1408 INTERNATIONAL JOURNAL OF ONCOLOGY 42: 1408-1416, 2013 PRAF2 stimulates cell proliferation and migration and predicts poor prognosis in neuroblastoma LISETTE P. YCO1,2,4, DIRK GEERTS5,6, JAN KOSTER5 and ANDRÉ S. BACHMANN1-4 1Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720; 2University of Hawaii Cancer Center and 3Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813; 4Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA; 5Department of Human Genetics, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam; 6Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands Received July 19, 2012; Accepted September 7, 2012 DOI: 10.3892/ijo.2013.1836 Abstract. Prenylated Rab acceptor 1 domain family, member 2 roblastomas, form the neuroblastic tumors. They are all derived (PRAF2) is a novel 19-kDa protein with four transmembrane- from migratory, primitive precursor cells of the developing spanning domains that belongs to the PRAF protein family. sympathetic nervous system. NB tumors are graded according to Neuroblastoma (NB) is the most common malignant extracra- INSS stage, where stages 1 and 2 represent low-risk tumors and nial solid tumor of childhood that originates in primitive cells stages 3-4 high-risk tumors (1,2). Children that are diagnosed of the developing sympathetic nervous system. We investigated with NB when they are over 1 year of age generally suffer from the correlation of PRAF2 mRNA expression to NB clinical the advanced stages of the disease. These NB tumors, especially and genetic parameters using Affymetrix expression analysis stage 4, can rapidly spread to regional lymph nodes and then to of a series of 88 NB tumors and examined the functional role distal bone and bone marrow, resulting in very poor patient prog- of PRAF2 in an NB cell line using RNA interference. We nosis. Infants diagnosed with NB can present a unique pattern found that high PRAF2 expression is significantly correlated to of metastatic spread (stage 4S, S = Special) to the liver and skin, several unfavorable NB characteristics: MYCN amplification, but only rarely to bone or bone marrow. Remarkably, these high age at diagnosis, poor outcome and high INSS stage. The metastasized tumors usually undergo spontaneous regression shRNA-mediated PRAF2 downregulation in the SK-N-SH NB and patients have excellent prognosis (1,2). Important prognostic cell line resulted in decreased cellular proliferation, migration features of NB tumors are therefore INSS stage and patient age and matrix-attachment. These findings were confirmed in at diagnosis. In addition, recurrent genomic aberrations like NB patient tumor samples, where high PRAF2 expression is MYCN gene amplification can be used for patient stratification significantly correlated to bone and bone marrow metastasis, (1,2). Unfortunately, these combined NB features are still too the main cause of death in NB patients. The present study limited to ensure early proper treatment stratification and new shows that PRAF2 plays an essential role in NB tumorigenesis prognostic markers are needed. In addition, NB treatment, espe- and metastasis. cially of metastasized tumors is still not sufficiently efficient and more insight is needed into the molecular pathways involved in Introduction NB metastasis. Prenylated Rab acceptor 1 domain family, member 2 Neuroblastoma (NB) is the most common and deadly extra- (PRAF2, initially described as JM4) is a 19-kDa protein with cranial solid tumor of childhood. NBs, together with the more four transmembrane-spanning domains. PRAF2 belongs to the differentiated, less aggressive ganglioneuromas and ganglioneu- PRAF family of vesicle transport-associated proteins. Members of the PRAF family are structurally related and contain a large prenylated Rab acceptor 1 (PRA1) domain. These PRAF proteins are localized in the ER, Golgi and vesicular structures of the cell (3,4). PRAF2 was first discovered as an interacting Correspondence to: Dr André S. Bachmann, Department of Phar- protein of human chemokine receptor 5 (CCR5) (3) and of maceutical Sciences, College of Pharmacy, University of Hawaii at human glycerophosphoinositol phosphodiesterase (GDE1/ Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA MIR16) (5). Human PRAF2 is highly expressed in numerous E-mail: [email protected] tissues: small intestine, lung, spleen, pancreas and most signifi- Key words: cell proliferation, metastasis, neuroblastoma, patient cantly in the brain. Furthermore, overexpression of PRAF2 is survival, pediatric cancer, prenylated Rab acceptor 1 domain family, observed in tumor tissue samples of breast, colon, lung and member 2 ovary, compared to