Minimal 16q Genomic Loss Implicates Cadherin-11 in Retinoblastoma Mellone N. Marchong,1,2 Danian Chen,1,6 Timothy W. Corson,1,3 Cheong Lee,1 Maria Harmandayan,1 Ella Bowles,1 Ning Chen,5 and Brenda L. Gallie1,2,3,4,5 1Divisions of Cancer Informatics and Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada; Departments of 2Medical Biophysics, 3Molecular and Medical Genetics, and 4Ophthalmology, University of Toronto, Toronto, Ontario, Canada; 5Retinoblastoma Solutions, Toronto, Ontario, Canada; and 6Department of Ophthalmology, West China Hospital, Faculty of Medicine, Sichuan University, Chengdu, People’s Republic of China Abstract compared with normal adult human retina. Our analyses Retinoblastoma is initiated by loss of both RB1 alleles. implicate CDH11, but not CDH13, as a potential tumor Previous studies have shown that retinoblastoma suppressor gene in retinoblastoma. (Mol Cancer Res tumors also show further genomic gains and losses. We 2004;2(9):495–503) now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using Introduction loss of heterozygosity (30 of 76 tumors) and quantitative Retinoblastoma is the most common intraocular tumor in multiplex PCR (71 of 76 tumors). The sequence-tagged children. Mutations of both alleles (M1 and M2) of the RB1 site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene at chromosome 13q14 are necessary (1) for retinoblastoma gene showed the highest frequency of loss (54%, 22 of tumor initiation but not sufficient for malignant transformation 41 samples tested). A second hotspot for loss (39%, 9 (2). G-banding analysis of retinoblastoma shows that addi- of 23 samples tested) was detected within intron 2 of tional chromosomal aberrations (3) always accompany RB1 À/À the cadherin-13 (CDH13) gene. Furthermore, deletion mutations. Moreover, RB1 murine retina does not sponta- of the exons of CDH11 and/or WI-5835 was shown by neously form retinoblastoma (4) unless one of the pRB-related quantitative multiplex PCR in 17 of 30 (57%) of previously proteins, p107 (5) or p130 (6), is also deleted. We hypothesize untested tumors. Immunoblot analyses revealed that that additional mutational events (M3 to Mn) are required for À/À 91% (20 of 22) retinoblastoma exhibited either a complete RB1 cells to progress into a fully malignant tumor (2). loss or a decrease of the intact form of CDH11 and 8 of To identify these mutational events, we have previously 13 showed a prevalent band suggestive of the variant analyzed chromosomal alterations in 50 retinoblastoma tumors form. Copy number of WI-5835 for these samples by comparative genomic hybridization (CGH) analysis (7). correlated with CDH11 protein expression. CDH11 The minimal region most frequently lost was detected at 16q22 staining was evident in the inner nuclear layer in early (14%; ref. 7). Cumulatively, 51 of 162 (31%) tumors in five mouse retinal development and in small transgenic different CGH studies showed a common region of loss at murine SV40 large T antigen–induced retinoblastoma 16q22 (7-11) as summarized in Fig. 1A. Allelic loss on 16q22 tumors, but advanced tumors frequently showed loss has also been frequently shown in liver (12), breast (13), pros- of CDH11 expression by reverse transcription-PCR, tate (14), and Wilms’ (15) tumors, suggesting the presence suggestive of a role for CDH11 in tumor progression or of important tumor suppressor gene(s) in this chromosomal metastasis. CDH13 protein and mRNA were consistently region. expressed in all human and murine retinoblastoma A cluster of cadherin genes is located at 16q22. Cadherins are calcium-dependent cell-cell adhesion molecules that play crucial roles in formation of gap junctions, in tissue morpho- genesis, and that have been implicated in cancer progression. Received 1/12/04; revised 7/14/04; accepted 7/19/04. For instance, epithelial cadherin (CDH1) has been documented Grant support: Vision Science Research Program of the University of Toronto as a tumor suppressor in a variety of carcinomas (16-21). Given (M.N. Marchong and T.W. Corson), Helen Keller Foundation for Research and Education (D. Chen), National Cancer Institute of Canada with funds from the the role of cadherins as regulators of cell adhesion and their Terry Fox Run and the Canadian Cancer Society (B.L. Gallie), Canadian Genetics apparent loss in a variety of cancers, it seems plausible that there Disease Network (B.L. Gallie), Canadian Institutes for Health Research (B.L. exists a role for cadherins in cancer progression. Gallie), Royal Arch Masons of Canada, Keene Annual Perennial Plant Sale, and Jeff Healy Christmas Concert. Detection of chromosomal loss using CGH has a limit of The costs of publication of this article were defrayed in part by the payment of resolution of f10 to 20 Mbp (22). We have been able to narrow page charges. This