Small Molecule Therapeutics Anti-hTERT siRNA-Loaded Nanoparticles Block the Growth of Anaplastic Thyroid Cancer Xenograft Giovanni E. Lombardo 1 , Valentina Maggisano 1 , Marilena Celano 1 , Donato Cosco 1 , Chiara Mignogna 1,2 , Federica Baldan 3 , Saverio M. Lepore 1 , Lorenzo Allegri 4 , Sonia Moretti 5 , Cosimo Durante 3 , Giuseppe Damante 4 , Massimo Fresta 1 , Diego Russo 1 , Stefania Bulotta 1 , and Efisio Puxeddu 5 Abstract The high frequency of hTERT-promoting mutations and the increased expression of hTERT mRNA in anaplastic thyroid cancer (ATC) make TERT a suitable molecular target for the treatment of this lethal neoplasm. In this study, we encapsulated an anti-hTERT oligonucleotide in biocompatible nanoparticles and analyzed the effects of this novel pharmaceutical prepara- tion in preclinical models of ATC. Biocompatible nanoparticles were obtained in an acidified aqueous solution containing chitosan, anti-hTERT oligoRNAs, and poloxamer 188 as a stabi- lizer. The effects of these anti-hTERT nanoparticles (Na-siTERT) were tested in vitro on ATC cell lines (CAL-62 and 8505C) and in vivo on xenograft tumors obtained by flank injection of CAL-62 cells into SCID mice. The Na-siTERT reduced the viabi- lity and migration of CAL-62 and 8505C cells after 48-hour incubation. Intravenous administration (every 48 hours for 13 days) of this encapsulated drug in mice hosting a xenograft thyroid cancer determined a great reduction in the growth of the neoplasm (about 50% vs. untreated animals or mice receiv- ing empty nanoparticles), and decreased levels of Ki67 associ- ated with lower hTERT expression. Moreover, the treatment resulted in minimal invasion of nearby tissues and reduced the vascularity of the xenograft tumor. No signs of toxicity appeared following this treatment. Telomere length was not modified by the Na-siTERT, indicating that the inhibitory effects of neo- plasm growth were independent from the enzymatic telomerase function. These findings demonstrate the potential suitability of this anti-TERT nanoparticle formulation as a novel tool for ATC treatment. Mol Cancer Ther; 17(6); 1187–95. Ó2018 AACR. Introduction An increased prevalence of thyroid cancer has been observed in the past decade (1, 2). Although the majority of these neoplasms are differentiated thyroid carcinomas (DTC) and have a good prognosis, poorly differentiated (PDTC) and ana- plastic thyroid cancers (ATC), which are often present with recurrent and metastatic lesions, are responsible for the mor- tality rate still observed for these tumors (1). Indeed, the current treatment (surgery and radioiodine administration) is effective only for those DTCs able to concentrate the radioiodine (3), so other strategies are currently under investigation and have shown promising results in patients with DTCs unresponsive to radioiodine, but a lesser degree of success has been had in the treatment of PDTCs and ATCs (4–6). In the search for novel molecular targets, valid help comes from the genomic analysis of a large number of human tumors. Studies on ATCs have revealed that telomerase reverse tran- scriptase (TERT) gene alterations are the most frequent event detected in this type of neoplasia (5, 7–10). In addition, overexpression of TERT has been reported in thyroid carcino- mas, lymph node metastases, and, more recently, even in ATCs (11, 12). Thus, hTERT has been proposed as an optimal target for the treatment of thyroid cancer, and in vitro studies have shown that the silencing of hTERT by oligoRNAs (siRNA) was able to reduce the growth, invasion, and migratory ability of ATC cells, which did not depend on whether or not they were carrying hTERT promoter mutations (12, 13). For potential application of this strategy in clinical practice, however, it is crucial that the rapid degradation of siRNA by extracellular RNases be avoided (14); so, various strategies have been tested to entrap genetic material within biocompatible systems able to promote efficient cellular uptake while preserving its pharmacologic activity (15). In this study, an anti-hTERT oligonucleotide was encapsu- lated in biocompatible nanoparticles made up of poly(D,L- 1 Department of Health Sciences, "Magna Graecia" University of Catanzaro, Catanzaro, Italy. 2 Interdepartmental Service Center, "Magna Graecia" University of Catanzaro, Catanzaro, Italy. 3 Department of Internal Medicine and Medical Specialties, University of Roma "Sapienza," Roma, Italy. 4 Department of Medical Area, University of Udine, Udine, Italy 5 Department of Medicine, University of Perugia, Perugia, Italy. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). G.E. Lombardo, V. Maggisano, and M. Celano contributed equally to this article. Corresponding Authors: Giuseppe Damante, Department of Medical Area, University of Udine, Udine 33100, Italy. Phone: 3904-3255-4359; Fax: 3904- 3255-4359; E-mail: giuseppe.damante@uniud.it; and Diego Russo, Department of Health Sciences, University of Catanzaro "Magna Graecia," Viale Europa, Germaneto 88100, Catanzaro, Italy. Phone: 3909-6136-4124; E-mail: d.russo@unicz.it doi: 10.1158/1535-7163.MCT-17-0559 Ó2018 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 1187 on May 20, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst March 21, 2018; DOI: 10.1158/1535-7163.MCT-17-0559