matched normal tissue samples (4,6,7). The other two members of PRAF protein family (PRAF1 and PRAF3) have already been extensively studied and their YCO et al: PRAF2 IN NEUROBLASTOMA TUMORIGENESIS AND METASTASIS 1409 biological function has been characterized. In fact, many of these against PRAF2 (shPRAF2) and GFP (scrambled; as a nega- studies associated PRAF1 and PRAF3 with several well-known tive control) were purchased from Origene Technologies cancer signaling pathways. For instance, PRAF1 (also known as (Rockville, MD). SK-N-SH cells were seeded in 6-well culture Rabac1, PRA1, prenylin and Yip3), a Golgi complex, post-Golgi plates (Greiner Bio-One Inc., Monroe, NC) at a concentration vesicle and endosomal transmembrane protein, was initially of 5.0x105 cells per well and transfected with 4 µg shPRAF2 identified to interact with small GTPase proteins that regulate or scrambled plasmid using Lipofectamine 2000 (Invitrogen). intracellular vesicle trafficking (8-10). In addition, PRAF1 has a Transfected cells were incubated for 24 h at 37˚C as described crucial role in colorectal tumorigenesis by regulating the nuclear above and subsequently selected using 1.0 µg/ml puromycin transport of β-catenin in TCF/β-catenin signaling (11). PRAF1 (Sigma-Aldrich). After selection for 2 weeks, cells retaining functions in Epstein-Barr virus (EBV)-induced transformation: the shPRAF2 (SK-shPRAF2) or scrambled expression vector it binds and modulates the anti-apoptotic activity of BHRF1, an (SK-Sc) were maintained at 100 ng/ml puromycin. EBV-encoded early protein and enhances nuclear import of the oncogenic EBV-encoded latent membrane protein 1 (LMP1) Western blot analysis. For total protein isolation, medium was (12,13). removed by aspiration, adherent cells were washed twice in ice- PRAF3 (also known as JWA, GTRAP3-18 and Arl6-IP5) cold DPBS (Dulbecco's phosphate buffered saline, Mediatech) is an integral ER membrane protein that is upregulated by and cell lysates prepared by scraping on ice into radioim- retinoic acid (RA) and then activates the excitation amino acid munoprecipitation assay (RIPA) buffer [20 mM Tris-HCl carrier 1 (EAAC1), a primary neuronal glutamate transporter (pH 7.5), 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium (14,15). PRAF3 is also defined as a potential inhibitor of cancer deoxycholate, 135 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) cell migration by regulation of the MAPK signaling pathway glycerol, 2 mM EDTA], supplemented with Complete Protease and F-actin cytoskeleton (16). PRAF3 was found to inhibit Inhibitor Cocktail (Roche Diagnostic Corp., Indianapolis, melanoma metastasis by suppressing integrin αvβ3 signaling, IN) and phosphatase inhibitors (20 mM sodium fluoride and a key factor in tumor metastasis. Downregulation of PRAF3 in 0.27 mM sodium vanadate). Lysate samples were resuspended melanoma cells significantly increased integrin αvβ3 expres- by rotation at 50 rpm and 4˚C for at least 30 min and clari- sion (both at mRNA and protein level) and accelerated cell fied by centrifugation for 15 min at 14,000 rpm and 4˚C. The adhesion, migration and invasion in vitro and enhanced mela- supernatant was stored at - 20˚C until further use. Total protein noma lung metastasis in vivo. concentration was determined by Bradford dye reagent protein Unlike PRAF1 and PRAF3, PRAF2 has not yet been assay (Bio-Rad Laboratories Inc., Richmond, CA). Laemmli widely studied and its functional role therefore remains largely buffer (Bio-Rad) containing 10% (v/v) β-mercaptoethanol unknown. Previously, our lab found PRAF2 as a candidate (VWR International, Brisbane, CA) solution was added to the prognostic marker of neuroblastic tumors. We observed high lysates and boiled for 5 min. Ten micrograms total protein was level of PRAF2 mRNA in most neuroblastic tumor samples. resolved by 12% sodium dodecyl sulfate-polyacrylamide gel We also found that PRAF2 protein is strongly expressed in all electrophoresis (SDS-PAGE) and electrotransferred onto a 24 NB cell lines tested, including SK-N-SH (17). Despite these polyvinylidene difluoride (PVDF) membrane (Immobilon-P, discoveries, the molecular role of PRAF2 in NB tumorigenesis, VWR International). Membranes were incubates with rabbit or in other tumors in which it is highly expressed, has remained polyclonal PRAF2, peptide-blocked rabbit polyclonal PRAF2 unexplored. Therefore, in this study, we generated NB cell (PRAF2-P) (QED Bioscience, San Diego, CA), or rabbit lines that stably downregulate endogenous PRAF2 expression monoclonal α-tubulin (Cell Signaling Technology, Danvers, by RNA interference (RNAi). We used these PRAF2 knock-