article must therefore be hereby marked advertisement in the limit of resolution to the level of single candidate tumor accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Brenda L. Gallie, Division of Cancer Informatics, Room suppressor genes by combining loss of heterozygosity (LOH) 8-415, Ontario Cancer Institute/Princess Margaret Hospital, University Health analysis with quantitative multiplex PCR (QM-PCR). Two Network, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2324; Fax: 416-946-4619. E-mail: [email protected] candidate tumor suppressor genes, cadherin-11 (CDH11) and Copyright D 2004 American Association for Cancer Research. cadherin-13 (CDH13), emerged from a study of retinoblastoma Mol Cancer Res 2004;2(9). September 2004 495 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. 496 Marchong et al. FIGURE 1. Loss of 16q22 in retinoblastoma. A. Loss of the long arm of chromosome 16, or even the entire chromosome, has been observed in five CGH studies of retinoblastoma (7, 8, 9, 10, and 11). Vertical bars to the left of the chromosome ideogram, region of loss in an individual tumor; vertical bars to the right of the chromosome ideogram, region of gain in an individual tumor. Cumulatively, 51 of 162 (31%) of tumors show loss of part or all of 16q, with a minimal common region of loss evident at 16q22 (dotted boxed region). B. Three regions of chromosome 16q allelic loss were defined in retinoblastoma. The percentage of tumors showing allelic loss is plotted against the distance (Mbp) of each marker from the 16p telomere. White bars, microsatellite polymorphic markers assayed for LOH; black bars, STS markers assayed by QM-PCR, with the STS name in italics. For each marker, the number and percentage of tumors showing loss compared with the number tested are indicated; the three hotspots are boxed. The region spanning 2.62 Mbp between STS WI-5835 and flanking markers was lost in 54% of retinoblastoma but, based on data for flanking markers, is consistent with loss in 59% of retinoblastoma (hatched bars). The approximate locations of the cadherin genes that cluster in 16q22-24 are indicated (CDH8, CDH11, CDH5, CDH16, CDH3, CDH1, CDH13). C. Schematic representation of the minimal region of loss, 2.62 Mbp, in which the most frequently lost marker, WI-5835 (59% loss), is located within intron 2 of the CDH11 gene. The two other predicted genes in the area are LOC390735, almost 1 Mbp away from CDH11, and LOC283867, which extends beyond the minimal region of loss. Markers D16S186 and SHGC-140394 that surround the hotspot marker showed only 19% and 27% loss, respectively. tumors. Expression studies in both human retinoblastoma and was analyzed in 41 retinoblastoma DNA samples by QM-PCR the TAg-RB transgenic murine model of retinoblastoma (mice (Fig. 1B). Sixty-eight percent (28 of 41) of the tumors showed expressing SV40 large T antigen in the retina) are consistent allelic loss (copy number V 1.2) of at least one of the five with a tumor suppressor gene role for CDH11 in retinoblastoma, markers. We have extensively applied QM-PCR technology to but CDH13 is excluded as a candidate tumor suppressor gene in the detection of exon copy number of RB1 in clinical tests for retinoblastoma. retinoblastoma families, in which we show that QM-PCR dis- tinguishes correctly between samples that have two, one, or no allele with 97.5% accuracy (23). We optimized the con- Results ditions for QM-PCR for five 16q22 STS markers in four sepa- Chromosome 16q Allelic Loss in Retinoblastoma rate runs. With nontumor DNA set to two copies, the one-copy To define the minimal region of loss at 16q in retinoblas- control (RB264) gave a calculated copy number for all 16q STS toma, 30 pairs of normal/tumor DNA were tested for LOH at loci of 0.78 F 0.22 (mean F SD). The frequency of allelic loss seven microsatellite markers spanning 16q21-23.3, which were for each locus varied from 19% to 54% in 41 tumors (Fig. 1B). heterozygous and therefore informative in a mean of 85% Marker WI-5835 (corresponding to position 16q22.1) showed (ranging from 59% for D16S496 to 100% for D16S514) of the highest frequency of loss (54%, 22 of 41). However, when the retinoblastoma patients. Of the 30 samples, 16 (53%) dis- the data from all markers in individual tumors were examined, played LOH for at least one of the seven markers analyzed it was evident that an additional five tumors not tested with (Fig. 1B). During these LOH studies, microsatellite instability WI-5835 showed a pattern of loss at adjacent markers, consis- (novel fragments in PCR products of tumor DNA compared tent with WI-5835 also being lost, so we might interpolate that with reference DNA) was observed for only two of the seven 59% (27 of 46) of tumors showed allelic loss for this marker microsatellite markers in only 3 of 210 (1.4%) assays.