Expression of a MYCN-Interacting Isoform of the Tumor Suppressor BIN1 Is Reduced in Neuroblastomas with Unfavorable Biological Features1

Vol. 9, 3345–3355, August 15, 2003 Clinical Cancer Research 3345

Expression of a MYCN-interacting Isoform of the Tumor Suppressor BIN1 Is Reduced in Neuroblastomas with Unfavorable Biological Features1

Tatsuro Tajiri, Xueyuan Liu, pression of BIN1 was lower in: NBs with MYCN amplifica- compared with those without, P < 0.03; in (10 ؍ Patricia M. Thompson, Shinji Tanaka, tion (n ؍ Sachiyo Suita, Huaqing Zhao, John M. Maris, International Neuroblastoma Risk Group high-risk NB (n 2 19) compared with low- or intermediate-risk NB, P < 0.01; ,compared with localized NB (21 ؍ George C. Prendergast, and Michael D. Hogarty and in metastatic NB (n Divisions of Oncology [X. L., P. M. T., J. M. M., M. D. H.] and P < 0.06. BIN1 inactivation by deletion or genomic rear- Biostatistics and Epidemiology [H. Z.], The Children’s Hospital of rangement was identified infrequently. Forced expression of Philadelphia, Philadelphia, Pennsylvania 19104-4318; Department of Pediatrics, University of Pennsylvania School of Medicine, BIN1 isoforms containing the Myc-binding domain (with or Philadelphia, Pennsylvania 19104 [J. M. M., M. D. H.]; Department of without exon 12A) inhibited colony formation in NB cell Pediatric Surgery, Graduate School of Medical Sciences, Kyushu lines with MYCN amplification (P < 0.01) but not in those University, Fukuoka, Japan 812-8582 [T. T., S. T., S. S.]; and without. Forced expression of BIN1 isoforms with a MBD Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096 [G. C. P.] deletion did not inhibit colony formation in any cell line assessed. Conclusions: These data support that reduced BIN1 ABSTRACT expression contributes to the malignant phenotype of child- Purpose: Amplification of the MYCN proto-oncogene is hood NB. As we reported previously, BIN1 may function to strongly correlated with poor outcome in neuroblastoma circumvent MycN-mediated apoptosis in NBs with MYCN (NB), although deregulated MYCN is a potent inducer of amplification. apoptosis. BIN1 (2q14) encodes multiple isoforms of a Myc- interacting adaptor protein that has features of a tumor INTRODUCTION suppressor, including the ability to inhibit Myc-mediated NB3 is the most common solid tumor of childhood and is cell transformation and to promote apoptosis. We hypothe- responsible for 15% of childhood cancer-related deaths (1, 2). sized that BIN1 may function as a suppressor gene in NB, The hallmark of NB is clinical heterogeneity with some tumors because Bin1 is highly expressed in neural tissues and binds regressing spontaneously, especially in infants, whereas others the Myc Box motifs that are conserved in MycN. differentiate into benign ganglioneuromas with or without treat- Experimental Design: Expression of MYCN, total BIN1, ment. Unfortunately, the majority of patients presenting with and BIN1 isoforms were determined in 56 primary NBs NB are over 1 year of age with locally aggressive and/or using the real-time PCR. Expression was correlated with disseminated disease that is rapidly progressive and resistant to biological and genetic features. To determine the functional therapy. These disparate tumor behaviors may in part be pre- significance of BIN1 expression we ectopically expressed dicted by clinical variables such as age (3), extent of disease (4), BIN1 isoforms in NB cell lines with and without MYCN and histopathologic classification (5); as well as by tumor- amplification, and assessed clonogenic growth. specific genetic characteristics such as amplification of MYCN Results: Four predominant BIN1 isoforms resulting (6, 7), TRK receptor expression (8), ploidy (9), loss of the distal from alternative splicing of exon 12A (a neural tissue-spe- short arm of chromosome 1 (10, 11), and unbalanced gain of cific exon) and exon 13 (a Myc-binding domain encoding 17q (12). Of these, MYCN amplification is the most robust exon) were variably expressed in the 56 primary NBs. Ex- predictor of disease behavior and the only specific genetic alteration used currently in the majority of risk-stratification schemas. MYCN amplification occurs in ϳ25% of primary NBs and Received 11/25/02; revised 4/7/03; accepted 4/8/03. is invariably accompanied by elevated MycN protein expression The costs of publication of this article were defrayed in part by the (13). It is strongly associated with the presence of metastatic payment of page charges. This article must therefore be hereby marked disease (6) and independently predictive of death from tumor advertisement in accordance with 18 U.S.C. Section 1734 solely to progression (7). The malignant phenotype MYCN amplification indicate this fact. 1 Supported by a Research Fellowship of the Uehara Memorial Foun- dation (to T. T.), by Career Development Awards from the Burroughs Wellcome Fund (to M. D. H.) and the American Society of Clinical Oncology (to M. D. H.), and by the Richard and Sheila Sanford Chair in 3 The abbreviations used are: NB, neuroblastoma; MBD, Myc-binding Pediatric Oncology. domain; INRG, International Neuroblastoma Risk Group; INSS, Inter- 2 To whom requests for reprints should be addressed, at The Children’s national Neuroblastoma Staging System; CMV, cytomegalovirus; Hospital of Philadelphia, Division of Oncology, 9 North ARC, 3615 Civic GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LOH, loss of het- Center Boulevard, Philadelphia, PA 19104-4318. Phone: (215) 590-2821; erozygosity; BIN1, bridging integrator-1; BAR, Bin1, Amphiphysin, Fax: (215) 590-3770; E-mail: [email protected]. RVS167; RT-PCR, reverse transcription-PCR.

