Minimal 16Q Genomic Loss Implicates Cadherin-11 in Retinoblastoma

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

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. The copy (Fig. 1B). Six tumors showed allelic loss of only WI-5835 and number of five sequence-tagged site (STS) markers on 16q no other marker (Fig. 2A).

Marker WI-5835 is located within intron 2 of the CDH11 Expression of Candidate Tumor Suppressor Genes gene (Fig. 1C). To better evaluate allelic loss and genomic Protein expression of candidate tumor suppressor genes aberrations of CDH11, QM-PCR was done on the first six exons was examined by immunoblot in healthy adult human retina of this gene in 45 additional retinoblastoma samples, 15 of (HR) and 17 retinoblastoma tumors and 5 retinoblastoma cell which were included in the initial QM-PCR analysis (Fig. 2A). lines. CDH11 has three isoforms: an intact form (i), a splice Overall, 54% (38 of 71) of tumors showed one copy of WI-5835 variant (v) that produces a truncated protein, and a secreted in intron 2 of CDH11. This number increased to 58% (41 of 71) form (s) that is most probably derived by proteolysis of the when those tumors showing loss of one or more exons, without intact form (24). WI-5835 loss, were included (Fig. 2B). Of the 30 tumors not CDH11(i) protein was expressed invariably in at least 10 previously tested, 57% (17 of 30) showed loss of WI-5835 and/ healthy HRs tested (Fig. 3A and B; data not shown). Expression or CDH11 exons (Fig. 2B). Moreover, three tumors in this data of CDH11(i) in retinoblastoma tumors RB1355 and RB1531 set showed deletions only of WI-5835, without flanking exonic was comparable with HR because the HR lane was relatively deletions (Fig. 2A, asterisks). However, of samples that had overloaded (Fig. 3A). However, most (91%, 20 of 22) retino- previously showed two copies of WI-5835, only 13% (2 of 15) blastoma tumors and cell lines showed either complete loss showed one copy of CDH11 exons (Fig. 2B). (6 of 22) or decreased expression (14 of 22) of CDH11(i) The polymorphic marker D16S422 within the CDH13 gene compared with HR (Fig. 3A and B). A lower band expressed in at 16q23.3 defined a second hotspot for loss (39%, 9 of 23 the majority of tumors and cell lines (8 of 13) corresponded to retinoblastoma tumors; Fig. 1B) and was the sole loss in two either the variant or the secreted form of CDH11 (Fig. 3A). tumors (data not shown). A third apparent region of high Copy number analysis showed that 10 of 12 retinoblastoma frequency loss was suggested by D16S398 (39%, 11 of 28 with two copies of WI-5835 expressed reduced/loss of CDH11, tumors; Fig. 1B). Each tumor showed an overall pattern whereas 9 of 9 retinoblastoma with WI-5835 allelic loss ex- consistent with D16S398 being lost in conjunction with pressed reduced/loss of CDH11 (Fig. 3A and B). All 22 retino- flanking markers within large deletions. Therefore, the evidence blastoma tumors and cell lines showed expression of CDH13 for a tumor suppressor gene close to D16S398 is less than that comparable with healthy HR (Fig. 3A and B), suggesting that for WI-5835 or D16S422. We found no difference in the CDH13 loss is most likely not involved in retinoblastoma frequency of 16q allelic loss in retinoblastoma tumors from tumorigenesis. males and females or patients with or without RB1 germline Mice have only an intact form of CDH11, which displays mutations (data not shown). 97% amino acid similarity to human CDH11(i) (25). TAg-RB

