The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms Federica Felicetti, 1 M. Cristina Errico, 1 Lisabianca Bottero, 1 Patrizia Segnalini, 1 Antonella Stoppacciaro, 2 Mauro Biffoni, 1 Nadia Felli, 1 Gianfranco Mattia, 1 Marina Petrini, 1 Mario P. Colombo, 3 Cesare Peschle, 1 and Alessandra Care ` 1 1 Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore Sanita ` and 2 Department of Histopathology, II Faculty of Medicine and Surgery, Sant’Andrea Hospital, University ‘‘La Sapienza,’’ Rome, Italy, and 3 Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Fondazione Istituti Di Ricovero e Cura a Carattere Scientifico Istituto Nazionale Tumori, Milan, Italy Abstract The incidence of cutaneous melanoma is steadily increasing. Although several molecular abnormalities have been associ- ated with melanoma progression, the mechanisms underlying the differential gene expression are still largely unknown and targeted therapies are not yet available. Noncoding small RNAs, termed microRNAs (miR), have been recently reported to play important roles in major cellular processes, including those involved in cancer development and progression. We have identified the promyelocytic leukemia zinc finger (PLZF) transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region. Spe- cifically, PLZF silencing in melanomas unblocks miR-221 and miR-222, which in turn controls the progression of the neoplasia through down-modulation of p27Kip1/CDKN1B and c-KIT receptor, leading to enhanced proliferation and differentiation blockade of the melanoma cells, respectively. In vitro and in vivo functional studies, including the use of antisense ‘‘antagomir’’ oligonucleotides, confirmed the key role of miR-221/-222 in regulating the progression of human melanoma; this suggests that targeted therapies suppressing miR-221/-222 may prove beneficial in advanced melanoma. [Cancer Res 2008;68(8):2745–54] Introduction Cutaneous melanoma is an aggressive neoplasm refractory to traditional therapies, especially at the metastatic stage. Further- more, its incidence is continuously increasing during the last decade (1). Melanomas develop through a multistep process that from normal melanocytes proceeds to nevi and to radial and vertical growth phase tumors (2). Although several molecular abnormalities have been associated with melanoma progression, as the loss of AP-2 transcription factor (3) or the high mutation rate of the B-RAF oncogene (4), the mechanisms underlying the differential gene expression are still largely unknown and the conventional histologic classification remains the best prognostic factor (5). A new class of small regulatory RNA sequences, termed micro- RNAs (miR), has recently been identified. Although relatively few miR targets have been experimentally validated, growing evidence indicates that miRs play important roles in major cellular processes (e.g., proliferation and differentiation, apoptosis, and angiogenesis) and, as a consequence, their abnormal expression may contribute to cancer development/progression (6, 7). MiR-221 and miR-222 are clustered on the X chromosome and possibly transcribed in a common precursor suggestive of a coor- dinate functional role. They have been reported to be overex- pressed in pancreatic cancer (8), papillary thyroid carcinoma (9), glioblastoma (10, 11), and prostate carcinoma (12). Considering that, in some cases, miR-221 and miR-222 exert their function through c-KIT receptor (9, 13), in view also of c-KIT down- regulation in the majority of invasive and metastatic melanomas (14), we tested whether miR-221 and miR-222 might be directly involved in melanoma pathogenesis. We show that the promyelo- cytic leukemia zinc finger (PLZF), previously reported as a tumor suppressor down-modulated in melanomas (15), is an upstream negative regulator of miR-221 and miR-222 expression. Moreover, we provide evidences of miR-221 and miR-222 capabilities to regulate two distinct but functionally convergent pathways of melanocyte transformation through the cyclin-dependent kinase inhibitor 1B (p27Kip1/CDKN1B) on one side and c-KIT and its downstream genes on the other. Materials and Methods Cell lines culture and transduction. The human melanoma cell lines used in the current study were stabilized from surgical specimens obtained from primary or metastatic tumors at the Istituto Nazionale Tumori in Milan (Italy). Cell lines were characterized for growth in soft agar and, whenever possible, their metastatic potential was evaluated into athymic nude mice. We included in Supplementary Table S1 the stage and a reference for each analyzed cell line. Normal human epidermal melanocytes from the foreskin were obtained from Promocell. The PLZF cDNA encompassing its complete coding sequence was cloned into the retroviral vector LXSN as described (15). ‘‘Control’’ cell lines are always empty vector transduced. Overexpression of miR-221 and miR-222 was obtained in melanoma cells by using a lentiviral vector system according to standard techniques (13). MiR-221 and miR-222 silencing by antagomir treatment. Chemically modified antisense oligonucleotides (antagomir) have been used to inhibit miR expression in vitro and in vivo (16, 17). The sequences of antagomir- 221 and antagomir-222 used are as follows: 5¶P-GAAACCCAGCAGAC- AAUGUAGCU-3¶-Chl and 5¶P-GAGACCCAGUAGCCAGAUGUAGCU-3¶-Chl, respectively; all the bases were 2¶-OMe modified. Antagomir oligonucleo- tides, deprotected, desalted, and purified by high-performance liquid Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Alessandra Care `, Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore Sanita `, Viale Regina Elena, 299-00161 Rome, Italy. Phone: 39-06-49902411; Fax: 39-06-49387087; E-mail: alessandra.care@iss.it. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-2538 www.aacrjournals.org 2745 Cancer Res 2008; 68: (8). 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