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1 Divisions of Cancer Informatics and Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada; Departments of 2 Medical Biophysics, 3 Molecular and Medical Genetics, and 4 Ophthalmology, University of Toronto, Toronto, Ontario, Canada; 5 Retinoblastoma Solutions, Toronto, Ontario, Canada; and 6 Department of Ophthalmology, West China Hospital, Faculty of Medicine, Sichuan University, Chengdu, People's Republic of China
Requests for reprints: Brenda L. Gallie, Division of Cancer Informatics, Room 8-415, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2324; Fax: 416-946-4619. E-mail: gallie{at}attglobal.net
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Introduction |
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To identify these mutational events, we have previously analyzed chromosomal alterations in 50 retinoblastoma tumors by comparative genomic hybridization (CGH) analysis (7). The minimal region most frequently lost was detected at 16q22 (14%; ref. 7). Cumulatively, 51 of 162 (31%) tumors in five different CGH studies showed a common region of loss at 16q22 (7-11) as summarized in Fig. 1A. Allelic loss on 16q22 has also been frequently shown in liver (12), breast (13), prostate (14), and Wilms' (15) tumors, suggesting the presence of important tumor suppressor gene(s) in this chromosomal region.
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Detection of chromosomal loss using CGH has a limit of resolution of 
10 to 20 Mbp (22). We have been able to narrow the limit of resolution to the level of single candidate tumor suppressor genes by combining loss of heterozygosity (LOH) analysis with quantitative multiplex PCR (QM-PCR). Two candidate tumor suppressor genes, cadherin-11 (CDH11) and cadherin-13 (CDH13), emerged from a study of retinoblastoma tumors. Expression studies in both human retinoblastoma and the TAg-RB transgenic murine model of retinoblastoma (mice expressing SV40 large T antigen in the retina) are consistent with a tumor suppressor gene role for CDH11 in retinoblastoma, but CDH13 is excluded as a candidate tumor suppressor gene in retinoblastoma.
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1.2) of at least one of the five markers. We have extensively applied QM-PCR technology to the detection of exon copy number of RB1 in clinical tests for retinoblastoma families, in which we show that QM-PCR distinguishes correctly between samples that have two, one, or no allele with 97.5% accuracy (23). We optimized the conditions for QM-PCR for five 16q22 STS markers in four separate runs. With nontumor DNA set to two copies, the one-copy control (RB264) gave a calculated copy number for all 16q STS loci of 0.78 ± 0.22 (mean ± SD). The frequency of allelic loss for each locus varied from 19% to 54% in 41 tumors (Fig. 1B). Marker WI-5835 (corresponding to position 16q22.1) showed the highest frequency of loss (54%, 22 of 41). However, when the data from all markers in individual tumors were examined, it was evident that an additional five tumors not tested with WI-5835 showed a pattern of loss at adjacent markers, consistent with WI-5835 also being lost, so we might interpolate that 59% (27 of 46) of tumors showed allelic loss for this marker (Fig. 1B). Six tumors showed allelic loss of only WI-5835 and no other marker (Fig. 2A).
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The polymorphic marker D16S422 within the CDH13 gene at 16q23.3 defined a second hotspot for loss (39%, 9 of 23 retinoblastoma tumors; Fig. 1B) and was the sole loss in two tumors (data not shown). A third apparent region of high frequency loss was suggested by D16S398 (39%, 11 of 28 tumors; Fig. 1B). Each tumor showed an overall pattern consistent with D16S398 being lost in conjunction with flanking markers within large deletions. Therefore, the evidence for a tumor suppressor gene close to D16S398 is less than that for WI-5835 or D16S422. We found no difference in the frequency of 16q allelic loss in retinoblastoma tumors from males and females or patients with or without RB1 germline mutations (data not shown).
Expression of Candidate Tumor Suppressor Genes
Protein expression of candidate tumor suppressor genes was examined by immunoblot in healthy adult human retina (HR) and 17 retinoblastoma tumors and 5 retinoblastoma cell lines. CDH11 has three isoforms: an intact form (i), a splice variant (v) that produces a truncated protein, and a secreted form (s) that is most probably derived by proteolysis of the intact form (24).