FIGURE 2. CDH11 is deleted in retinoblastoma. A. Schematic of allelic loss of 16q22 in 71 retinoblastoma. Each row is one tumor. Black cells, allelic loss for the marker tested (columns with S for ‘‘SHGC’’) by QM- PCR for STS markers or exons of CDH11 (copy number V 1.2); gray cells, two copies were present (copy number > 1.2). QM-PCR was initially (Fig. 1B) done on 41 samples (a + b), of which 15 (b) were also assessed for copy number for the first six exons of CDH11. Subsequently, 30 more tumors were assessed for WI-5835 and exons of CDH11 (c). Three tumors (*) have loss of only the intron 2 marker, WI-5835. B. Fifty-eight percent of 71 samples (a + b + c) showed one copy of the STS WI-5835 in intron 2 and/or exons of CDH11. Of 15 samples that showed two copies of WI-5835 (b), 2 (13%) were one copy for CDH11 exons. Of 30 previously untested tumors, 17 (57%) showed one copy of CDH11.

FIGURE 3. CDH11 and CDH13 expression in retinoblastoma. A and B. Immunoblot analysis showed expression of CDH11(i) in healthy adult HR, whereas 20 of 22 retinoblastoma tumors and cell lines showed either a decrease compared with the HR (14 of 22) or a complete loss of CDH11(i) (6 of 22). h-tubulin was probed as loading control. Tumors RB1355 and RB1531 showed normal levels of CDH11. WI-5835 copy number, determined by QM-PCR, is above the tumor names. DNA was unavailable for RB1807. Ten of 12 retinoblastoma with two copies of WI-5835 expressed reduced/loss of CDH11, whereas 9 of 9 retinoblastoma with WI-5835 allelic loss expressed reduced/loss of CDH11. All retinoblastoma tumors and cell lines examined showed normal uniform expression of CDH13 similar to healthy retina. A. CDH11 was detected with OB-cadherin monoclonal antibody. A lower band expressed in most tumors and cell lines (8 of 13) corresponded to either the variant (v) or the secreted (s) form of CDH11 running at 85 or 80 kDa, respectively (not shown on B). B. CDH11 was detected with mouse monoclonal CDH11 (CDH113H); all three antibodies were probed on the same nitrocellulose membrane separately. C. By RT-PCR, three of eight TAg-RB tumors showed reduced or loss of expression of CDH11. Loading control: TATA box binding protein (TBP). D. By RT-PCR, adult mouse retina expressed CDH13 mRNA at a low level; eight of eight TAg-RB tumors expressed CDH13 at levels similar to normal mouse brain. -RT, control containing no reverse transcriptase; loading control: glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

tumors were studied for expression of CDH11 and CDH13. intense expression of CDH11 in small TAg-RB at age 4 weeks Three of eight advanced TAg-RB tumors exhibited little or (Fig. 4E) and a decreased expression of CDH11 was seen by no CDH11 mRNA by reverse transcription-PCR (RT-PCR; 21 weeks (Fig. 4F). Two human retinoblastomas were studied Fig. 3C), whereas all eight tumors displayed similar CDH13 for expression of CDH11. One showed expression of CDH11 in mRNA levels (Fig. 3D). all tumor areas, whereas the other showed expression in some tumor regions, with loss in focal areas (data not shown).

Developmental Expression of CDH11 in the Retina CDH11 mRNA expression levels, measured by RT-PCR, decreased during murine retinal development from embryonic Discussion day E15 to postnatal day P4, suggesting that CDH11 plays an 16q22 Loss in Retinoblastoma Narrowed to Candidate important role in the developing murine retina (Fig. 4A). Genes CDH11 and CDH13 CDH11 protein levels remained relatively constant up to post- Cytogenetic studies show several recurrent chromosomal natal day P7 (Fig. 4B), when levels seemed to decrease and abnormalities in human retinoblastoma: 6p+ (45%), 1q+ (44%), then increase again in adult retina. A longer protein than monosomy 13 (21%), monosomy 16 (18%), 1p+ (13%), and mRNA half-life may explain the presence of protein several homogeneously staining regions and double minutes (9%; days after the loss of message. ref. 3). CGH studies of retinoblastoma corroborate the cytogenetic studies (7-11). The most common genomic loss in retinoblastoma detected by CGH is at chromosome 16. This Immunohistochemistry of CDH11 loss may represent monosomy of the entire chromosome, or Both the ganglion cell layer and the inner nuclear layer loss of part of, or the entire long arm (Fig. 1A). of healthy adult human (Fig. 4C) and murine (Fig. 4D) retina The combination of LOH and QM-PCR with the STS used stained for CDH11 protein. Immunohistochemistry showed here indicated 59% allelic loss within a 2.62 Mbp minimal