portends has been presumed to be a result of unencumbered cell expression is reduced in NB cell lines with MYCN amplification proliferation driven by the transcriptional activity of deregulated compared with NB cell lines without MYCN amplification (27). MycN. Yet it is increasingly clear that deregulated Myc proteins This deficit was of functional significance as forced expression (including MycN) are potent inducers of programmed cell death of full length BIN1 inhibited colony formation selectively in when expressed in the absence of mitogenic signals or in the MYCN amplified cell lines through the induction of pro- presence of genotoxic stress (14). This apoptotic signal may be grammed cell death. blocked by constitutive survival signals or rendered ineffectual In the present study we have identified the BIN1 isoforms by loss of apoptotic pathways, as has been shown frequently in that are expressed in primary NBs. The expression level of BIN1 malignant cells with deregulated Myc expression. We similarly isoforms has been determined in 56 primary NBs diagnosed hypothesize that loss of engagement or execution of this apop- both clinically and through a urine catecholamine-screening totic signal is a requirement for neoplastic neuroblasts with program. We have correlated BIN1 expression with MYCN deregulated MYCN to survive and maintain a proliferative state expression, and with clinical and biological features of the after genotoxic stress or in a mitogen-poor environment. The tumors (including INRG classification, INSS tumor stage, age at mechanisms whereby deregulated MYC primes cells for apo- diagnosis, and MYCN gene status and expression level). The ptosis remain poorly characterized but may involve direct tran- functional role of these isoforms was investigated after transient scriptional activation of proapoptotic genes such as BAX, as well transfection into NB cell lines with and without amplification of as indirect activation of BIM and/or p14/ARF (15, 16). These the MYCN proto-oncogene, and initial mutation analyses were critical pathways have not been fully characterized with dereg- performed. We present data correlating reduced expression of ulated MYC and are less well understood for MYCN. MycN-interacting BIN1 isoforms with unfavorable features in BIN1 maps to the long arm of human chromosome 2 (2q14) primary NB including amplification of the MYCN proto-onco- and encodes multiple tissue-specific isoforms of a Myc-inter- gene, INRG high-risk disease, age Ͼ1-year, and metastatic acting adaptor protein implicated in tumor suppression and cell disease. This reduction in BIN1 expression may be of functional death processes in malignant human cells. BIN1 has homology significance, as forced ectopic expression of these isoforms to amphiphysin (a breast carcinoma-related autoantigen respon- (containing an intact MBD but not those deleting this domain) sible for “stiff man” syndrome) and RVS167, a cell-cycle reg- potently and selectively inhibited colony formation in NB cell ulator in yeast (17, 18). This homology is restricted to the lines with MYCN amplification. NH2-terminal BAR domain (Fig. 1). Additionally, BIN1 contains a neuron-specific endocytic-function domain (NTS) encoded by exons 12A, 12B, 12C, and 12D; a MBD encoded by MATERIALS AND METHODS exons 13 and 14; and a COOH-terminal SH3 domain encoded Patients and Tumor Sample Preparation. Fifty-six tu- by exons 15 and 16 (18, 19). Multiple ubiquitous and tissue- mor samples were obtained from previously untreated patients specific alternatively spliced isoforms of BIN1 have been iden- with NB (Department of Pediatric Surgery, Kyushu University). tified. The ubiquitously expressed isoforms omit exon 10 (which The clinical features of the 56 NB patients are shown in Table is expressed only within muscle-derived tissues) and exons 1. Ten tumors (18%) demonstrated amplification of the MYCN 12A–D (which encode a domain important in mediating synap- proto-oncogene by Southern blot methodology as described tic vesicle function and are found mainly in brain; also called previously (28). Twenty-nine cases (52%) were detected amphiphysin-II isoforms; Refs. 18, 20–23). Alternate splicing through mass screening at 6 months of age. All of the studies also contributes to the transcription of isoforms omitting exon were approved by the Institutional Review Board at The Chil- 13 predicted to abrogate direct Myc or MycN interactions, dren’s Hospital of Philadelphia. Tumors were snap-frozen at the because it encodes approximately half of the MBD. This may be time of surgery, and total RNA was extracted using Isogen LS relevant to tumorigenesis, because Myc-interacting isoforms (Nippon Gene, Osaka, Japan) according to the manufacturer’s have been demonstrated to have tumor suppressor function, instructions. Human fetal brain RNA (Clontech, Palo Alto, CA) and may mediate cell differentiation and death decisions (17, was used as a control for expression analyses. Reverse- 21, 24). transcribed cDNA was generated from 1 ␮g of total RNA in a BIN1 isoforms, which include the MBD, have been shown final volume of 20 ␮l using the First-Strand cDNA Synthesis kit to physically interact with Myc in vitro and in vivo (17, 21). with random hexamer priming according to the manufacturer’s BIN1 potently inhibits transformation when coexpressed with instructions (Amersham Pharmacia, Piscataway, NJ). MYC and activated RAS in rat embryo fibroblasts (17). BIN1 Real-time PCR for Total BIN1 and MYCN Expression. expression is reduced or absent in many tumor-derived cell lines Expression of total BIN1 (all of the isoforms together) and and primary tumors (including melanoma, breast cancer, and MYCN were determined using real-time PCR (TaqMan; PE prostate cancer), and forced re-expression of BIN1 in these cell Biosystems, Foster, CA). Primers and probes for BIN1 and lines results in apoptosis and reduced clonogenicity (20, 25, 26). MYCN were designed using Primer Express v2.0 (PE Biosys- Together these data suggest that BIN1 may function as a tumor tems). We targeted sequences within exon 9 and 11 to analyze suppressor through inhibition or alteration of specific Myc func- total BIN1 expression, because this region shares no homology tions. Because BIN1 is highly expressed in neural tissues, func- with amphiphysin. The forward BIN1 primer (5Ј-AAG GCC tionally interacts with Myc within a domain with 100% identity CAG CCC AGT GAC-3Ј) is complimentary to sequences at the to MycN, and inhibits Myc-mediated transformation, we hy- boundary of exons 9 and 11, whereas the reverse BIN1 primer pothesized that BIN1 might function as a NB suppressor through (5Ј-GAG CCA TCT GGA GGC GAA G-3Ј) is complimentary interactions with MycN. We have shown previously that BIN1 to sequences within exon 11. The BIN1 probe is carboxyfluo-

