[CANCER RESEARCH 64, 1730 –1736, March 1, 2004] Inhibition of Connective Tissue Growth Factor (CTGF/CCN2) Expression Decreases the Survival and Myogenic Differentiation of Human Rhabdomyosarcoma Cells Stefania Croci, 1 Lorena Landuzzi, 1,2 Annalisa Astolfi, 1 Giordano Nicoletti, 1,2 Angelo Rosolen, 3 Francesca Sartori, 3 Matilde Y. Follo, 1 Noelynn Oliver, 4 Carla De Giovanni, 1 Patrizia Nanni, 1 and Pier-Luigi Lollini 1 1 Cancer Research Section, Department of Experimental Pathology, University of Bologna, Bologna, Italy; 2 Istituti Ortopedici Rizzoli, Bologna, Italy; 3 Section of Pediatric Hemato-Oncology, Department of Pediatrics, University of Padua, Padua, Italy; and 4 FibroGen Inc., South San Francisco, California ABSTRACT Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microar- ray screen of genes expressed by human rhabdomyosarcoma cells. Rhab- domyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomy- osarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhab- domyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhab- domyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleo- tide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma. INTRODUCTION Connective tissue growth factor (CTGF or CCN2) is a cysteine-rich protein originally identified in conditioned medium of human umbil- ical vein endothelial cells (1). It belongs to the CCN family of genes, which is composed of five other members: Cyr61 (cysteine-rich protein 61), Nov (nephroblastoma overexpressed gene), WISP (Wnt- 1-induced secreted protein)-1, WISP-2, and WISP-3 (2, 3). These proteins share an NH 2 -terminal secretory signal peptide and four conserved domains with sequence similarities to insulin-like growth factor (IGF)-binding proteins, von Willebrand type C factor, throm- bospondin 1, and a cysteine knot characteristic of other growth fac- tors, including platelet-derived growth factor, nerve growth factor, and transforming growth factor (TGF)- (4). CTGF is involved in many biological processes such as cell pro- liferation, survival, migration, differentiation, and angiogenesis and plays a role in osteogenesis, in chondrogenesis, in the development of vasculature, in placentation (5), and in wound healing (6). Moreover, CTGF is expressed in various pathological conditions: fibrosis (7, 8), scleroderma (9), atherosclerosis (10), and renal diseases (11), in which it mainly acts as a TGF- downstream mediator. The function of CTGF in human cancer is still unclear (12). Its angiogenic activity suggests a role for CTGF in tumor growth and vascularization (13). CTGF expression has been found in various tumors of mesenchymal, epithelial, and lymphoid origins (14 –24) possibly with different roles in each tumor type. Overexpression of CTGF gene in primary breast cancer was associated with an advanced stage of disease (21); however, functional studies in the human breast cancer cell line MCF-7 showed that CTGF promotes apoptosis (25). In cartilaginous tumors and in fibroblast- and endothelial-cell-derived tumors, inverse correlations were reported between malignant pheno- type and the level of CTGF expression (14, 19). CTGF emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells previously conducted in our labora- tory (26). Rhabdomyosarcoma is the most common soft tissue sar- coma of childhood. It is committed to the myogenic lineage but cannot complete the myogenic developmental program (27). The role of CTGF in human rhabdomyosarcoma or skeletal muscle cells is pres- ently unknown. In this paper, we investigated the presence of an autocrine loop based on CTGF in human rhabdomyosarcoma and its effects on proliferation, survival, and myogenic differentiation. MATERIALS AND METHODS Cell Lines. Six human rhabdomyosarcoma cell lines of the two main histotypes were used: RD/12 and RD/18, two clones of the RD cell line (28), and CCA (29), derived from tumors of the embryonal histotype; and RMZ- RC2 (30), SJ-RH4, and SJ-RH30 (31), derived from tumors of the alveolar histotype. Cells were routinely cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum in a 7% CO 2 humidified atmosphere at 37°C. Culture media were purchased from Invitrogen (Milan, Italy). Myogenic Differentiation. RD/18 and RMZ-RC2 can differentiate along the myogenic pathway if cultured in a medium with a low-serum supplement (DMEM plus 2% horse serum; Refs. 26, 30). The level of myogenic differ- entiation was determined by indirect immunofluorescence on cytocentrifuged samples, fixed with ethanol:acetone (3:7) at -20°C, using the antiembryonic myosin monoclonal antibody BF-G6 (32) as reported previously (30). The percentage of myosin-positive cells was determined in a Leica DM micro- scope. For each sample, at least 400 cell elements in random fields were analyzed. Expression of CTGF. Total RNA was extracted from cultured cells and from surgical specimens by Tri-Zol reagent (Invitrogen, Milan, Italy). One g of RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase in the presence of oligo-dT and dNTP (Invitrogen). cDNA (1 l) was amplified using TaqPlatinum DNA polymerase in a final volume of 25 l with 1 mM MgCl 2 and dNTP (0.2 M each) and specific primer pairs (0.5 M each) for CTGF (direct, 5'-GCATCCGTACTCCCAAAATCTC-3'; reverse, 5'-ATGTCTCTCACTCTCTGGCTTC-3') and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech, Palo Alto, CA). The amplification was carried out as follows: 95°C for 30 s, 60°C for 30 s, 72°C for 1 min (20 –25 cycles for GAPDH and 25–30 cycles for CTGF). The amplification products (452 bp for GAPDH and 304 bp for CTGF) were visualized on ethidium bromide-stained agarose gels. CTGF expression was also analyzed by quantitative real-time PCR using an ABI Prism 5700 sequence detection system (Applied Biosystems, Milan, Italy). Real-time PCR was performed using Taqman Universal PCR Master Mix Reagents (Applied Biosystems). CTGF primers and probes were designed using Primer Express software version 2.0: direct, 5'-AGCCGCCTGTG- CATGGT-3'; reverse, 5'-GGGAGTACGGATGCACTTTTTG-3'; and MGB- probe 5'-FAM-CCTTGCGAAGCTGAC-minor groove binder/nonfluorescent quencher 3'. GAPDH was used as an endogenous reference gene (TaqMan Received 12/12/02; revised 12/24/03; accepted 1/7/04. Grant support: Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, and the Italian Ministry of University and Research. S. Croci and A. Astolfi are recipients of fellowships from the Italian Foundation for Cancer Research (F.I.R.C.), Milan, Italy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Pier-Luigi Lollini, Sezione di Cancerologia, Viale Filopanti 22, I-40126 Bologna, Italy. E-mail: pierluigi@lollini.dsnet.it. 1730 Research. on May 1, 2017. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from