region between markers flanking WI-5835 at 16q22.1 and with one copy of WI-5835 seemed more likely (9 of 9) not to 39% allelic loss in two other separate regions at 16q22-23.3 show expression of protein than tumors with two copies (10 of (Fig. 1B). The QM-PCR for STSs was much more efficient 12; Fig. 3A and B). If CDH11 is a tumor suppressor gene, one than LOH studies of polymorphic markers (Fig. 1B) because allele may be deleted and the other may contain an internal LOH is dependent on the constitutional cells of the patient being mutation that renders the residual expressed protein inactive. heterozygous; therefore, many samples are uninformative. We hypothesize that those samples showing loss of protein The most commonly deleted individual marker, WI-5835 expression without copy number loss at WI-5835 may have (54%, 38 of 71 total tumors), is within intron 2 of CDH11 point mutations or small intragenic deletions undetectable by (Fig. 1C). CDH11 is the only cloned gene from the 2.62 Mbp the QM-PCR protocol used here. Sequencing of the CDH11 minimal region, although there are two other predicted genes in gene is under way for these samples. the region (Fig. 1C), LOC390735 (similar to ribosomal pro- In the present study, we found that CDH11 mRNA levels tein S15a) and LOC283867 (hypothetical protein LOC283867; decreased in the maturing murine retina (Fig. 4A and B), National Center for Biotechnology Information Human suggesting an important role during retinal development, as has Genome Build 34.3). By combining samples studied with been shown for neuronal cadherin. Neuronal cadherin is an QM-PCR of STS and CDH11 exons, we estimate that 54% to essential cell adhesion molecule, with a role necessary for 59% of retinoblastoma tumors showed loss within CDH11. The retinal lamination (26). Previous studies in chicken reveal its three tumors that showed deletion of WI-5835, within intron importance for the maintenance of the overall structure of the 2ofCDH11, without exonic deletions (Fig. 2A) are a tantaliz- undifferentiated retina (27). ing suggestion of small intragenic deletions of CDH11 in some The laminar structure of the retina consists of three layers retinoblastoma. Such deletions could cause aberrant splicing of cell bodies named from surface toward center of the eye: and truncated proteins. outer nuclear layer, inner nuclear layer, and ganglion cell layer, separated by the outer and inner plexiform layers. Retinoblas- toma is initiated in the inner nuclear layer of the retina in a cell CDH11 Expression in Retinoblastoma and Retinal of origin that is as yet unidentified. Here, we show that Development CDH11 is highly expressed in a subset of cells of the inner Of human retinoblastoma examined, 91% displayed either nuclear layer of the retina (Fig. 4C and D). These expression a decrease or a complete loss of CDH11(i) protein expression studies concur with data reported by Honjo et al. (28), who compared with healthy HR (Fig. 3A and B). Correlation of suggested that CDH11 is expressed by Mu¨ller glia cells, copy number analysis with immunoblot data showed a pattern whereby it may contribute to regulation of retinal architecture consistent with the loss of a tumor suppressor gene. Tumors as a cell-cell adhesion molecule. Mu¨ller glia cells, with cell