Fig. 1 The organization of the BIN1 polypeptide with respect to functional protein domains is shown, as well as the exon structure encoding these domains (A). U1 and U2, unique regions of functionally undefined sequence that are not conserved in amphiphysin or RVS167; NTS, neural-tissue specific domain (encoded within exons 12A-12D); SH3, Src homology-3 domain. B, alternate splicing of exons 10, 12A, and 13. Four prevalent BIN1 isoforms were identified in NB cell lines and primary tumors. No BIN1 isoforms containing exon 10, which is expressed uniquely within muscle tissue, could be detected. BIN1(Ϫ10Ϫ13) and BIN1(Ϫ10) are ubiquitously expressed isoforms. Inclusion of exon 12A, as occurs with two additional isoforms expressed in the majority of NBs, is typically demonstrated in tissues of neural origin (isoforms containing exons 12A-12D, which were infrequently demonstrated in NBs, are also referred to as amphiphysin II). C, representative RT-PCR using BIN1 primers as described, demonstrating the relative expression of various BIN1 isoforms in NBs and fetal brain cDNA. Absolute expression values were derived from these isoform ratios and total BIN1 expression obtained using real-time PCR, as described in “Materials and Methods.”

rescein-5Ј-CGC GCC TGC AAA AGG GAA CAA GA-3Ј- forms were detected in any NB cell line or primary tumor carboxytetramethylrhodamine. These primers and probe were analyzed previously using RT-PCR with primers flanking or designed to quantitate all of the BIN1 isoforms except those within exon 10 (data not shown). containing exon 10 (spiking experiments using pCMV-BIN1 The MYCN forward primer (5Ј-GAC CAC AAG GCC containing exon 10 confirmed that this assay did not detect exon CTC AGT ACC-3Ј) is complimentary to sequences within 10 containing isoforms). Of note, no exon 10 containing iso- MYCN exon 2, whereas the reverse MYCN primer (5Ј-TGA

␮ ␮ Table 1 Clinical features of 56 primary neuroblastoma patients 1.5 mM MgCl2, 0.4 M of each primer, and 1 l of cDNA. The Clinical Screening Total samples were first denatured at 95°C for 10 min, followed by 30 diagnosis diagnosis (n ϭ 56) cycles of 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s. The Age (years) final cycle was followed by a 5-min extension step at 72°C. Ͻ1 7 27 34 (61%) Each PCR sample was electrophoresed through a nondenaturing Ն1 20 2 22 (39%) 7% polyacrylamide gel, transferred to a nylon membrane (Hy- INSS stage bond Nϩ; Amersham Pharmacia), and immobilized by UV 1 3 20 23 (41%) cross-linking. Chemiluminescent detection of biotin-labeled 2 1 5 6 (11%) 3 3 3 6 (11%) PCR products was performed using the Southern Light Detec- 4 15 0 15 (26%) tion System (Tropix, Bedford, MA) and captured on 4S 5 1 6 (11%) X-Omat-AR film (Eastman Kodak, Rochester, NY). Band im- MYCN status ages were captured with a Model 4910 high-performance CCD Not amplified 18 28 46 (82%) Amplified 9 1 10 (18%) camera and analyzed using NIH Image v1.55 software. For each INRG risk group isoform, expression was calculated as the relative proportion of Low or intermediate 9 28 37 (63%) that isoform determined by densitometry using RT-PCR as High 18 1 19 (34%) above (intensity of specific isoform divided by the sum of the intensities of all present BIN1 isoforms) times the total BIN1 expression value for that sample determined by the real-time PCR assay. CCA CGT CGA TTT CTT CCT-3Ј) is complimentary to se- Cell Culture, Plasmids, and Colony Formation Assays. quences within exon 3. The MYCN probe (carboxyfluorescein- The NB cell lines used have been described previously (29–32). 5Ј-CCG GAG AGG ACA CCC TGA GCG A-3Ј-carboxytetra- All of the cell lines were grown in RPMI 1640 (Life Technol- methylrhodamine) hybridizes within exon 2. GAPDH was used ogies, Inc., Gaithersburg, MD) supplemented with 10% fetal as an internal control for these analyses. PCR primers and probe bovine serum, 2 mML-glutamine, 1% OPI media supplement for GAPDH (TaqMan GAPDH Control Reagent) were pur- (Sigma-Aldrich, USA) 100 units/ml of penicillin, and 100 chased from ABI and are available from the manufacturer. ␮g/ml streptomycin. Tissue culture conditions were 37°Cina

TaqMan PCR was carried out in a reaction volume of 25 humidified atmosphere of 5% CO2. The BIN1 expression vec- ␮l. Each reaction mixture contained 0.1 pmol/␮l fluorogenic tors pCMV-BIN1, pCMV-BIN1(Ϫ10), pCMV-BIN1(Ϫ10 ϩ probe, 0.2 pmol/␮l of each primer, 1ϫ TaqMan Universal PCR 12A), and pCMV-BIN1(⌬MBD) have been described previously Mastermix (PE Biosystems), and 1.0 ␮l of cDNA. Each sample (17). The pCMV-BIN1(Ϫ10) vector contains the coding se- was analyzed at least in duplicate with good reproducibility. quence of the most abundant BIN1 isoform (which excludes Thermal cycling was started with a 2-min incubation at 50°C, exons 10 and 12A-12D; GenBank accession no. U68485) in followed by a first denaturation step of 10 min at 95°C, and then pcDNA3 (Invitrogen, Carlsbad, CA). Similarly, pCMV- 40 cycles of two-step PCR consisting of 95°C for 15 s and 60°C BIN1(Ϫ10 ϩ 12A) excludes exons 10 and 12B-D but includes for 1 min. A quantitative analysis was performed using the ABI exon 12A. pCMV-BIN1⌬MBD is an artificial construct encod- PRISM 7700 Sequence Detection System and software (PE ing a Bin1 protein with a deletion in the MBD (amino acid Biosystems). For analysis of total BIN1 expression, pCMV- residues 270–377). Empty vector pcDNA3 was used to control BIN1(Ϫ10) a plasmid encoding the ubiquitous BIN1 isoform for transfection efficiency. that excludes exon 10, was serially diluted to establish the For clonogenic growth assays, NB cells were seeded into calibration curve. For MYCN and GAPDH, the cDNA from T25 culture flasks and grown to ϳ60% confluence. Transfection the NB cell line SMS-KAN was serially diluted to establish the with 8 ␮g of pCMV-BIN1, pCMV-BIN1(Ϫ10), pCMV- calibration curve. The expression level of BIN1 and MYCN were BIN1(Ϫ10 ϩ 12A), pCMV-BIN1(⌬MBD), or pcDNA3 was defined as the ratio of the signal obtained for target gene (BIN1 performed using FuGENE 6 Transfection Reagent (Roche Mo- or MYCN) divided by that for GAPDH, then normalized to a lecular Biochemicals) at 2 mcl transfection reagent/␮g plasmid reference cDNA. DNA in serum-free medium according to the manufacturer’s Semiquantitation of BIN1 Isoform Expression Using recommendations. Approximately 1 ϫ 105 cells were plated into RT-PCR. To determine the relative expression of individual 35-mm wells, and selection for neomycin resistance using 0.5 BIN1 isoforms in primary NBs we designed PCR primers that mg/ml G418 was initiated 24 h later. Medium with G418 was span exons 11 to 14 and reliably amplify all of the alternatively changed every 2–3 days, and cell colonies were scored after spliced BIN1 isoforms. The forward primer (5Ј-TCC CCA AGT 100% methanol fixation and 0.005% crystal violet staining after CCC CAT CTC AG-3Ј) is complimentary to sequences within ϳ21 days. Each colony formation assay was performed in exon 11 of BIN1, whereas the reverse primer (5Ј-CTC CAC triplicate and was repeated from at least three independent GGT GCC ATT CAC AG-3Ј) is complimentary within exon 14. transfection reactions. Colony formation was expressed relative These primers were biotinylated at their 5Ј ends. Assays using to colonies obtained with control vector transfection. BIN1 primers flanking exons 9–14 as well as exons 11–16 BIN1 Mutation Analyses. Constitutional DNA was not demonstrated no isoforms not identified with the BIN1 exon obtained routinely from patients in our study group. Therefore, 11–14 primers (data not shown). PCR was carried out in a final BIN1 LOH studies were performed using a separate group of 67 volume of 20 ␮l, containing 1 unit of Taq Gold DNA polym- paired primary NB and constitutional-tissue derived DNA sam- erase (PE Biosystems), 100 ␮M deoxynucleoside triphosphates, ples and seven NB cell lines (KCN, CHP-904, CHP-901, LA-

