Abstract

BackgroundOvarian 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.MethodsUsing 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.ResultsWe 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.ConclusionsThese 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.

Highlights

  • Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women

  • We first confirmed the differential effect of Olaparib treatment on OC cell lines depending on BRCA status, by performing a dose- and time-curve evaluation of cell viability through MTT assay in the BRCA1-null UWB1.289 cell line (UWB), the UWB1.289 + BRCA1 cells (UWBBRCA), in which BRCA1 expression was permanently restored, and the BRCA wild-type SKOV3 cell line

  • We demonstrated that Olaparib could significantly upmodulate Neuropilin 1 (NRP1) mRNA and protein only in UWB-BRCA cells, suggesting that in these cells NRP1 expression was sufficient to modulate drug sensitivity, this restricting the effect of Olaparib on cell viability and apoptosis induction

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Summary

Introduction

Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Recognition of the role of inherited mutations in the DNA repair genes BRCA1 and BRCA2 in a proportion of OC patients led to the introduction of new therapeutic strategies targeting other DNA repair pathways, using poly-ADP ribose polymerase (PARP) inhibitors, such as Olaparib [7]. PARPi act by blocking the catalytic domain of PARP enzymes, but these agents can trap PARP proteins on the double-stranded DNA helix, this leading to cytotoxic lesions [9]. This strategy has been approved as treatment option for OC patients bearing BRCA1/2 gene mutation [10]. The potential mechanisms involved in PARPi resistance are represented by DNA repair restoration [13, 14], PI3K/AKT pathway activation [15] and miRNA dysregulation [16,17,18], but further investigation is needed to clarify the complexities of pathways underlying PARPi-related clinical resistance

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