FIGURE 4. Temporal and spatial expression of CDH11 in the retina and TAg-RB. A. RT-PCR analyses showed that expression of CDH11 mRNA decreased during mouse retinal development. Positive control: CDH11 cDNA; loading control: glyceraldehyde 3-phosphate dehydrogenase (GAPDH). B. CDH11 protein levels seem to be consistent at earlier stages but decrease at P7 and then increase again in adulthood. Positive control: adult mouse retinal (AMR) protein; loading control: h-tubulin. C and D. Retinal sections were stained with antibodies against CDH11. Adult HR (C) and adult murine retina (D) showed expression of CDH11 in a subset of cells in the inner nuclear layer (INL) and ganglion cell layer (GCL). ONL, outer nuclear layer. E and F. TAg-RB eyes double stained for CDH11 and TAg. E. Tumors in 4-week-old mice showed the highest level of expression of CDH11 coex- pressing with TAg. F. This level decreases in tumors of 21-week-old mice.

bodies in the inner nuclear layer but processes spanning CDH13 Is Excluded as a Candidate Tumor Suppressor the entire retina, are crucial to the development of retinal Gene in Retinoblastoma architecture; disaggregated developing retina forms laminated The second most frequently deleted (39%) 16q marker in rosettes only in the presence of Mu¨ller glia cells or Mu¨ller glia retinoblastoma was D16S422, within intron 2 of the CDH13 cell factors (29). gene. Because D16S422 was the most telomeric marker exam- Early-stage tumors of TAg-RB mice showed surprisingly ined, the telomeric boundary of this minimal region of loss high levels of expression of CDH11 (Fig. 4E). Larger tumors, at cannot be determined from our data. However, the centromeric 21 weeks, showed decreased CDH11 expression (Fig. 4F) and boundary is the flanking marker D16S511. In the 1.2 Mbp much more advanced tumors showed complete loss/decrease region between D16S511 and D16S422 lies one predicted gene, of CDH11 mRNA expression in three of eight tested (Fig. 3C). LOC388301, and five cloned genes aside from CDH13: CMIP, These analyses suggest that the cell of origin of retinoblastoma PLCG2, HSPC105, HSD17B2, and MPHOSPH6 (National is a CDH11-expressing cell and that loss of CDH11 occurs at Center for Biotechnology Information Human Genome Build a late stage of tumor progression. This assertion is supported 34.3). We have shown that all human and murine retinoblastoma by focal loss of CDH11 expression in one of two human tumors or cell lines tested expressed normal levels of CDH13 retinoblastoma examined (data not shown). Analysis of more (Fig. 3A, B, and D). We conclude that loss of CDH13 is not advanced tumors is necessary to confirm that CDH11 loss is a involved in human or murine retinoblastoma development. late event. However, the data obtained thus far are consistent However, we cannot currently rule out a role for loss of a with the hypothesis that CDH11 is present in the growing tumor closely linked gene, such as one of the candidates above, in and is lost in some advanced tumors. retinoblastoma.

