Published OnlineFirst October 17, 2019; DOI: 10.1158/1078-0432.CCR-19-2154 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors Mari C. Vazquez-Borrego 1,2,3,4, Vandana Gupta5, Alejandro Ibanez-Costa~ 1,2,3,4, Manuel D. Gahete1,2,3,4, Eva Venegas-Moreno6, Alvaro Toledano-Delgado1,3,7, David A. Cano6, Cristobal Blanco-Acevedo1,3,7, Rosa Ortega-Salas1,3,8, Miguel A. Japon 9, Ana Barrera-Martín1,3,10, Alexandre Vasiljevic11,12,13, Jason Hill5, Shengwen Zhang5, Heather Halem5, Juan Solivera1,3,7,Gerald Raverot11,12,14, María A. Galvez 1,3,10, Alfonso Soto-Moreno6, Marcelo Paez-Pereda5, Michael D. Culler5, Justo P. Castano~ 1,2,3,4, and Raul M. Luque1,2,3,4 ABSTRACT ◥ fi Purpose: Somatostatin analogues (SSA) are ef cacious and in response to SST3-agonists. Tumor growth was assessed in a safe treatments for a variety of neuroendocrine tumors, especially preclinical PitNET mouse model treated with a SST3-agonist. fi fi pituitary neuroendocrine tumors (PitNET). Their therapeutic Results: We successfully identi ed the rst SST3-agonist pep- effects are mainly mediated by somatostatin receptors SST2 and tides. SST3-agonists lowered cell viability and chromogranin-A in vitro SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are secretion, increased apoptosis , and reduced tumor growth either nonselective or activate mainly SST2. However, nonfunc- in a preclinical PitNET model. As expected, inhibition of cell fi tioning pituitary tumors (NFPTs), the most common PitNET viability in response to SST3-agonists de ned two NFPT popula- fi type, mainly express SST3 and nding peptides that activate this tions: responsive and unresponsive, wherein responsive NFPTs particular somatostatin receptor has been very challenging. expressed more SST3 than unresponsive NFPTs and exhibited a Therefore, the main objective of this study was to identify profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT SST3-agonists and characterize their effects on experimental signaling pathways upon SST3-agonist treatments. Concurrently, NFPT models. SSTR3 silencing increased cell viability in a subset of NFPTs. Experimental Design: Binding to SSTs and cAMP level deter- Conclusions: This study demonstrates that SST3-agonists acti- minations were used to screen a peptide library and identify SST3- vate signaling mechanisms that reduce NFPT cell viability and agonists. Key functional parameters (cell viability/caspase activ- inhibit pituitary tumor growth in experimental models that ity/chromogranin-A secretion/mRNA expression/intracellular expresses SST3, suggesting that targeting this receptor could be an signaling pathways) were assessed on NFPT primary cell cultures efficacious treatment for NFPTs. (PitNET), where they reduce/normalize hormonal levels, shrink Introduction tumors, and improve clinical symptoms (1, 4). Most of their ther- Somatostatin receptors (SST1–5) comprise a family of seven trans- apeutic actions are assumed to be mediated through SST2 and SST5 membrane G-protein–coupled receptors able to bind and be activated activation (1). Although SSAs are able to bind other SSTs, such by somatostatin (1, 2). SST activation has been widely associated to as SST3, this binding capacity is significantly lower in comparison multiple effects, most of them inhibitory actions on hormone secre- with SST2/SST5 (1). Remarkably, some NETs types express SST3 tion and cellular processes such as proliferation in normal and tumor at higher or similar levels than SST2 or SST5. This is the case for tissues (1). Thus, SSTs are considered as an attractive therapeutic some gastroenteropancreatic NETs (5), pheochromocytomas (6) target to treat different tumor pathologies (1, 3). Clinically available and specially nonfunctioning pituitary tumors (NFPTs), which rep- somatostatin analogues (octreotide/lanreotide/pasireotide) are used resent a heterogeneous group of tumors that constitute 30% of all to treat neuroendocrine tumors (NET), including pituitary NETs PitNETs (7–10). 1Maimonides Institute of Biomedical Research of Cordoba (IMIBIC), Cordoba, Spain. Note: Supplementary data for this article are available at Clinical Cancer 2Department of Cell Biology, Physiology and Immunology, University of Cordoba, Research Online (http://clincancerres.aacrjournals.org/). Cordoba, Spain. 3Reina Sofia University Hospital (HURS), Cordoba, Spain. 4CIBER Physiopathology of Obesity and Nutrition (CIBERobn), Cordoba, Spain. 5IPSEN M.C. Vazquez-Borrego and V. Gupta contributed equally to this article. 