N-5, CHLA-118, CHLA-123, and CHLA-150) with matched lated based on log scale values transformed back to expression constitutional DNA available from the Pediatric Oncology levels. The ␹2 test was used to test for significance in compar- Group, The Children’s Hospital of Philadelphia Tumor bank and isons of BIN1 alternative splice isoforms and biological features. a generous gift from Dr. C. Patrick Reynolds. PCR was performed to amplify an ϳ125 bp fragment containing a polymor- RESULTS phic (GT)n microsatellite within intron 5 of the BIN1 locus to assess for LOH. The forward primer BIN1(GT)-F is 5Ј-CCA MYCN Expression as Determined by Real-Time PCR. TTC CTT TCC TGT CCC TTT G-3Ј, and the reverse primer All 56 of the primary NBs expressed detectable levels of MYCN BIN1(GT)-R is 5Ј-CCT TCC ACA ATC ATA TAG CCA RNA with a mean of 72.7 Ϯ 290.9 units (range, 0.1–2034). As GCC-3Ј. PCR reactions were performed in 20-␮l volumes using expected, expression levels for tumors with MYCN amplifica- conditions as described above for the RT-PCR. Forward primers tion (n ϭ 10) were higher (325 Ϯ 653; range, 0.1–2034) than for for each reaction were ␥-33P end-labeled using T4 polynucle- MYCN single-copy tumors (18 Ϯ 31; range, 0.3–189; P Ͻ 0.01). otide kinase (New England Biolabs, Inc., Beverly, MA). PCR For purposes of comparison, MYCN expression in human fetal products were separated by electrophoresis through an 8% poly- brain cDNA was 35 units using this assay. MYCN expression acrylamide denaturing sequencing gel (Burst-Pak; Owl Scien- from INRG high-risk NBs (n ϭ 19) was 175 Ϯ 490 and from tific, Inc., Woburn, MA), and analyzed by autoradiography and low- or intermediate risk NBs was 20 Ϯ 34 (P ϭ 0.06). There densitometry as described. The threshold for LOH was defined was likewise a trend but not a statistically significant difference by an allelic intensity reduction of Ͼ60% for one allele, and all in MYCN expression comparing localized NB (INSS stages 1, 2, of the samples with LOH were analyzed in triplicate for con- or 3) to metastatic NB (INSS stages 4 or 4S; 22 Ϯ 37 and 157 Ϯ firmation. 467; P ϭ 0.09). Eleven NBs had MYCN expression levels To assess for homozygous deletion within the BIN1 locus greater than that of fetal brain (Ͼ35 units): 6 arising in children a set of 46 unique NB cell lines demonstrating unique genotypes Ͼ1-year-old, and 5 arising in infants. Of the 6 high MYCN at three highly polymorphic microsatellite markers were used expressing NBs in children, all demonstrated MYCN amplifica- and have been described previously (33). The 46 cell lines, a tion and were high risk using INRG criteria. In contrast, of the normal DNA control, and a negative control containing no 5 highly MYCN expressing NBs arising in infants, none had added template were amplified by PCR essentially as above MYCN amplification or was high-risk (P Ͻ 0.01), and 4 were using the BIN1(GT)-F and -R primer pair, and 14.4 ng template stage 1 NBs detected through the screening program. This is DNA. Twenty ␮l of reaction solution was analyzed by electro- consistent with prior reports that NBs in infants frequently have phoresis on an enhanced sensitivity gel system (Visigel; Strat- higher levels of MYCN expression in the absence of gene agene, La Jolla, CA), with products detected by ethidium bro- amplification or other high-risk NB features, possibly reflecting mide staining. normal developmental regulation of the gene, rather than dereg- Southern analysis to assess for genomic rearrangements ulated overexpression (34, 35). within the BIN1 locus was performed on 21 NB cell lines (11 Of the 56 patients, 27 were clinically diagnosed (17 after with MYCN amplification and 10 without) and 11 primary NBs having had a negative urine catecholamine screen at 6 months of (all with MYCN amplification). Ten ␮g of genomic DNA was age, and 10 diagnosed before the 6-month screening), and 29 restriction digested with PstI (Promega, Madison, WI), and were diagnosed through the screening program. Nine (33%) of fractionated on 0.6% agarose and transferred to Hybond-Nϩ the 27 clinically diagnosed tumors had MYCN amplification membranes followed by UV cross-linking and hybridized with compared with only 1 (3%) of the tumors diagnosed through the two BIN1 probes sequentially. Probes used were an ϳ1.5 kb screening program, although MYCN expression was not statis- EcoRI fragment from pCMV-BIN1 including most of the coding tically different between the two groups (132 Ϯ 414 and 18 Ϯ region of BIN1, and a PCR-generated 1.2 kb fragment encom- 21, respectively; P ϭ 0.15). A similar low incidence of MYCN passing ϳ0.8 kb of the BIN1 promoter region and exon 1 and amplification in NBs detected during mass screening programs intron 1 sequences (primer sequences available by request). On has been described in multiple prior studies as a result of the basis of the organization of the BIN1 gene this latter probe ascertainment bias. is Ͼ20 kb from the coding sequence probe (18). Probe radiola- Expression of BIN1 Isoforms in Primary NBs Using beling was performed with the Rediprime Random Labeling kit Real-Time PCR and RT-PCR. BIN1 isoforms were detected using Redivue [␣-32P]dCTP (Amersham Pharmacia). Hybrid- in all 56 of the NBs assayed. Values for total BIN1 expression ization conditions were as described previously (33), and detec- ranged from 1.4 to 1304 units (mean 149 Ϯ 275). BIN1 expres- tion was by autoradiography on Biomax-MS film (Eastman sion for fetal brain cDNA was 8.1 units with this assay. RT-PCR Kodak) at Ϫ80°C from 1 to 3 days. demonstrated four predominant BIN1 isoforms in NB: Statistical Analyses. Analyses correlating MYCN ex- BIN1(Ϫ10Ϫ13), BIN1(Ϫ10), BIN1(Ϫ10 ϩ 12A-13), and pression with clinical and biological variables, as well as results BIN1(Ϫ10 ϩ 12A); Fig. 1. These same four isoforms were also for colony formation assays, were performed using the two- detected in all 12 of the NB-derived cell lines assayed (Fig. 1C; tailed Student’s t test. BIN1 expression levels in the tumor data not shown). Of interest, isoforms containing exons 12B- samples were skewed (non-normal distribution) and, therefore, 12D were not detectable in either primary NBs or human fetal all of the statistical analyses for BIN1 were done on the natural brain cDNA, although they are expressed in murine brain cDNA logarithm of the expression level using two-sample t tests. (18) and are considered to be neural-tissue restricted in their Two-sample Wilcoxon rank-sum tests were used to confirm expression. Nineteen primary NBs (34%) expressed all four of these results. Means and 95% confidence intervals were calcu- the BIN1 isoforms, whereas 37 NBs had undetectable expression

