MiR-200c sensitizes Olaparib-resistant ovarian cancer cells by targeting Neuropilin 1

Abstract

Background: Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance.

Methods: Using three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons' correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells.

Results: We observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells.

Conclusions: These data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

Keywords: Drug resistance; NRP1; Ovarian cancer; PARP inhibitors; miR-200c; miRNAs.

Fig. 1

Differential effect of Olaparib on clonogenic ability of OC cell lines. UWB, UWB-BRCA and SKOV3 cells were treated with Olaparib for 144 h, then seeded at low concentration and allowed to grow for 12 days. The effect of Olaparib on cell clonogenicity was determined by colony formation assay. The images are representative pictures of colonies stained with crystal violet. Colony forming efficiency was calculated by crystal violet absorbance. Mean values obtained from two independent experiments, each performed in triplicate, are reported in graph. Error bars represent standard deviations. **, p < 0.005, ***, p < 0.0005 vs. control (DMSO)

Fig. 2

Effects of Olaparib treatment on apoptosis/autophagy induction in OC cell lines. UWB, UWB-BRCA and SKOV3 cells were treated for 144 h with Olaparib. a) The percentages of early apoptotic and late apoptotic cells were obtained by flow cytometry quadrant analysis with annexin A5 FITC/7-AAD double staining, and expressed as histograms. b) The expression of the apoptosis related proteins phAKT (Ser473), AKT, cleaved Caspase 3 and cleaved PARP1 was determined by Western blot analysis. Tubulin expression was used as internal control. The images are representative of at least two independent experiments. CF, cleaved form of PARP1. c) The expression of the autophagy related proteins LC3 and P62 was assessed by Western blot analysis. d) UWB and UWB-BRCA cells were treated for 144 h with Olaparib (5 μM), Chloroquine (CQ, 10 μM) or a combination of them, and the expression of LC3 and P62 was assessed. The intensity of the LC3-II and P62 bands was evaluated by densitometric analysis, normalized with LC3-I and Tubulin, respectively, and reported in graph. Error bars represent standard deviations. *, p < 0.05, **, p < 0.005 vs. control (DMSO)

Fig. 3

Effect of Olaparib on NRP1 expression in OC. a) The expression pattern of NRP1 was assessed by qRT-PCR assays in OC tumor tissues from 28 patients and noncancerous ovarian tissues from 12 healthy donors. ***, p < 0.0005. b, c) Basal NRP1 protein (b) and mRNA (c) levels in UWB, UWB-BRCA and SKOV3 cells were assessed by Western blot analysis and qRT-PCR, respectively. d) The expression pattern of NRP1 was assessed by qRT-PCR assays in OC tumor biopsies from 16 patients obtained before and after platinum-based chemotherapy (CHT). **, p < 0.005. e, f) NRP1 protein (e) and mRNA (f) expression after Olaparib treatment for 144 h were evaluated by Western blot analysis and qRT-PCR, respectively. For Western blot analysis, tubulin expression was used as internal control, and the images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to control (DMSO). For qRT-PCR, each experiment was performed in triplicate and mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. *, p < 0.05 vs. UWB cells (c) or vs control (DMSO) (f)

Fig. 4

Effect of NRP1 silencing on Olaparib resistance. SKOV3 cells were transfected with NRP1-specific siRNA (siNRP) or nonspecific control siRNA (siNC). a, b) NRP1 expression was assessed at 48, 72 or 144 h from transfection (a), and after Olaparib treatment at 144 h (b) by Western blot analysis. Tubulin expression was used as internal control. The images are representative of at least three independent experiments. c, d) The effect of Olaparib on cell viability and clonogenicity of silenced SKOV3 cells was determined by MTT assay and colony formation assay, respectively. Mean values obtained from two independent experiments, each performed in triplicate, are reported in graph. Error bars represent standard deviations. **, p < 0.005, ***, p < 0.0005 vs. siNC-transfected cells or vs. control (DMSO). e) The expression of the apoptosis related proteins phospho-AKT (Ser473), AKT, cleaved caspase 3 and cleaved PARP1 in silenced SKOV3 cells treated or not with Olaparib was assessed by Western blot analysis. Tubulin expression was used as internal control. The images are representative of at least two independent experiments

Fig. 5

miR-200c-mediated regulation of NRP1 expression in OC cell lines. a) Schematic representation of the conserved target sites for miR-141/200a-3p (red) and miR-429/200bc-3p (green) in the 3′ UTR of human NRP1, and Targetscan prediction of miR-200c-3p binding site within the NRP1 3′-UTR. b) The expression pattern of miR-200c was assessed by qRT-PCR assays in OC tumor tissues from 28 patients and noncancerous ovarian tissues from 12 healthy donors. ***, p < 0.0005. c) Correlation analysis between NRP1 and miR-200c expression was performed in OC tumor tissues from 28 patients. d, e) UWB, UWB-BRCA and SKOV3 cells were analyzed at basal conditions (d) or after Olaparib treatment (e), and miR-200c expression was determined by qRT-PCR analysis. Each experiment was performed in triplicate, and miRNA levels were normalized to U6 expression. Error bars represent standard deviations. *, p < 0.05, **, p < 0.005, ***, p < 0.0005 vs. UWB cells (d) or vs control (DMSO) (e)

Fig. 6

Effect of miR-200c overexpression on NRP1. SKOV3 cells were stably transfected with a plasmid carrying the precursor of miR-200c (miR-200c) and its corresponding vector control (CTRL). miR-200c expression was assessed by qRT-PCR analysis (a). NRP1 protein (b) and mRNA (c) expression were evaluated by Western blot analysis and qRT-PCR, respectively. For Western blot analysis, tubulin expression was used as internal control, and the images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to control (CTRL). For qRT-PCR, each experiment was performed in triplicate. miRNA levels were normalized to U6 expression, while mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. **, p < 0.005 vs. CTRL

Fig. 7

Effect of miR-200c overexpression on SKOV3 response to Olaparib. SKOV3 cells were stably transfected with a plasmid carrying the precursor of miR-200c (miR-200c) and its corresponding vector control (CTRL), then treated for 144 h with Olaparib. a, b) Cell viability and colony forming efficiency were determined by MTT assay and clonogenic assay, respectively. Mean values obtained from two independent experiments, each performed in triplicate, are reported in graph. Error bars represent standard deviations. ***, p < 0.0005 vs. CTRL cells or vs. control (DMSO). c) The presence of γH2AX foci (red) was assessed by immunofluorescence analysis. Nuclei (blue) were visualized with 4′, 6-diamidino-2-phenylindole (DAPI). Images were captured under ApoTome microscope at 40x magnification. Quantification of γH2AX foci was determined by measuring red fluorescence intensity with ImageJ software. Mean values obtained from measurements of five microscopic fields randomly taken from three independent experiments are reported in graph. Error bars represent standard deviations. **, p < 0.005 vs. control (DMSO)

Fig. 8

Effect of miR-200c overexpression on Olaparib-induced apoptosis. SKOV3 cells were stably transfected with a plasmid carrying the precursor of miR-200c (miR-200c) and its corresponding vector control (CTRL), then treated for 144 h with Olaparib. a) The percentages of early apoptotic and late apoptotic cells were obtained by flow cytometry quadrant analysis with annexin A5 FITC/7-AAD double staining, and expressed as histograms. b) The expression of the apoptosis related proteins phAKT (Ser473), AKT, cleaved Caspase 3 and cleaved PARP1 was determined by Western blot analysis. Tubulin expression was used as internal control. The images are representative of at least two independent experiments. CF, cleaved form of PARP1