2780 cells and OVCAR5 cells. VNOVA, Dunnett’s Multiple Comparison Test, mean ± SEM, n = 3, *p < 0.05, ***p < 0.001. (B,C) Immunoblotting analysis of geminin protein levels in A2780 cells (B) and OVCAR5 cells (C) under a 10 μM LPA time gradient stimulus. Quantification of three independent experiments was performed, normalized to tubulin, and expressed as a ratio of 0 h, respectively. Mean ± SEM, VNOVA, Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001. (D,E) Immunoprecipitation analysis with tyrosine-phosphorylated EGFR antibodies (PY) in A2780 cells (D) and OVCAR5 cells (E) treated with 0.1% DMSO (vehicle), 10 μM LPA or 10 ng/mL EGF for 5 min. (F,G) IP analysis with tyrosine-phosphorylated EGFR antibodies (PY) in A2780 cells (F) and OVCAR5 cells (G) pretreated with or without 10 μM BB94 for 30 min, and then stimulated with 0.1% DMSO (vehicle), 10 μM LPA or 10 ng/mL EGF for 5 min. (H) The mRNA expression of LPARs in OVCAR5 cells. Mean ± SEM, n = 3, VNOVA, Dunnett’s Multiple Comparison Test, ***p < 0.001. (I) IB analysis of geminin protein levels in OVCAR5 cells as indicated. Cells were pretreated with 0.1% DMSO (vehicle), 10 μM Ki16425, 10 μM BB94, 10 μM AG1478, 10 μM LY294002 or 100 nM Rapamycin for 30 min, and then stimulated with 10 μM LPA time gradient. (J) Quantificantion of IB in (I). Data are analyzed with OriginPro 2021, mean ± SEM, n = 3, VNOVA, Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

LPA potentiates geminin stability by targeting Aurora-A^Thr288 in HGSOC cells. (A,B) Immunoblotting analysis of geminin protein levels in A2780 cells (A) and OVCAR5 cells (B) after pretreated with or without CCT137690 (25 μM) for 120 min, and then stimulated with 0.1% DMSO (vehicle) or 10 μM LPA. Mean ± SEM, n = 3, VNOVA, Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001. (C) IB analysis of Aurora-A protein levels in A2780 cells and OVCAR5 cells. Mean ± SEM, n = 3, VNOVA, Dunnett’s Multiple Comparison Test, ns, non-significant. (D,E) Immunoprecipitation analysis with anti-phospho-Aurora Kinase (Thr288) antibodies in A2780 cells (D) and OVCAR5 cells (E) treated with 0.1% DMSO (vehicle), 10 μM LPA or 10 ng/mL EGF for 5 min (A2780 cells) or 30 min (OVCAR5 cells). (F) Correlation of gene AURKA and EGFR in TCGA ovarian cancer cohort. log2 fold changes of gene expression on the axis, and the Spearman Method was used for calculating the correlation coefficient. (G) The protein-protein associations between Aurora-A and EGFR protein. Data from STRING. (H–K) IP analysis with anti-phospho-Aurora Kinase (Thr288) antibodies in A2780 cells (H,J) and OVCAR5 cells (I,K) after pretreated with or without 10 μM BB94 (H,I) or 10 μM AG1478 (J,K) for 30 min, then treated with 0.1% DMSO (vehicle), 10 μM LPA or 10 ng/mL EGF for 5 min (A2780 cells) or 30 min (OVCAR5 cells).

FIGURE 4

LPA signal mediates DNA replication, cell-cycle progression, and cell proliferation of HGSOC cells. (A) Representative images of immunolluorescence staining with anti-BrdU (green) and DAPI/nuclei (blue). Some increased green puncta in the nucleus are identified with white arrows. OVCAR5 cells. Scale bar 10 μm. All images were obtained with the same acquisition conditions. (B) The cell proliferation of OVCAR5 cells. Cells were pretreated with specified inhibitors for 30 min and then grown in 0.1% DMSO, 10 µM LPA or 10 ng/ml EGF. Mean ± SEM, n = 3, VNOVA, Dunnett’s multiple comparison test, **p < 0.01, ***p < 0.001. (C) Representative phase-contrast images of bioreactor expanded OVCARS cells at indicated days of cultivation. Scale bar 20 μm. (D) Graphical illustration of the average aggregate size measured at the indicated day of cultivation. Day 8, DMSO n = 140, LPA n = 139; Day 16, DMSO n = 153, LPA n = 138. Images were analyzed using Nikon NIS Elements D software, mean ± SD, Student’s unpaired t-test, ***p < 0.001.

FIGURE 5

The signaling pathway of LPA-mediated DNA replication initiation, cell-cycle progression, and autophagy in HGSOC cells.