Fig. 2 Expression of BIN1(Ϫ10) in 56 primary NBs determined by real-time and RT-PCR as described in “Materials and Meth- ods.” Statistical comparisons are for: A, MYCN amplified NB versus MYCN single-copy tumors; B, INRG high-risk versus low- or intermediate-risk NB; C, metastatic (INSS stage 4 or 4 s) versus localized NB; and D, age at diagnosis Ͻ1 year versus Ͼ1 year. Data are represented as box plots: filled square denotes the mean value, box represents 25th percentile through the 75th percentile (50th percentile denoted by line through box), and whiskers above and below extend to the 90th and 10th percentiles, respectively.

(Յ1.0 unit) of at least one isoform. Six tumors (11%) did not those diagnosed through a screening program (P ϭ 0.02). express BIN1(Ϫ10Ϫ13); 9 (16%) did not express BIN1(Ϫ10); BIN1(Ϫ10) was not detectable (defined as expression Յ1 unit) 23 (41%) did not express BIN1(Ϫ10 ϩ 12A-13); and 32 (57%) in 9 primary NBs. Of these, 5 (56%) had MYCN amplification, did not express BIN1(Ϫ10 ϩ 12A). Expression of these isoforms and 7 (78%) were INRG high-risk tumors [compared with 5 of was 89 Ϯ 172 units (range, 0.6–717) for BIN1(Ϫ10Ϫ13);37Ϯ 47 (11%) with MYCN amplification and 12 of 47 (26%) high- 68 (range, 0.1–295 units) for BIN1(Ϫ10);15Ϯ 38 units (range, risk in NBs with detectable BIN1(Ϫ10); P Ͻ 0.01]. Thus, 0–220) for BIN1(Ϫ10 ϩ 12A-13); and 8 Ϯ 31 units (range, reductions in the major MycN-interacting BIN1 isoform were 0–220) for BIN1(Ϫ10 ϩ 12A). Thus, the BIN1(Ϫ10Ϫ13) and demonstrated in NBs with multiple unfavorable biological fea- BIN1(Ϫ10) isoforms were the most commonly and highly ex- tures. An association of reduced BIN1(10–13) expression was pressed BIN1 isoforms in primary NBs. apparent only for INRG high-risk versus low- or intermediate- Because of the skewed distribution of BIN1 expression risk disease (Table 2). Of the less abundant BIN1(Ϫ10 ϩ values (as demonstrated in Fig. 2, box plots) the data were log 12A-13) and BIN1(Ϫ10 ϩ 12A), there was a correlation between transformed to achieve a normal distribution for statistical anal- lower BIN1(Ϫ10 ϩ 12A-13) levels and MYCN amplification, as yses. Expression of each identified BIN1 isoform was then well as INRG high-risk disease (P Ͻ 0.05). correlated with specific clinical and biological features of NB We next assessed whether the pattern of alternative splic- (Table 2). Reduced expression of BIN1(Ϫ10) was correlated ing could be correlated with biological features of NB. The least with unfavorable biological features of NB including the pres- highly expressed isoforms, BIN1(Ϫ10 ϩ 12A) and BIN1(Ϫ10 ϩ ence of MYCN amplification (P Ͻ 0.03), high-risk disease based 12A-13), which both contain the neural-tissue restricted exon on INRG classification (P Ͻ 0.01), and age at diagnosis Ͼ1 year 12A, were not detectable in 57% and 39% of the primary NBs (P Ͻ 0.04). There was a strong trend toward lower BIN1(Ϫ10) examined, respectively, and were not the predominant BIN1 expression in metastatic NB compared with localized NB as isoform in any tumor. BIN1(Ϫ10 ϩ 12A) accounted for Ͻ20% well (P ϭ 0.058). In addition, BIN1(Ϫ10) expression was sig- of BIN1 in all of the samples, and BIN1(Ϫ10 ϩ 12A-13) nificantly lower in NBs diagnosed clinically compared with accounted for Ͻ20% in 40 of 56 NBs. No correlations between