Possible Role for CDH11 Isoforms in Tumor Invasiveness Summary and Conclusion Loss of CDH11, cell-cell adhesion, and subsequent The expression studies presented here are qualitative, but they downstream signals to apoptosis may play a role in progression do concur with a more quantitative technique, QM-PCR. By QM- of some RB1À/À developing retinal cells to malignancy. Studies PCR, we were able to show that, of a total of 71 tumors tested, of CDH11 knockout mice suggest a role for CDH11 in regu- more than half showed loss within the CDH11 gene. This high lation of osteoblast differentiation; no retinal phenotype was proportion strongly supports a role for CDH11 deletion as one of reported (30). These mice do not seem to have increased tumor the possible M3 to Mn events in retinoblastoma. Moreover, our predisposition, but CDH11 knockout mice crossed with TAg- expression and developmental data suggest that early retinoblas- RB mice are yet to be described. We do not propose that toma tumors are CDH11 positive, having both copies of the CDH11 loss would initiate retinoblastoma, only that it is a later CDH11 gene, but as these tumors progress, loss of CDH11, mutational event (M3 to Mn) that may contribute to tumor manifested as deletion of one allele or LOH, occurs. This idea is progression after mutation of both RB1 alleles, resulting in consistent with the features identifying tumor suppressor genes. earlier, faster developing tumors. Although our data do not yet provide functional evidence that CDH11(v) is generated from a splice variant that causes CDH11 acts as a tumor suppressor gene in retinoblastoma, they do a frameshift, resulting in a truncated protein. It shows no provide strong genomic expression and developmental evidence homophilic cell-cell adhesion property but is not considered toward this conclusion. Despite the rarity of retinoblastoma, it once a dominant negative because it has been shown to assist in cell- again offers a unique opportunity to define genes that may be cell adhesion of the intact form and does not disrupt the important in human malignancy. intact form when both forms are coexpressed at similar levels (24, 31). It has been suggested that CDH11(s) is derived from Materials and Methods the intact form via proteinase cleavage, although, to date, it Clinical Samples and Retinoblastoma Cell Lines has no known function (24, 31). Low levels of CDH11(i) With approval of the research ethics boards of the Wellesley have been detected in osteosarcoma compared with osteoblasts, Hospital, the Hospital for Sick Children, and the University of which normally express high levels of CDH11(i), interpreted Toronto, parental consent was obtained to use tumor and blood to suggest that reduced expression is associated with local for research. Retinoblastoma tumor cells were obtained from invasion of osteosarcoma (24). CDH11(v) is highly expressed eyes removed as part of therapy. For some children, tumor was in invasive breast cancer and is associated with promotion first used to determine the RB1 mutation for the purpose of of invasiveness (31). Other cadherins (CDH1 and neuronal genetic counseling. Normal HRs were obtained from the cadherin) do contribute to tumor cell invasion (32-34). We Eye Bank of Canada, with research ethics board approval for hypothesize that loss of CDH11(i) contributes to loss of cell- anonymous use of discarded specimens. Analyses were done on cell adhesion in the developing retina contributing to tumor subsets of 91 retinoblastoma tumors and 30 matched lympho- development and progression. Because a prominent clinical cyte specimens from retinoblastoma patients as well as five feature of retinoblastoma is dissociation of tumor cells and dis- retinoblastoma cell lines: Y79, WERI-RB1, RB247, RB409, persion within the eye, for example, as vitreous ‘‘seeds,’’ gain and RB1021. The samples were from patients (44 male, 52 of CDH11(v) expression may also promote retinoblastoma female), 21 with bilateral retinoblastoma and 75 with unilateral invasiveness, metastasis, and vitreous seeding. The most com- retinoblastoma. CGH results from 31 of the tumors (7) and mon secondary malignant neoplasm seen in patients with RB1 the karyotypes of tumors/cell lines RB247, RB383, RB386, germline mutations is osteosarcoma (35), so potentially CDH11 RB409, RB445 (3), Y79 (36), and WERI-RB1 (37) were plays a similar role in both tumors. published previously.