CDH11(i) protein was expressed invariably in at least 10 healthy HRs tested (Fig. 3A and B; data not shown). Expression of CDH11(i) in retinoblastoma tumors RB1355 and RB1531 was comparable with HR because the HR lane was relatively overloaded (Fig. 3A). However, most (91%, 20 of 22) retinoblastoma tumors and cell lines showed either complete loss (6 of 22) or decreased expression (14 of 22) of CDH11(i) compared with HR (Fig. 3A and B). A lower band expressed in the majority of tumors and cell lines (8 of 13) corresponded to either the variant or the secreted form of CDH11 (Fig. 3A). Copy number analysis showed that 10 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 (Fig. 3A and B). All 22 retinoblastoma tumors and cell lines showed expression of CDH13 comparable with healthy HR (Fig. 3A and B), suggesting that CDH13 loss is most likely not involved in retinoblastoma tumorigenesis.
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Developmental Expression of CDH11 in the Retina
CDH11 mRNA expression levels, measured by RT-PCR, decreased during murine retinal development from embryonic day E15 to postnatal day P4, suggesting that CDH11 plays an important role in the developing murine retina (Fig. 4A). CDH11 protein levels remained relatively constant up to postnatal day P7 (Fig. 4B), when levels seemed to decrease and then increase again in adult retina. A longer protein than mRNA half-life may explain the presence of protein several days after the loss of message.
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The combination of LOH and QM-PCR with the STS used here indicated 59% allelic loss within a 2.62 Mbp minimal region between markers flanking WI-5835 at 16q22.1 and 39% allelic loss in two other separate regions at 16q22-23.3 (Fig. 1B). The QM-PCR for STSs was much more efficient than LOH studies of polymorphic markers (Fig. 1B) because LOH is dependent on the constitutional cells of the patient being heterozygous; therefore, many samples are uninformative.
The most commonly deleted individual marker, WI-5835 (54%, 38 of 71 total tumors), is within intron 2 of CDH11 (Fig. 1C). CDH11 is the only cloned gene from the 2.62 Mbp minimal region, although there are two other predicted genes in the region (Fig. 1C), LOC390735 (similar to ribosomal protein S15a) and LOC283867 (hypothetical protein LOC283867; National Center for Biotechnology Information Human Genome Build 34.3). By combining samples studied with QM-PCR of STS and CDH11 exons, we estimate that 54% to 59% of retinoblastoma tumors showed loss within CDH11. The three tumors that showed deletion of WI-5835, within intron 2 of CDH11, without exonic deletions (Fig. 2A) are a tantalizing suggestion of small intragenic deletions of CDH11 in some retinoblastoma. Such deletions could cause aberrant splicing and truncated proteins.
CDH11 Expression in Retinoblastoma and Retinal Development
Of human retinoblastoma examined, 91% displayed either a decrease or a complete loss of CDH11(i) protein expression compared with healthy HR (Fig. 3A and B). Correlation of copy number analysis with immunoblot data showed a pattern consistent with the loss of a tumor suppressor gene. Tumors with one copy of WI-5835 seemed more likely (9 of 9) not to show expression of protein than tumors with two copies (10 of 12; Fig. 3A and B). If CDH11 is a tumor suppressor gene, one allele may be deleted and the other may contain an internal mutation that renders the residual expressed protein inactive. We hypothesize that those samples showing loss of protein expression without copy number loss at WI-5835 may have point mutations or small intragenic deletions undetectable by the QM-PCR protocol used here. Sequencing of the CDH11 gene is under way for these samples.
In the present study, we found that CDH11 mRNA levels decreased in the maturing murine retina (Fig. 4A and B), suggesting an important role during retinal development, as has been shown for neuronal cadherin. Neuronal cadherin is an essential cell adhesion molecule, with a role necessary for retinal lamination (26). Previous studies in chicken reveal its importance for the maintenance of the overall structure of the undifferentiated retina (27).