Table 2 Expression of BIN1 isoforms in 56 primary neuroblastomas: correlation with clinical and biological features BIN1 isoform expressiona BIN1(Ϫ10Ϫ13) BIN1(Ϫ10) BIN1(Ϫ10ϩ12A-13) BIN1(Ϫ10ϩ12A) n Mean (95% CI)b P Mean (95% CI) P Mean (95% CI) P Mean (95% CI) P MYCN status 0.12 0.03 0.05 0.65 Not amplified 46 21.3 (12.4–36.6) 10.0 (5.8–17.2) 0.9 (0.3–2.5) 0.2 (0.1–0.8) Amplified 10 7.7 (1.7–34.9) 2.3 (0.5–10.2) 0.1 (0.0–1.6) 0.1 (0.0–2.0) INRG risk group 0.05 0.01 0.05 0.14 Low/intermed 37 25.3 (13.6–47.1) 12.8 (6.9–23.5) 1.1 (.31–3.8) 0.4 (0.1–1.3) High 19 8.9 (3.7–21.3) 2.8 (1.2–6.8) 0.1 (0.0–0.8) 0.1 (0.0–0.4) INSS stage 0.14 0.06 0.60 0.82 1, 2, or 3 35 23.7 (11.9–46.9) 11.2 (5.5–22.8) 0.7 (0.2–2.7) 0.2 (0.1–0.8) 4 or 4s 21 11.0 (5.2–23.5) 4.1 (2.0–8.3) 0.4 (0.1–1.7) 0.3 (0.1–1.0) Age (years) 0.17 0.04 0.17 0.13 Ͻ1 34 23.5 (12.1–45.6) 11.7 (6.3–21.6) 0.9 (0.2–3.7) 0.4 (0.1–1.5) Ն1 22 11.5 (5.1–26.2) 4.0 (1.6–9.9) 0.2 (0.0–1.1) 0.1 (0.0–0.4) Screening diagnosis 0.08 0.02 0.87 0.49 Yes 29 27.2 (12.9–57.5) 13.4 (6.9–26.3) 0.6 (0.1–2.9) 0.3 (0.1–1.4) No 27 11.2 (5.6–22.3) 4.2 (1.9–9.1) 0.5 (0.1–1.9) 0.2 (0.0–0.6) a Natural logarithm of BIN1 isoform expression relative to GAPDH expression (see “Materials and Methods”). b Mean and 95% confidence intervals (CI) based on log scale values transformed back to expression levels.

the proportion of these exon 12A containing BIN1 isoforms and rally expressing an inducible MYCN-ER construct. BIN1 expres- biological features of NB were found. In contrast, there was sion was invariant in the presence or absence of induced MYCN more variability in the proportion of the abundantly expressed activity refuting a direct transcriptional effect (data not shown). BIN1(Ϫ10) and BIN1(Ϫ10Ϫ13). BIN1(Ϫ10) was detectable in Thus, BIN1 expression is higher in NBs with high levels of 47 of 56 NBs (84%) and ranged from 0 to 68% of all of the BIN1 MYCN expression (in the absence of MYCN amplification) in isoforms present. BIN1(Ϫ10Ϫ13) was present in 50 of 56 NBs infants but is reduced in NBs with unfavorable biological fea- (89%) and accounted for a range from 10 and 90% of all of the tures such as MYCN amplification and deregulated expression BIN1 isoforms present. Fifteen of 30 (50%) NBs with Ͼ50% of (only 1 patient in our cohort had MYCN amplification and age BIN1 as BIN1(Ϫ10Ϫ13), were INRG high-risk tumors (7 with Ͻ1 year so we could not assess the relationship between MYCN MYCN amplification), whereas only 4 of 26 (15%) tumors with and BIN1 expression in this subset). Because MYCN is more Ͻ50% BIN1(Ϫ10Ϫ13) isoforms were high-risk (3 with MYCN highly expressed in the developing peripheral nervous system amplification; P Ͻ 0.01) suggesting that a possible mechanism than in mature tissues, we hypothesize that these findings in for down-regulation of MYCN-interacting BIN1 isoforms in infant NBs reflect higher levels of MYCN regulated in a devel- unfavorable NBs is through alternate exon splicing to exclude opmentally appropriate manner with no impetus to inactivate exon 13 (encoding a portion of the MBD), in addition to BIN1 (which may also be developmentally regulated). In con- transcriptional regulation. An increase in isoforms omitting trast, tumors with MYCN amplification have deregulated MYCN exon 13 (with a reciprocal decrease in exon 13 containing expression, and inactivation of BIN1 may provide a survival isoforms through alternative splicing at this junction) could advantage by abrogating MycN-mediated apoptosis after geno- effectively limit MycN-Bin1 interactions through omission of toxic stressors. the MBD of Bin1. Effect of Ectopic Expression of Bin1 Isoforms on Col- Correlations between BIN1 Expression and MYCN Ex- ony Formation in NB Cell Lines. As described previously, pression. Despite having demonstrated reduced BIN1 expres- BIN1 is subject to tissue-specific splicing in normal tissues (18) sion in NBs with MYCN amplification (among other unfavor- as well as aberrant tumor-specific splicing (20). The NB-derived able biological features), we found no correlation between cell line SK-N-AS does not have amplification of the MYCN MYCN and BIN1 expression in the study cohort overall (corre- locus and expresses BIN1 at levels comparable with fetal brain, lation coefficient r ϭ 0.14; P ϭ 0.32). In fact, after excluding whereas IMR-5 has MYCN amplification and markedly reduced the 10 tumors with MYCN amplification (with marked deregu- BIN1 expression (27). Although total BIN1 expression differs, lated overexpression) there was a moderate direct correlation both SK-N-AS and IMR-5 express all four of the isoforms of between MYCN and BIN1 expression (r ϭ 0.67; P Ͻ 0.001). BIN1 seen in the majority of primary NBs (Fig. 1C; data not This correlation between MYCN and BIN1 expression in single- shown). copy tumors results from a strong correlation in tumors arising To determine the functional effect of ectopic expression of in children Ͻ1 year of age alone. In these cases (n ϭ 33), BIN1 BIN1 isoforms on these cell lines, transient transfection studies expression was correlated with MYCN expression (r ϭ 0.68; using pCMV-BIN1, pCMV-BIN1(Ϫ10), pCMV-BIN1(Ϫ10 ϩ P Ͻ 0.001), whereas in children Ͼ1 year of age (n ϭ 13) there 12A), pCMV-BIN1⌬MBD, or pcDNA3 (transfection control) was no correlation (r ϭ 0.32; P ϭ 0.28). To exclude a direct were performed, and in vitro colony formation was assessed. transcriptional effect of MYCN on BIN1 expression we used the Forced expression of BIN1, BIN1(Ϫ10),orBIN1(Ϫ10 ϩ 12A), human neuroectoderm-derived cell line hTERT-RPE1 retrovi- each of which includes the unique BAR domain as well as an

