Inhibition of PKM2 Enhances Sensitivity of Olaparib to Ovarian Cancer Cells and Induces DNA Damage

Poly (ADP-ribose) polymerase inhibitors (PARPi) have showed clinical benefit as maintenance therapy in advanced ovarian cancer by impairing the homologous recombination (HR) pathway. Pyruvate kinase M2 (PKM2), the significant cancer metabolic biomarker, integrates with DNA damage to directly promote HR. We aimed to investigate the role and molecular mechanism of PKM2 downregulation on sensitization of ovarian cancer cells to PARPi. Inhibitory effects in vitro were assessed by cell viability, clone formation, transwell assay, and flow cytometry. Downregulation of PKM2 by siRNA or small molecular inhibitor shikonin (Sk) enhanced anti-tumour activity of olaparib (Ola) in ovarian cancer cells. Silencing PKM2 or Sk synergized with Ola and reduced cell growth, colony formation and migration, and induced apoptosis. Western blot and immunofluorescence demonstrated that inhibition of PKM2 amplified Ola-induced γH2AX and phospho-ATM (p-ATM) activation and interfered with BRCA1 accumulation in the nucleus. A xenograft animal model demonstrated in vivo antitumor combination effect of Sk and Ola. Furthermore, Western blot and immunofluorenscent analyses of tissue samples revealed that treatment of Sk increased DNA damage, reduced expression of BRCA1 and PKM2. Therefore, this study identified that PKM2 downregulation is a novel therapeutic strategy to enhance Ola effectiveness in treating ovarian cancer.


Rescue experiment
Transfection of plasmid or siRNA was performed using Lipo6000™. According to the instructions of transfection reagent, SKOV3 cells (2*10 5 cells/well) were transfected with PKM2 siRNA 3# for 6 hours, then cells were cultured in complete medium for 24 hours. Above cells were continued transfected with plasmids pCMV3-PKM-GFPSpark vector or pCMV3 -GFP Spark vector for 6 hours, the samples were collected at 48 hours for western blot after transfection of plasmid.

Cologenic assay
Cells (8×10 3 cells/well) were plated evenly (24-well plate) and given different drugs for 7 days. The next, the culture medium was discarded. Cells were carefully rinsed twice with PBS and fixed with 10% formalin for 2 hours. Appropriate amount of 0.1% crystal violet solutions was added to dye for 2 hours. Quantitative analysis was performed at 550 mm using a microplate reader.

Transwell migration assay
Serum-free cell suspensions (2×10 4 cells/well) were plated on the upper chamber and complete culture solutions were generally added to the lower chamber for 24 hours.
Cells were fixed with 10% formaldehyde and stained with 0.1% crystal violet. For the cell migration assay in vitro, we removed the stained cells on the upper surface first.
Then, the stained cells on the lower surface were photographed using microscopy. The number of migrated cells in three random fields was counted. Table S1. The detailed score analysis for immunohistochemical staining of human specimens. quantification of the western blot bands of PKM2. One-way ANOVA was used to compare the means of different groups. Differences between the means were considered to be significant at P<0.05 by using Tukey multiple comparison tests; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the siCtrl-transfected cells.  A, Different concentrations of olaparib alone or combined with siPKM2 (si 2# or si 3#) treatment using cell viability assay. B-C, Colony formation assay results of si 2# or si 3# combined with olaparib treatment with 7-days treatment. D-E, Transwell assay results of si 3# combined with olaparib treatment, scar bar = 100 μM. Data represented the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, two-sided Student's t-test. Figure S4, Combination of PKM2 inhibitor shikonin with olaparib exerts synergistic effect on SKOV3 and OVCAR3 cells in vitro.