LOH Analysis The areas under all STS marker peaks were measured and The chromosomal location and the relative order of each subsequent calculations were done with Gene Objects 3.1 marker used in LOH or QM-PCR analysis were obtained from the software (Visible Genetics). The copy number of each 16q STS Human Map View of the National Center for Biotechnology marker was estimated from the calculated ratio of the areas under Information Web site (http://www.ncbi.nlm.nih.gov). For LOH the 16q peaks to the area of the 10q peak control STS marker, analysis, seven microsatellite markers located on chromosome which was set to two copies, compared with similar ratios in a 16q21-23.3 (marker name, distance from the chromosome 16p normal DNA sample. Two external controls were included in telomere based on National Center for Biotechnology Information each QM-PCR run: a two-copy control DNA from a normal male Human Genome Build 34.3: D16S514, 62,113 kbp; D16S186, and one retinoblastoma (RB264), which had loss of 16q11-22 by 65,358 kbp; D16S398, 65,906 kbp; D16S496, 68,725 kbp; CGH (7) and LOH of 16q22. We defined allelic loss as occurring D16S515, 76,297 kbp; D16S511, 81,481 kbp; D16S422, 82,691 when the copy number for an STS marker was V1.2. kbp) were assayed by PCR using a Robocycler (Stratagene, La To analyze copy number of the CDH11 exons, a similar Jolla, CA) with conditions and magnesium concentrations QM-PCR protocol was employed on 45 retinoblastoma tumors, optimized for each marker. The majority of markers were 15 of which were included in the initial QM-PCR study. WI-5835 copy number in the remaining 30 samples, as well as samples amplified for 10 minutes at 94jC for one cycle followed by a used in immunoblot analyses (Fig. 3A and B), was determined three-step amplification of 30 seconds at 94jC, 23 seconds at the in independent assays. We used the following CDH11 exon- appropriate annealing temperature, and 23 seconds at 72jC for 30 specific primers: exon 1, forward 5V-CCTCAGCAAGACC- cycles. A final extension at 72jC was for 4 minutes. The products ACCGT-3V and reverse 5V-ACAGACGACTATTCCAATG-3V; were denatured for 5 minutes at 94jC and run on a 6% poly- exon 2, forward 5V-GAGTTTCCCCACTGCTTT-3V and reverse acrylamide gel (8 mol/L urea, 1Â Tris-borate EDTA) at 200 V 5V-CCACCACAGAGACACCAG-3V; exon 3, forward 5V-TGC- on a vertical gel electrophoresis system (Bethesda Research CTTCTTCAGGGTTAA-3V and reverse 5V-GGCCTGGGACT- Laboratories, Inc., Carlsbad, CA). The gel was stained in 0.12% TCTTATT-3V; exon 4, forward 5V-GGCATGGGATATTTTA- AgNO3 for 30 minutes and developed in a solution of 3% NaOH TTCAG-3V and reverse 5V-AGGAATAGGTTAGAGGAGGG-3V; and 0.18% formaldehyde for 30 to 50 seconds (38). The stained gel exon 5, forward 5V-ACAGCTGCCAAATAAAAGAG-3V and was dried using a gel-drying apparatus (Promega, Madison, WI). reverse 5V-CCCTGTTTCTTGCAGTGT-3V; and exon 6, forward Lymphocyte and tumor DNA from each patient were analyzed in 5V-CGGGTCTTTCTCTTCCAT-3V and reverse 5V-GT- adjacent lanes. Heterozygous alleles in lymphocyte DNA were TCCTACAGGGCTTTCC-3V. We amplified the first 6 of the 14 considered informative and the tumor was scored for LOH. CDH11 exons and the 10q internal control marker simultaneously in a 25 AL total reaction volume containing 250 ng of Quantitative Multiplex PCR template DNA, 30 to 50 ng of each primer, 1Â Taq polymerase We selected five STS from the National Center for buffer, 2.5 mmol/L deoxynucleoside triphosphates, and 1 unit Biotechnology Information UniSTS (http://www.ncbi.nlm.nih. Taq polymerase. The PCR conditions included an initial gov/entrez/query.fcgi?db=unists) database (site designations, denaturing step at 94jC for 2 minutes and 19 cycles of 94jC distance from the chromosome 16p telomere: SHGC-140394, for 30 seconds, 54jC for 30 seconds, and 70jC for 1 minute 62,742 kbp; WI-5835, 64,856 kbp; A005R23, 68,377 kbp; followed by a final extension at 70jC for 4 minutes. SHGC-84108, 73,559 kbp; SHGC-155721, 76,281 kbp). The Electrophoresis and analysis were done as above. selected markers were without known polymorphisms and had PCR products longer than 200 bp. For an internal control, the RT-PCR Analyses STS marker WI-7221 at 10q21 was selected because our TAg-RB tumor (mice were a gift from Dr. Joan O’Brien previous CGH work indicated that chromosome 10 was not (39) and B6 wild-type mouse retina total RNA were prepared by involved in gains or losses in retinoblastoma (7); therefore, this TRIzol extraction. One microgram was used for the production of STS could be expected to consist of two copies in each single-stranded cDNA using oligo(dT) primers and SuperScript II retinoblastoma cell genome. All forward primers for the STS reverse transcriptase (Invitrogen, Carlsbad, CA). The primers markers were labeled with the fluorescent dye Cy 5.0 used for the detection of murine CDH11 cDNA by PCR were (Integrated DNA Technologies, Inc., Coralville, IA). forward 5V-ATGAAGGAGAACTACTGTTTA-3V and reverse 5V- We amplified all five STS 16q markers and the 10q internal TTAAGAGTCATCATCAAAGTG-3V,whereasthoseforCDH13 control marker simultaneously. Reactions were done in a 20 AL were forward 5V-AGTCGATAGCGACAGACC-3Vand reverse 5V- total reaction volume containing 400 ng of template DNA, 4 pmol CACTGTCAAGTTGACAAT-3V. Glyceraldehyde 3-phosphate of each primer, 2Â Taq polymerase buffer, 4 mmol/L dehydrogenase (forward primer 5V-ACTGGTGCTGC- deoxynucleoside triphosphates, and 1 unit Taq polymerase. CAAGGCT-3V and reverse primer 5V-TGGAGGCCATGTAGG The template was amplified for only 18 cycles to maintain CCA-3V) and TATA box binding protein (forward primer 5V- linearity of generation of the PCR products. The PCR conditions CAGCCTTCCACCTTATGCTC-3V and reverse primer 5V-TGG- included an initial denaturing step at 94jC for 10 minutes and 18 TCTTCCTGAATCCCTTT-3V) were used as loading controls. cycles of 94jC for 30 seconds, 60jC for 30 seconds, and 72jC for 30 seconds using a PTC 100 thermal cycler (MJ Research, Watertown, MA). The PCR products were resolved on a 6% Immunoblot Analyses denaturing polyacrylamide gel using a Micro-Gene Clipper Total protein was extracted from normal HR, 17 human retin- sequencer (Visible Genetics, Inc., Toronto, Ontario, Canada). oblastoma tumors, 5 human retinoblastoma cell lines, B6 wild-type