The laminar structure of the retina consists of three layers of cell bodies named from surface toward center of the eye: outer nuclear layer, inner nuclear layer, and ganglion cell layer, separated by the outer and inner plexiform layers. Retinoblastoma is initiated in the inner nuclear layer of the retina in a cell of origin that is as yet unidentified. Here, we show that CDH11 is highly expressed in a subset of cells of the inner nuclear layer of the retina (Fig. 4C and D). These expression studies concur with data reported by Honjo et al. (28), who suggested that CDH11 is expressed by Müller glia cells, whereby it may contribute to regulation of retinal architecture as a cell-cell adhesion molecule. Müller glia cells, with cell bodies in the inner nuclear layer but processes spanning the entire retina, are crucial to the development of retinal architecture; disaggregated developing retina forms laminated rosettes only in the presence of Müller glia cells or Müller glia cell factors (29).
Early-stage tumors of TAg-RB mice showed surprisingly high levels of expression of CDH11 (Fig. 4E). Larger tumors, at 21 weeks, showed decreased CDH11 expression (Fig. 4F) and much more advanced tumors showed complete loss/decrease of CDH11 mRNA expression in three of eight tested (Fig. 3C). These analyses suggest that the cell of origin of retinoblastoma is a CDH11-expressing cell and that loss of CDH11 occurs at a late stage of tumor progression. This assertion is supported by focal loss of CDH11 expression in one of two human retinoblastoma examined (data not shown). Analysis of more advanced tumors is necessary to confirm that CDH11 loss is a late event. However, the data obtained thus far are consistent with the hypothesis that CDH11 is present in the growing tumor and is lost in some advanced tumors.
Possible Role for CDH11 Isoforms in Tumor Invasiveness
Loss of CDH11, cell-cell adhesion, and subsequent downstream signals to apoptosis may play a role in progression of some RB1–/– developing retinal cells to malignancy. Studies of CDH11 knockout mice suggest a role for CDH11 in regulation of osteoblast differentiation; no retinal phenotype was reported (30). These mice do not seem to have increased tumor predisposition, but CDH11 knockout mice crossed with TAg-RB mice are yet to be described. We do not propose that CDH11 loss would initiate retinoblastoma, only that it is a later mutational event (M3 to Mn) that may contribute to tumor progression after mutation of both RB1 alleles, resulting in earlier, faster developing tumors.
CDH11(v) is generated from a splice variant that causes a frameshift, resulting in a truncated protein. It shows no homophilic cell-cell adhesion property but is not considered a dominant negative because it has been shown to assist in cell-cell adhesion of the intact form and does not disrupt the intact form when both forms are coexpressed at similar levels (24, 31). It has been suggested that CDH11(s) is derived from the intact form via proteinase cleavage, although, to date, it has no known function (24, 31). Low levels of CDH11(i) have been detected in osteosarcoma compared with osteoblasts, which normally express high levels of CDH11(i), interpreted to suggest that reduced expression is associated with local invasion of osteosarcoma (24). CDH11(v) is highly expressed in invasive breast cancer and is associated with promotion of invasiveness (31). Other cadherins (CDH1 and neuronal cadherin) do contribute to tumor cell invasion (32-34). We hypothesize that loss of CDH11(i) contributes to loss of cell-cell adhesion in the developing retina contributing to tumor development and progression. Because a prominent clinical feature of retinoblastoma is dissociation of tumor cells and dispersion within the eye, for example, as vitreous "seeds," gain of CDH11(v) expression may also promote retinoblastoma invasiveness, metastasis, and vitreous seeding. The most common secondary malignant neoplasm seen in patients with RB1 germline mutations is osteosarcoma (35), so potentially CDH11 plays a similar role in both tumors.
CDH13 Is Excluded as a Candidate Tumor Suppressor Gene in Retinoblastoma
The second most frequently deleted (39%) 16q marker in retinoblastoma was D16S422, within intron 2 of the CDH13 gene. Because D16S422 was the most telomeric marker examined, the telomeric boundary of this minimal region of loss cannot be determined from our data. However, the centromeric boundary is the flanking marker D16S511. In the 1.2 Mbp region between D16S511 and D16S422 lies one predicted gene, LOC388301, and five cloned genes aside from CDH13: CMIP, PLCG2, HSPC105, HSD17B2, and MPHOSPH6 (National Center for Biotechnology Information Human Genome Build 34.3). We have shown that all human and murine retinoblastoma tumors or cell lines tested expressed normal levels of CDH13 (Fig. 3A, B, and D). We conclude that loss of CDH13 is not involved in human or murine retinoblastoma development. However, we cannot currently rule out a role for loss of a closely linked gene, such as one of the candidates above, in retinoblastoma.
