Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Netrin-1 and its receptor DCC are causally implicated in melanoma progression Amina Boussouar1, Antonin Tortereau1,2, Ambroise Manceau1, Andrea Paradisi1, Nicolas Gadot2, Jonathan Vial1, David Neves3, Lionel Larue4-6, Maxime Battistella7, Christophe Leboeuf7, Celeste Lebbé8, Anne Janin7, and Patrick Mehlen1,2$ 1Apoptosis, Cancer and Development Laboratory - Equipe labellisée ‘La Ligue’, LabEx DEVweCAN, Centre de Recherche en Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, 69008 Lyon, France. 2Department of Translational Research and Innovation, Centre Léon Bérard, 69008 Lyon. 3Netris Pharma, 69008 Lyon. 4Institut Curie, PSL Research University, INSERM U1021, Normal and Pathological Development of Melanocytes, Orsay, France, 5Univ Paris-Sud, Univ Paris- Saclay, CNRS UMR3347, Orsay, France, 6Equipe Labellisée Ligue Contre le Cancer, Orsay, France. 7Université Paris Diderot, Inserm, UMR_S1165, Paris, France, Laboratoire de pathologie, Hôpital Saint Louis, APHP, Paris, France. 8Service de dermatologie, Hôpital Saint Louis, APHP, Paris, France. Running title: Netrin-1 and DCC in melanoma Corresponding author: Patrick Mehlen, University of Lyon-CRCL, Centre Léon Bérard, 28 rue Laennec, Lyon 69008, France. Phone: 334-7878-2870; Fax: 334-7878-2887; E-mail: [email protected] Conflict of Interest: PM declares to have a conflict of interest in the study as a founder and shareholder of Netris Pharma 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Deleted in Colorectal Cancer (DCC), the receptor for the multifunctional cue netrin-1, acts as a tumor suppressor in intestinal cancer and lung metastasis by triggering cancer cell death when netrin-1 is lowly expressed. Recent genomic data highlighted that DCC is the third most frequently mutated gene in melanoma, we therefore investigated whether DCC could act as a melanoma tumor suppressor. Re- expressing DCC in human melanoma cell lines promoted tumor cell death and tumor growth inhibition in xenograft mouse models. Genetic silencing of DCC pro- death activity in a BRAFV600E mouse model increased the proportion of mice with melanoma, further supporting that DCC is a melanoma tumor suppressor. Netrin-1 expression was elevated in melanoma compared to benign melanocytic lesions. Upregulation of Netrin-1 in the skin cells of a BRAFV600E-mutated murine model reduced cancer cell death and promoted melanoma progression. Therapeutic antibody blockade of netrin-1 combined with dacarbazine increased overall survival in several mouse melanoma models. Together these data support that interfering with netrin-1 could be a viable therapeutic approach in patients with netrin-1- expressing melanoma. Significance: Netrin-1 and its receptor DCC regulate melanoma progression, suggesting therapeutic targeting of this signaling axis as a viable option for melanoma treatment. 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Vogelstein and colleagues identified Deleted in Colorectal Cancer (DCC) as a gene frequently silenced in cancer because of loss of heterozygosity (LOH) on chromosome 18q. DCC expression is markedly reduced in more than 50% of colorectal tumors, as well as in many other neoplasms (1). Moreover, loss of DCC is associated with poor prognosis and potentially decreased response to adjuvant chemotherapy in colorectal cancer patients. Finally, restoration of DCC expression can suppress tumorigenic growth properties in vitro and in nude mice (1). These findings led to the proposal that DCC expression is a constraint for tumor progression, and thus that DCC functions as a tumor suppressor gene. However, the localization of the DCC gene close to well-established tumor suppressors such as Smad4 (2) and the absence of increased tumor susceptibility in a mouse model in which DCC was inactivated (3) created skepticism about its potential role as a tumor suppressor gene (4). However the more recent use of conditional DCC mutant mouse model have demonstrated that DCC inactivation is associated with increased tumoral progression (5). DCC, a large 200 kDa transmembrane receptor, belongs to the family of dependence receptors (6). Such receptors induce apoptosis when their trophic ligands are absent, thus conferring a state of cellular dependence on ligand availability for survival (7), and this has been proposed to confer a tumor suppressive activity to these dependence receptors (7,8). To address this hypothesis, we generated a mouse model in which the DCC locus is point-mutated in Asp1290 to specifically silence the pro-death activity of the receptor without impacting DCC non-cell death signaling (9). These mice spontaneously develop more colorectal cancers and lymphomas than age-matched control animals and are more prone to develop metastatic intestinal adenocarcinoma when backcrossed in an 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. APC mutant background (9,10). Of interest, while loss of DCC has been extensively described in cancer, studies are have emerged showing that the ligand of DCC, netrin-1, is also implicated in cancer progression. Netrin-1 was initially discovered as a navigation cue during the development of the nervous system, as a key factor for brain wiring (11). Although initially described as a diffusible cue guiding, via long distance processes, different types of axons (12), netrin-1 is currently regarded as a laminin-related sticky protein acting at short distance (13) via its main receptors, DCC and UNC5-Homolog (UNC5H- i.e., UNC5A, UNC5B, UNC5C, UNC5D) (14). Indeed, netrin-1 appears to be a multifunctional secreted protein that plays key roles in neuronal navigation, angiogenesis, and cell survival (15), and as a consequence, has been associated with numerous diseases including diabetes, cardiovascular diseases, and cancers (15). In the latter case, netrin-1 is often reported to be up-regulated, and this up-regulation was proposed to act as a selective mechanism blocking apoptosis induced by the dependence receptors DCC and UNC5H (15). Consistently, netrin-1 expression in brain metastases is an independent poor prognostic factor for patient survival (16). Moreover, netrin-1 detection in blood or urine has been proposed to be a predictive marker for cancers (17,18). Netrin-1 up-regulation has been reported not only in cancer cells but also in cancer-associated fibroblasts (19). Efforts to develop drugs that inhibit the interaction of netrin-1 with its receptors are therefore ongoing. Several preclinical proof-of-concept studies have shown that candidate drugs interfering with netrin-1-receptor interactions, either used alone or in combination with conventional chemotherapies or epidrugs, markedly inhibit tumor growth and metastasis development (18,20,21). A humanized anti-netrin-1 monoclonal antibody (Net1-mAb), also called NP137, has been developed (20) and the interim results of a phase I clinical trial have recently been reported (22), showing promising signs of anti-tumor activity in monotherapy in patients with advanced solid tumors. 4 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. While the DCC gene was initially thought to be rarely mutated in cancers, Krauthammer and colleagues identified the DCC gene as the third most frequently mutated gene in sun-exposed melanoma (23). Based on this observation and as melanoma still represent a significant clinical challenge (24-26), we investigated the role of the netrin-1/DCC pair in this cancer. We reveal that the netrin-1/DCC pair is causally implicated in disease progression and propose that netrin-1 interference could be an attractive therapeutic perspective for treating melanoma. 5 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-18-1590 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods: Patients and tumor samples: 241 patients underwent surgery for skin melanocytic lesions between January 2007 and January 2011 and had enough tissue sample remaining after the diagnosis had been established. According to the French Bioethics law of August 2004, the patients were informed of the research