Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Neurotensin receptor 1 antagonist SR48692 improves response to carboplatin by enhancing apoptosis and inhibiting drug efflux in ovarian cancer Jin Liu 1, Mikael Agopiantz2,3, Joel Poupon4, Zherui Wu1, Pierre-Alexandre Just5, Bruno Borghese6, Evelyne Segal-Bendirdjian1, Guillaume Gauchotte3,7, Anne Gompel1,8 and Patricia Forgez1* 1 INSERM UMRS 1007, Paris Descartes University, 75270 Paris cedex 06, France, 2 Department of Medical Gynecology, CHRU Nancy, University of Lorraine, Vandœuvre-lès-Nancy, France 3 INSERM U 954, Faculty of Medicine, University of Lorraine, Vandœuvre-lès-Nancy, France 4 Toxicology Laboratory, Lariboisière Hospital, AP-HP, Paris, France 5 Department of Pathology, Paris Descartes University, AP-HP, HUPC, Paris, France 6 Department of Gynecology Obstetrics II and Reproductive Medicine, Paris Descartes University, AP-HP, Paris, France 7 Department of Pathology, CHRU Nancy, University of Lorraine, France 8 Department of Gynecological Endocrinology, Paris Descartes University, APHP, HUPC, Paris, France. *Corresponding author: Patricia Forgez Mailing address: INSERM UMR-S 1007, Université Paris Descartes, 45, rue des Saints-Pères, 75270 Paris cedex 06, France Email: [email protected] Running title: NTSR1 blockade improves platinum response in ovarian cancer Keywords: Neurotensin; Neurotensin receptor 1; ovarian cancer; platinum salt-based chemotherapy; sensitization. 1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Financial support: This work was supported by INSERM TRANSFERT MAT-PI-07563-A-09 (PI. Forgez), SATT idf INNOV Paris France (PI. Forgez), Fondation de France 2013 00038286 (E. Segal-Bendirdjian). China Scholarship council supported Dr. J. Liu’s PhD study. SATT idf INNOV Paris France supported Dr. Z. Wu. 2 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: The high affinity receptor 1 (NTSR1) and its agonist, neurotensin (NTS), is correlated with tumor cell aggressiveness in most solid tumors. As chemo-resistance and tumor aggressiveness are often related; we decided to study the role of the NTSR1 complex within platinum-based chemotherapy responses. In an ovarian model, we studied carboplatin because it is the main standard of care for ovarian cancer (OC). Experimental Design: Experimental tumors and in vitro studied were performed using SKOV3 and A2780 cells treated with carboplatin, with or without a very specific NTSR1 antagonist, SR48692. We measured the effects of these treatments on cell apoptosis and apoptosis related proteins, platinum accumulation in the cell and nucleus, and the expression and localization of platinum transporters. NTS and NTSR1 labelling was measured in patients with ovarian cancer. Results: SR48692 enhanced the response to carboplatin in OC cells and experimental tumors. When SR48692 is combined to carboplatin, we noted a major improvement of platinum induced DNA damage and cell death, as well as a decrease in tumor growth. The relationship of these results to clinic studies was made by the detection of NTS and NTSR1 in 72% and 74% of OC, respectively. Furthermore, in a large series of high grade OC, NTSR1 mRNA was shown to correlate with higher stages and platinum resistance. Conclusions: This study strongly suggests that the addition of NTSR1 inhibitor in combination with platinum salt-based therapy will improve the response to the drug. 3 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational relevance Platinum salt-based therapy is used to treat almost 50% of cancer patients. It is proposed as adjuvant, main line, or in palliative therapy. Unfortunately, many patients relapse because of intrinsic, acquired resistance, or a weak sensitivity to the treatment. New therapeutic strategies to optimize the current treatment will add a real benefit to patient therapy. Neurotensin receptor 1 (NTSR1) is overexpressed in a large number of solid cancers, and contributes to tumor aggressiveness. We report that blocking NTS/NTSR1 complex improves the effect of carboplatin in ovarian cancer by enhancing the drug to target ratio (platinum binding to DNA), and consequently the toxic effect of the drug. A combined platinum salt-based therapy with NTS/NTSR1 inhibitor would be a suitable alternative to the current standard of care without adding toxicity to the treatment. 4 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Ovarian cancer (OC) is the seventh most common cancer in the world and the eighth most frequent cause of cancer death among women (1). In the United States, 22,280 new cases and 14,270 deaths were estimated for 2016. This represents about 2.4 percent of all cancer deaths. Because this disease has no obvious specific symptoms, the majority of females with OC are diagnosed at advanced stages, and the percent surviving at 5 year rate is 46.2% (2). Consequently, OC is one of the most lethal gynecological malignancies in women. The standard treatment for advanced ovarian cancer patients is primarily debulking surgery and platinum-based chemotherapy combined with taxane (3,4). The combination of carboplatin plus paclitaxel, results in a complete response rate in 40-60 % of the cases. However, more than 90% of these patients relapse after 2 years and recurrent patients in most cases become incurable due to the development of chemo-resistance (5). OC remains a real clinical challenge, and despite the development of new therapeutic strategies, there is an urgent need to optimize the current treatment. To this regard, we suggest, here, the use of neurotensin blockage to improve the sensitivity to platinum-based chemotherapy Neurotensin (NTS) is a 13-amino-acid peptide acting as a neurotransmitter in the central nervous system and local hormone in gastrointestinal tract (6). NTS activates three subtypes of receptors. NTSR1 and NTSR2 belong to G-protein coupled receptors family (7). NTSR3 (gp95/sortilin) is a sorting protein member of the family of Vps10p-domain receptor family (8). The complex of Neurotensin (NTS) and its high affinity receptor 1 (NTSR1) was shown to contribute to cancer progression (9). NTSR1 was found overexpressed in several types of solid cancers (10), in association with the dysregulation of the beta-catenin pathway or epigenetic regulation (11-13). Moreover, the presence of NTS/NTSR1 complex enhanced the tumor growth and metastasis process in many solid cancers (7,14-19). Our group demonstrated that this 5 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0861 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cell aggressiveness was due to the establishment of the epidermal growth factor receptor autocrine activation, occurring under the sustained stimulation of NTSR1 (7,12,14). Independently of the contribution of NTS/NTSR1 complex to cancer progression, it was recently shown that NTS plasma concentration is increased in rats treated with cisplatin and oxaliplatin for several weeks, causing specific damage of the sciatic nerve, and producing variable effects in motor and behavioral tests (20). We hypothesized, that NTS/NTSR1 complex may contribute to chemotherapy resistance and that the blockage of NTS/NTSR1 may improve the response to platinum salt-based chemotherapy. Up to now the most studied approach to antagonize NTSR1 activation is the use of the non-peptidic component meclinertant (SR-48692) (21). This drug was developed to counteract the action of NTS on neurotransmitters in the brain (22). Meclinertant exhibits a nanomolar affinity for different tissues and cells from various species (21). This compound crosses the blood brain barrier. It was reported to reverse most intracellular mediator signals linked to NTSR1 activation, such as intracellular Ca2+ mobilization; inositol monophosphate; cyclic GMP; cyclic AMP activation (23). This component showed agonist effects on NTSR2, which may explain its partial or non-response on particular central nervous system effect induced by NTS as hypothermia or analgesia (24,25). Meclinertant was also tested on non-selected patients with lung cancer, after a first line of chemotherapy using cisplatin combined with etoposide,