6 Bioscience, Cambridge, Massachusetts. Metabolism and Nutrition Unit, Hospital Corresponding Authors: Raul M. Luque, Maimonides Institute of Biomedical Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS), Sevilla, Research of Cordoba (IMIBIC), Cordoba 14004, Spain. Phone: þ34-957-213-740; 7 8 Spain. Service of Neurosurgery, HURS, Cordoba, Spain. Anatomical Pathology Fax: þ34-957-213-740; E-mail: [email protected]; and Justo P. Castano,~ 9 Service, HURS, Cordoba, Spain. Department of Pathology, Hospital Universitario [email protected] Virgen del Rocío, Sevilla, Spain. 10Service of Endocrinology and Nutrition, IMIBIC, 11 HURS, Cordoba, Spain. FacultedeMedecine Lyon Est, UniversiteLyon1,Lyon, Clin Cancer Res 2020;XX:XX–XX France. 12INSERM U1052, CNRS UMR5286, Cancer Research Centre of Lyon, Lyon, France. 13Centre de Pathologie et de Biologie, Groupement Hospitalier Est, Hospices doi: 10.1158/1078-0432.CCR-19-2154 Civils de Lyon, Lyon, France. 14Fed erationd ’endocrinologie, Groupement Hospitalier Est, Hospices Civils de Lyon, Bron, France. Ó2019 American Association for Cancer Research. AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 17, 2019; DOI: 10.1158/1078-0432.CCR-19-2154 Vazquez-Borrego et al. SST3-agonists through the screening of a peptide library; and (ii) Translational Relevance in vitro/in vivo determine the therapeutic potential of SST3 by fi fi fi fi Our results provided the rst successful identi cation of studying: (iiA) the direct effect of the identi ed SST3-speci c fi speci c/functional somatostatin receptor subtype-3 (SST3)- agonists/antagonists on key functional parameters for NFPTs peptidic agonists and antagonists, which demonstrated a key (i.e., cell viability/caspase activity/chromogranin-A (CgA) secre- pathologic function and therapeutic potential of SST3 in non- tion/signaling pathways/mRNA expression) and the functional functioning pituitary neuroendocrine tumor (NFPT) cells consequences of SSTR3 silencing on cell viability; and (iiB) the in vitro in vivo and . Indeed, treatment with SST3-peptidic agon- effect of a selected SST3-specific agonist on tumor growth in a ists and antagonists altered key pathophysiologic parameters in preclinical mouse model of PitNETs. NFPTs in vitro, including cell viability, apoptosis, chromogranin- A release, and expression levels of gonadotropin hormones (through the modulation of relevant signaling pathways), as well Materials and Methods as tumor growth in vivo, in a preclinical model. Importantly, the Reagents results obtained revealed two distinct populations of NFPTs All reagents used in this study were purchased from Sigma-Aldrich, fi (responsive vs. unresponsive NFPTs) in terms of inhibition of unless otherwise speci ed. Subtype selective SST3-agonists (L- aggressiveness features upon SST3-agonist treatment, wherein 796,778/BIM-355/BIM-071) and antagonists (BIM-839/BIM-152, responsive NFPTs expressed more SST3 and exhibited a pro- previously known as BN81658), were generously provided by Merck found reduction of MAPK/PI3K-AKT/mTOR/JAK/STAT sig- & Co., Inc. (L-796,778) or IPSEN Bioscience, Inc. (BIM compounds). naling pathways in response to SST3-agonists. Altogether, the Specificity of the MERCK human agonist was reported previously (20). translational research implications of these findings indicate that Detailed information of the affinity profile [expressed as Ki (nmol/L)] SST3 could have potential value as a therapeutic target in NFPTs and structure of the human SST3 agonists/antagonists is described and in tumor pathologies expressing SST3. in Fig. 1; Supplementary Table S1. Library screening fi The identi cation of compounds that act as selective SST3-agonists Most NFPTs are silent gonadotropinomas characterized by lack of was based on the examination of IPSEN's extensive collection of hormone hypersecretion, which usually determines a delay in diag- synthetic peptides: see Supplementary Methods. nosis (10) and, therefore, are mostly detected as macroadenomas with consequently associated severe comorbidities related to mass effect Radioligand binding assays (i.e., headaches/visual defects/hypopituitarism; refs. 11, 12). Trans- The affinities of the compounds of interest for the different sphenoidal surgery is the first-line therapy and the only current human and mouse SSTs were determined by radioligand binding curative approach of NFPTs; however, it is often not definitive due assays in HEK-293 cells (ATCC) stably transfected with each K to the invasion of surrounding structures (sphenoidal/cavernous receptor. The inhibition constants ( i) were calculated from the K ¼ þ K sinuses) and tumor relapses are frequent even when resection seems following equation: i IC50/(1 L/ d) where L is the radioligand – fi K total (20% 30% of relapses at 10