this intragenic polymorphism. Of these informative cases, 3 (6%) demonstrated LOH with reductions in allelic intensity of 61%, 63%, and 76%, and all were from primary tumor specimens. Two of these were stage 1, and 1 was stage 4 (none had MYCN amplification). Information on the DNA content (or ploidy) of these tumor specimens was not available, nor was RNA/cDNA available to assess expression of BIN1 isoforms in those samples demonstrating LOH. As 2 of the 3 primary NBs demonstrating LOH were stage 1, and there is an association with low-stage NB and near-triploidy, it is possible that in these cases the allelic imbalance seen could be accounted for by differences in whole chromosome number, i.e. trisomy for chromosome 2 rather than chromosome 2q deletion events. We next assessed for homozygous deletion at the BIN1 Fig. 3 Colony formation assays after transient transfection of NB cell locus in a panel of 46 unique NB-derived cell lines (a full listing lines to express BIN1 constructs. The data are presented as the percent- of the panel of cell lines used is found in Ref 33). Homozygous age of colonies obtained relative to transfection of the control vector deletion has been observed occasionally in cell lines or primary without insert, pcDNA3. Datapoints are the mean of at least three independent transfection experiments each plated in triplicate; bars, tumors from a variety of malignancies, and, although rare, their ϮSE. IMR5 has MYCN amplification and low endogenous BIN1 ex- characterization has been instrumental in the identification of pression; SK-N-AS has single-copy MYCN and endogenous BIN1 ex- several tumor suppressor genes, including RB1, WT1, and significantly different from CDKN2A (38–40). DNA was amplified via PCR using the same ,ء .pression comparable with fetal brain Ͻ control transfection, P 0.01 by Student’s t test. primers and conditions described above to amplify an intragenic BIN1 marker. A product of the predicted size was visible for each of the 46 NB cell lines tested but not in negative control lanes (data not shown). intact MBD, resulted in markedly reduced colony formation in To assess for genomic rearrangements or larger genomic IMR-5 but had no effect on colony formation of SK-N-AS (Fig. deletions elsewhere within the BIN1 locus, Southern analysis 3). Because we hypothesized that BIN1 acts as an adapter was performed using DNA from 21 NB-derived cell lines and protein mediating MycN function, we repeated these assays with 11 primary NBs (all primary tumors had MYCN amplification pCMV-BIN1 or pcDNA3 (transfection control) in two addi- and were INSS stage 3 or 4). Two BIN1 probes were sequen- tional NB-derived cell lines with MYCN amplification, NGP and tially hybridized, one encompassing a portion of the promoter SMS-KAN. Similar marked reductions in colony formation (to region and exon 1, and a second including the majority of the 11% and 42% of control transfectants, respectively) were seen BIN1 coding region (this latter probe is ϳ20 kb from the (P Ͻ 0.05). Additionally supporting a role for the requirement former). All of the tumors and cell lines demonstrated of direct Bin1-MycN interactions in inhibiting colony formation the anticipated single hybridization band of ϳ3.4 kb when is the inability of pCMV-BIN1⌬MBD to similarly inhibit colony hybridized with the BIN1 exon1/promoter probe, and multiple formation in either cell line tested (Fig. 3). It should be noted identical bands at ϳ2.8 kb, 2.0 kb, 1.6 kb, 1.0 kb, and 0.8 kb that pCMV-BIN1⌬MBD is an artificial BIN1 construct lacking when hybridized with BIN1 coding sequences (data not shown). the entire MBD through the exclusion of amino acid residues No evidence for genomic rearrangement or deletion was ob- 270–377 resulting in an isoform with a larger internal deletion tained. Taken together, these experimental results suggest that than the naturally occurring splice isoforms BIN1(Ϫ10Ϫ13) and BIN1 loss occurs in NB through epigenetic rather than genetic BIN1(Ϫ10 ϩ 12A-13), which omit amino acid residues 315–345 alterations. from the MBD. BIN1 Mutation Analyses in Primary NBs and in NB- derived Cell Lines. In the present study we have correlated DISCUSSION reduced BIN1 expression with MYCN amplification, INRG We reported previously that expression of BIN1 is reduced high-risk disease, and the presence of metastatic disease in in NB cell lines with MYCN amplification compared with cell primary NBs. As BIN1 maps to the long arm of human chro- lines with single-copy MYCN and hypothesized that BIN1 may mosome 2 (2q14; Ref. 36) that has been reported to be hemizy- function as a tumor suppressor through MycN interactions (20, gously deleted in up to 30% of primary NBs (37), we assessed 25–27). However, multiple alternatively spliced BIN1 tran- for LOH within the BIN1 locus that might account for reduced scripts have been described, including isoforms that demonstrate BIN1 expression. A polymorphic dinucleotide repeat within neural-tissue restricted expression (inclusion of exons 12A-D) intron 5 (defined in Ref. 25) was amplified from 74 paired or that lack the putative MBD (exon 13), and it is likely that samples representing tumor and constitutional DNA. these alterations have functional consequences. We have now Sixty-seven samples were from primary tumor specimens, determined the isoforms of BIN1 expressed in primary NBs, and and an additional 7 were from NB-derived cell lines. Twenty- have demonstrated that reduced expression of the predominant one of the 67 primary NBs (31%) and 6 of the 7 NB-derived cell MycN-interacting BIN1 isoform [BIN1(Ϫ10)] is correlated with lines (86%) had MYCN amplification. Forty-eight of the 74 total unfavorable biological features including amplification of the constitutional DNA samples tested (65%) were heterozygous at MYCN proto-oncogene, INRG high-risk disease, age Ͼ1 year,

