Published OnlineFirst August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Cancer Therapy: Preclinical Clinical Cancer Research Neurotensin Receptor 1 Antagonist SR48692 Improves Response to Carboplatin by Enhancing Apoptosis and Inhibiting Drug Efflux in Ovarian Cancer Jin Liu1, Mikael€ Agopiantz2,3,Joel€ Poupon4, Zherui Wu1, Pierre-Alexandre Just5, Bruno Borghese6, Evelyne Segal-Bendirdjian 1, Guillaume Gauchotte3,7, Anne Gompel1,8, and Patricia Forgez1 Abstract Purpose: The high affinity receptor 1 (NTSR1) and its agonist, Results: SR48692 enhanced the response to carboplatin in neurotensin (NTS), are correlated with tumor cell aggressiveness ovarian cancer cells and experimental tumors. When SR48692 in most solid tumors. As chemoresistance and tumor aggres- is combined with carboplatin, we noted a major improvement siveness are often related, we decided to study the role of the of platinum-induced DNA damage and cell death, as well as a NTSR1 complex within platinum-based chemotherapy responses. decrease in tumor growth. The relationship of these results In an ovarian model, we studied carboplatin because it is the main to clinical studies was made by the detection of NTS and standard of care for ovarian cancer. NTSR1 in 72% and 74% of ovarian cancer, respectively. Fur- Experimental Design: Experimental tumors and in vitro studies thermore, in a large series of high-grade ovarian cancer, NTSR1 were performed using SKOV3 and A2780 cells treated with mRNA was shown to correlate with higher stages and platinum carboplatin, with or without a very specific NTSR1 antagonist, resistance. SR48692. We measured the effects of these treatments on cell Conclusions: This study strongly suggests that the addition apoptosis and apoptosis-related proteins, platinum accumula- of NTSR1 inhibitor in combination with platinum salt–based tion in the cell and nucleus, and the expression and localization of therapy will improve the response to the drug. Clin Cancer Res; platinum transporters. NTS and NTSR1 labeling was measured in 23(21); 6516–28. Ó2017 AACR. patients with ovarian cancer. Introduction obvious specific symptoms, the majority of females with ovarian cancer are diagnosed at advanced stages, and the 5-year survival Ovarian cancer is the seventh most common cancer in the rate is 46.2% (2). Consequently, ovarian cancer is one of the most world and the eighth most frequent cause of cancer-related death lethal gynecologic malignancies in women. among women (1). In the United States, 22,280 new cases and The standard treatment for advanced ovarian cancer patients is 14,270 deaths were estimated for 2016. This represents about primarily debulking surgery and platinum-based chemotherapy 2.4% of all cancer-related deaths. Because this disease has no combined with taxane (3, 4). The combination of carboplatin plus paclitaxel results in a complete response rate in 40% to 60% of the cases. However, more than 90% of these patients relapse 1 2 INSERM UMRS 1007, Paris Descartes University, Paris, France. Department after 2 years, and patients with recurrence become incurable due œ of Medical Gynecology, CHRU Nancy, University of Lorraine, Vand uvre to the development of chemoresistance (5). Ovarian cancer -les-Nancy, France. 3INSERM U 954, Faculty of Medicine, University of Lorraine, Vandœuvre-les-Nancy, France. 4Toxicology Laboratory, Lariboisiere Hospital, remains a real clinical challenge, and despite the development AP-HP, Paris, France. 5Department of Pathology, Paris Descartes University, of new therapeutic strategies, there is an urgent need to optimize AP-HP, HUPC, Paris, France. 6Department of Gynecology Obstetrics II and the current treatment. To this regard, we suggest the use of Reproductive Medicine, Paris Descartes University, AP-HP, Paris, France. neurotensin blockage to improve the sensitivity to platinum- 7 Department of Pathology, CHRU Nancy, University of Lorraine, France. based chemotherapy. 8 Department of Gynecological Endocrinology, Paris Descartes University, APHP, Neurotensin (NTS) is a 13-amino acid peptide acting as a HUPC, Paris, France. neurotransmitter in the central nervous system and local hor- Note: Supplementary data for this article are available at Clinical Cancer mone in gastrointestinal tract (6). NTS activates three subtypes of Research Online (http://clincancerres.aacrjournals.org/). receptors. NTSR1 and NTSR2 belong to G-protein–coupled recep- Corresponding Author: Patricia Forgez, INSERM UMR-S 1007, Universite Paris tor family (7). NTSR3 (gp95/sortilin) is a sorting protein member Descartes, 45 rue des Saints-Peres, Paris 75270, France. Phone: 331-428-622; 33 of the family of Vps10p-domain receptor family (8). The complex 142 862 227; E-mail: [email protected] of Neurotensin (NTS) and its high affinity receptor 1 (NTSR1) was doi: 10.1158/1078-0432.CCR-17-0861 shown to contribute to cancer progression (9). NTSR1 was found Ó2017 American Association for Cancer Research. overexpressed in several types of solid cancers (10), in association 6516 Clin Cancer Res; 23(21) November 1, 2017 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 NTSR1 Blockade Improves Platinum Response in Ovarian Cancer from normal ovary samples, whereas these makers are present in Translational Relevance approximately 70% of ovarian cancer. Platinum salt–based therapy is used to treat almost 50% of cancer patients. It is proposed as an adjuvant, first-line, or in Materials and Methods palliative therapy. Unfortunately, many patients relapse Cell culture because of intrinsic, acquired resistance, or a weak sensitivity Ovarian adenocarcinoma cells SKOV3 was purchased from to the treatment. New therapeutic strategies to optimize the ATCC and A2780 was purchased from European Collection current treatment will add a real benefit to patient therapy. of Authenticated Cell Cultures (ECACC). Cells were cultured Neurotensin receptor 1 (NTSR1) is overexpressed in a large in RPMI 1640 supplemented with 10% FBS (Gibco) and number of solid cancers, and contributes to tumor aggres- 2 mmol/L glutamine. SKOV3 was used at passages between siveness. We report that blocking NTS/NTSR1 complex 38 and 48 and A2780 was used at passages between 25 and 35. improves the effect of carboplatin in ovarian cancer by enhanc- The corresponding NTSR1-overexpressing clone A2780-R1 was ing the drug-to-target ratio (platinum binding to DNA), and cultured in RPMI 1640 supplemented with 10% FBS, 2 mmol/L consequently the toxic effect of the drug. A combined plati- glutamine, and 0.5 mg/mL G418. All cells used in our research num salt–based therapy with NTS/NTSR1 inhibitor would be were mycoplasma-free as confirmed by the EZ-PCR Mycoplas- a suitable alternative to the current standard of care without ma Test Kit (Biological Industries). adding toxicity to the treatment. Transfection procedure A2780 cells were transfected with NTSR1 expressing pcDNA3 vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Selection was performed with 1 mg/mL with the dysregulation of the b-catenin pathway or epigenetic of G418 (Invitrogen). Stable transfectants were screened for NTS – regulation (11 13). Moreover, the presence of NTS/NTSR1 com- and NTSR1 expression by RT-PCR and immunofluorescence. plex enhanced the tumor growth and metastasis process in many – solid cancers (7, 14 19). Our group demonstrated that this cell Cell viability assay aggressiveness was due to the establishment of the EGFR autocrine Tumor cell growth was evaluated by PrestoBlue Cell Viability activation, occurring under the sustained stimulation of NTSR1 Reagent (Invitrogen). Cell suspension (100 mL) containing 8,000 (7, 12, 14). cells were seeded in 96-well plates, and the cells were treated Independently of the contribution of NTS/NTSR1 complex to accordingly. After 72 hours, 10-mL Presto blue was added to each cancer progression, it was recently shown that NTS plasma con- well and the cells were incubated for 2 hours at 37C. The centration is increased in rats treated with cisplatin and oxaliplatin fluorescence was measured by a multimode plate reader (EnSpire, fi for several weeks, causing speci c damage of the sciatic nerve, and PerkinElmer). producing variable effects in motor and behavioral tests (20). We hypothesized that NTS/NTSR1 complex may contribute to che- Tumor xenografts motherapy resistance and that the blockage of NTS/NTSR1 may A2780 cells (2.5  106) were suspended in PBS and Matrigel improve the response to platinum salt–based chemotherapy. (50%) (BD Biosciences) then inoculated in the right flank of Until now, the most studied approach to antagonize NTSR1 nude mice (NMRI-Nude Foxn1). All the procedures were in activation is the use of the nonpeptidic component meclinertant accordance with the Guide of the Care and Use of laboratory (SR48692; ref. 21). This drug was developed to counteract the Animals. Institutional Review Board approval was obtained by action of NTS on neurotransmitters in the brain (22). Mecliner- Le Comite d'Ethique en Experimentation Animale Charles tant exhibits a nanomolar affinity for different tissues and cells Darwin # B751201. Fourteen days after injection, four groups from various species (21). This compound crosses the blood– of six to seven mice were randomized as follows: 116 Æ 10 mm3 brain barrier. It was reported to reverse most intracellular medi- for control group, 120 Æ 11 mm3 for SR48692