Summary and Conclusion
The expression studies presented here are qualitative, but they do concur with a more quantitative technique, QM-PCR. By QM-PCR, we were able to show that, of a total of 71 tumors tested, more than half showed loss within the CDH11 gene. This high proportion strongly supports a role for CDH11 deletion as one of the possible M3 to Mn events in retinoblastoma. Moreover, our expression and developmental data suggest that early retinoblastoma tumors are CDH11 positive, having both copies of the CDH11 gene, but as these tumors progress, loss of CDH11, manifested as deletion of one allele or LOH, occurs. This idea is consistent with the features identifying tumor suppressor genes. Although our data do not yet provide functional evidence that CDH11 acts as a tumor suppressor gene in retinoblastoma, they do provide strong genomic expression and developmental evidence toward this conclusion. Despite the rarity of retinoblastoma, it once again offers a unique opportunity to define genes that may be important in human malignancy.
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Materials and Methods |
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LOH Analysis
The chromosomal location and the relative order of each marker used in LOH or QM-PCR analysis were obtained from the Human Map View of the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov). For LOH analysis, seven microsatellite markers located on chromosome 16q21-23.3 (marker name, distance from the chromosome 16p telomere based on National Center for Biotechnology Information Human Genome Build 34.3: D16S514, 62,113 kbp; D16S186, 65,358 kbp; D16S398, 65,906 kbp; D16S496, 68,725 kbp; D16S515, 76,297 kbp; D16S511, 81,481 kbp; D16S422, 82,691 kbp) were assayed by PCR using a Robocycler (Stratagene, La Jolla, CA) with conditions and magnesium concentrations optimized for each marker. The majority of markers were amplified for 10 minutes at 94°C for one cycle followed by a three-step amplification of 30 seconds at 94°C, 23 seconds at the appropriate annealing temperature, and 23 seconds at 72°C for 30 cycles. A final extension at 72°C was for 4 minutes. The products were denatured for 5 minutes at 94°C and run on a 6% polyacrylamide gel (8 mol/L urea, 1x Tris-borate EDTA) at 200 V on a vertical gel electrophoresis system (Bethesda Research Laboratories, Inc., Carlsbad, CA). The gel was stained in 0.12% AgNO3 for 30 minutes and developed in a solution of 3% NaOH and 0.18% formaldehyde for 30 to 50 seconds (38). The stained gel was dried using a gel-drying apparatus (Promega, Madison, WI). Lymphocyte and tumor DNA from each patient were analyzed in adjacent lanes. Heterozygous alleles in lymphocyte DNA were considered informative and the tumor was scored for LOH.
Quantitative Multiplex PCR
We selected five STS from the National Center for Biotechnology Information UniSTS (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unists) database (site designations, distance from the chromosome 16p telomere: SHGC-140394, 62,742 kbp; WI-5835, 64,856 kbp; A005R23, 68,377 kbp; SHGC-84108, 73,559 kbp; SHGC-155721, 76,281 kbp). The selected markers were without known polymorphisms and had PCR products longer than 200 bp. For an internal control, the STS marker WI-7221 at 10q21 was selected because our previous CGH work indicated that chromosome 10 was not involved in gains or losses in retinoblastoma (7); therefore, this STS could be expected to consist of two copies in each retinoblastoma cell genome. All forward primers for the STS markers were labeled with the fluorescent dye Cy 5.0 (Integrated DNA Technologies, Inc., Coralville, IA).