and the presence of metastases at diagnosis. Furthermore, we pattern for this gene within the developing peripheral nervous have shown that forced expression of BIN1 isoforms that con- system. In contrast, high levels of MYCN persist in a deregulated tain the MBD potently inhibit colony formation in NB cell lines fashion after MYCN amplification, and it is the inability to with MYCN amplification. No effect on colony formation was down-regulate MycN that generates an apoptosis signal after seen with forced expression of a MBD-deleted construct nor genotoxic stress or in the setting of limited mitogenic factors. In when BIN1 isoforms were expressed in NB cell lines without these cells, the inability to engage MYCN-primed apoptosis, as MYCN amplification. However, it will be necessary to confirm may occur through loss of MycN-Bin1 signaling, would provide that deletion of that portion of the MBD encoded within exon 13 a survival advantage (17, 21, 24, 27). Therefore, BIN1 loss of alone results in a similar inability to inhibit colony formation in function would be selected for in-tumor cells with deregulated MYCN-amplified NB cell lines, as the MBD deleted construct MYCN, but not necessarily in those cells with higher levels of used in these experiments lacked the entire MBD. We hypoth- MYCN that are normally regulated, consistent with our findings. esize that BIN1 isoforms with an intact MBD interact with In the present study we have not explored the mechanistic MYCN to induce apoptosis when MYCN remains forcibly over- aspects of Bin1 function, although we have shown previously expressed (as occurs in NBs with MYCN amplification). A that re-expression of Bin1 in MYCN-amplified cell lines with similar finding of reduced BIN1 expression in MYC-overex- reduced endogenous expression results in an increase in pro- pressing neural tumors has been reported previously. Gene grammed cell death (27). This apoptotic effect was more pro- expression profiling of 11 primary diffuse astrocytomas dem- nounced in the setting of genotoxic stress (low-dose doxorubicin onstrated 8 with MYC overexpression, and all of these had exposure) or mitogen deprivation (serum-free culture condi- Ͼ50% reductions in BIN1 expression (41). tions), and we hypothesize that Bin1 signaling plays a role in the As shown in other tissues as well, the most abundantly engagement of MYCN-primed apoptosis when MycN expression expressed BIN1 isoforms in NB were BIN1(Ϫ10) and is aberrantly regulated. Although the relative proportion of BIN1(Ϫ10Ϫ13), rather than those isoforms specific to neural individual BIN1 isoforms generally trended together in tumor tissues. These isoforms localize predominantly to the nuclear samples, suggesting transcriptional regulation, we provide evi- and cytosolic compartments, respectively. Differential cytolo- dence that altered splicing (to omit a MycN-interacting func- calization may be responsible for governing the differential tional domain encoded partially within exon 13) may contribute functional effects of these isoforms, particularly in mediating to BIN1 loss of function. Fifteen of 19 high-risk NBs (79%) had MycN interactions (21). Inclusion of the neural tissue-specific Ͼ50% of their BIN1 transcripts as BIN1(Ϫ10Ϫ13) and a recip- exons 12A-12D was rarely observed in NBs despite the genesis rocal decrease in the putative MycN-interacting BIN1(Ϫ10) of this tumor within the peripheral nervous system. BIN1 tran- isoform. That aberrant splicing may play a role in tumor sup- scripts containing exons 12B-12D were not detected, and iso- pressor inactivation has been suggested previously for DCC, forms containing exon 12A were in low abundance, which may FHIT, and BIN1 (20, 44, 45). We found no evidence for reflect the primitive differentiation state of neuroblasts. The genomic rearrangements to account for loss of BIN1 expression, 12A-12D exons encode determinants necessary for synaptic and only infrequently was there evidence for BIN1 deletion vessicle endocytosis (42, 43), and these isoforms are located using LOH analysis (6%). This is in contrast to prostate cancer preferentially within the cytosol. It has been reported previously in which LOH within BIN1 is seen in 40% of tumors (25), but that aberrant splicing to include exon 12A in melanoma cells consistent with prior studies of breast carcinoma in which loss may be a mechanism of BIN1 inactivation, as this isoform was of BIN1 expression was frequent (60% of cases) in the absence incapable of inhibiting Myc-mediated transformation or cell of gross genomic rearrangements (26). The finding that reduc- viability (20) in these cells, possibly through aberrant localiza- tions in BIN1 expression occur frequently in the absence of tion outside the nuclear compartment. In contrast, we have demonstrable genomic alterations suggests that epigenetic fac- found that forced expression of BIN1(Ϫ10 ϩ 12A) in the NB tors may play a role in regulating BIN1 expression. cell line IMR-5 did inhibit colony formation, although immu- nohistochemical studies to assess localization of the protein ACKNOWLEDGMENTS were not performed. In most neural cells exon 12A is not We thank Dr. Garrett Brodeur for support and helpful discussion in included in BIN1 in the absence of 12B-12D (which encode the preparation of this manuscript. We also thank the Pediatric Oncol- clathrin binding determinants); it may be that exon 12A is ogy Group, The Children’s Hospital of Philadelphia Tumor Bank, and insufficient for cytosolic localization in neuroblasts. Dr. C. Patrick Reynolds for providing tumor, cell line, and/or constitu- Overall there was a positive correlation between BIN1 and tional DNA, as well as Dr. Dai Sakamuro for the generation and MYCN expression levels in primary NBs without MYCN ampli- provision of BIN1 plasmid clones for these studies. fication, although there is no evidence that BIN1 is a MYCN (or MYC) target gene. Therefore, it is more striking that BIN1 is REFERENCES reduced significantly in NBs with MYCN amplification and 1. Landis, S. H., Murray, T., Bolden, S., and Wingo, P. A. Cancer deregulated overexpression. These findings may be explained statistics. CA: Cancer J. Clin., 49: 8–31, 1999. by the large proportion of tumors in our cohort diagnosed during 2. Gurney, J. G., Ross, J. A., Wall, D. A., Bleyer, W. A., Severson, infancy through a mass screening program. BIN1 isoforms were R. K., and Robison, L. L. Infant cancer in the US: histology-specific incidence and trends, 1973 to 1992. J. Ped. Hem. 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Tatsuro Tajiri, Xueyuan Liu, Patricia M. Thompson, et al.

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