mouse retina at seven time points (embryonic days E15 and E18; Acknowledgments postnatal days P0, P2, P4, and P7; and adult), and TAg-RB. We thank Suzanne Richter, Dr. Sanja Pajovic, Dr. Vivette Brown, and Clarellen Spencer for technical advice and Dr. Kirk Vandezande for assistance in the Samples were mixed with cold TNE buffer [2% NP40, 20 mmol/L analysis of QM-PCR. Tris (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L NaN3, 0.1 mmol/L phenylmethylsulfonyl fluoride, 2 Ag/mL A leupeptin, and 20 g/mL aprotinin] and incubated for 1 hour at References 4jC on a rotor mixer. After centrifugation at 12,600 Â g for 20 1. Godbout R, Dryja TP, Squire J, Gallie BL, Phillips RA. Somatic inactivation minutes, supernatants were recovered and protein concentrations of genes on chromosome 13 is a common event in retinoblastoma. Nature 1983; were determined using a Bradford protein assay (Bio-Rad, 304:451 – 3. Hercules, CA). Proteins (50 Ag) were separated by 7% SDS-PAGE 2. 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Mol Cancer Res 2004;2(9). September 2004 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. Minimal 16q Genomic Loss Implicates Cadherin-11 in Retinoblastoma 11Vision Science Research Program of the University of Toronto (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 Terry Fox Run and the Canadian Cancer Society (B.L. Gallie), Canadian Genetics Disease Network (B.L. Gallie), Canadian Institutes for Health Research (B.L. Gallie), Royal Arch Masons of Canada, Keene Annual Perennial Plant Sale, and Jeff Healy Christmas Concert.

Mellone N. Marchong, Danian Chen, Timothy W. Corson, et al.

Mol Cancer Res 2004;2:495-503.

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