We amplified all five STS 16q markers and the 10q internal control marker simultaneously. Reactions were done in a 20 µL total reaction volume containing 400 ng of template DNA, 4 pmol of each primer, 2x Taq polymerase buffer, 4 mmol/L deoxynucleoside triphosphates, and 1 unit Taq polymerase. The template was amplified for only 18 cycles to maintain linearity of generation of the PCR products. The PCR conditions included an initial denaturing step at 94°C for 10 minutes and 18 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds using a PTC 100 thermal cycler (MJ Research, Watertown, MA). The PCR products were resolved on a 6% denaturing polyacrylamide gel using a Micro-Gene Clipper sequencer (Visible Genetics, Inc., Toronto, Ontario, Canada). The areas under all STS marker peaks were measured and subsequent calculations were done with Gene Objects 3.1 software (Visible Genetics). The copy number of each 16q STS marker was estimated from the calculated ratio of the areas under the 16q peaks to the area of the 10q peak control STS marker, which was set to two copies, compared with similar ratios in a normal DNA sample. Two external controls were included in each QM-PCR run: a two-copy control DNA from a normal male and one retinoblastoma (RB264), which had loss of 16q11-22 by CGH (7) and LOH of 16q22. We defined allelic loss as occurring when the copy number for an STS marker was 1.2.
To analyze copy number of the CDH11 exons, a similar QM-PCR protocol was employed on 45 retinoblastoma tumors, 15 of which were included in the initial QM-PCR study. WI-5835 copy number in the remaining 30 samples, as well as samples used in immunoblot analyses (Fig. 3A and B), was determined in independent assays. We used the following CDH11 exon-specific primers: exon 1, forward 5'-CCTCAGCAAGACCACCGT-3' and reverse 5'-ACAGACGACTATTCCAATG-3'; exon 2, forward 5'-GAGTTTCCCCACTGCTTT-3' and reverse 5'-CCACCACAGAGACACCAG-3'; exon 3, forward 5'-TGCCTTCTTCAGGGTTAA-3' and reverse 5'-GGCCTGGGACTTCTTATT-3'; exon 4, forward 5'-GGCATGGGATATTTTATTCAG-3' and reverse 5'-AGGAATAGGTTAGAGGAGGG-3'; exon 5, forward 5'-ACAGCTGCCAAATAAAAGAG-3' and reverse 5'-CCCTGTTTCTTGCAGTGT-3'; and exon 6, forward 5'-CGGGTCTTTCTCTTCCAT-3' and reverse 5'-GTTCCTACAGGGCTTTCC-3'. We amplified the first 6 of the 14 CDH11 exons and the 10q internal control marker simultaneously in a 25 µL total reaction volume containing 250 ng of template DNA, 30 to 50 ng of each primer, 1x Taq polymerase buffer, 2.5 mmol/L deoxynucleoside triphosphates, and 1 unit Taq polymerase. The PCR conditions included an initial denaturing step at 94°C for 2 minutes and 19 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 70°C for 1 minute followed by a final extension at 70°C for 4 minutes. Electrophoresis and analysis were done as above.
RT-PCR Analyses
TAg-RB tumor (mice were a gift from Dr. Joan O'Brien (39) and B6 wild-type mouse retina total RNA were prepared by TRIzol extraction. One microgram was used for the production of single-stranded cDNA using oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). The primers used for the detection of murine CDH11 cDNA by PCR were forward 5'-ATGAAGGAGAACTACTGTTTA-3' and reverse 5'-TTAAGAGTCATCATCAAAGTG-3', whereas those for CDH13 were forward 5'-AGTCGATAGCGACAGACC-3' and reverse 5'-CACTGTCAAGTTGACAAT-3'. Glyceraldehyde 3-phosphate dehydrogenase (forward primer 5'-ACTGGTGCTGCCAAGGCT-3' and reverse primer 5'-TGGAGGCCATGTAGGCCA-3') and TATA box binding protein (forward primer 5'-CAGCCTTCCACCTTATGCTC-3' and reverse primer 5'-TGGTCTTCCTGAATCCCTTT-3') were used as loading controls.
Immunoblot Analyses
Total protein was extracted from normal HR, 17 human retinoblastoma tumors, 5 human retinoblastoma cell lines, B6 wild-type mouse retina at seven time points (embryonic days E15 and E18; postnatal days P0, P2, P4, and P7; and adult), and TAg-RB. Samples were mixed with cold TNE buffer [2% NP40, 20 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L NaN3, 0.1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, and 20 µg/mL aprotinin] and incubated for 1 hour at 4°C on a rotor mixer. After centrifugation at 12,600 x g for 20 minutes, supernatants were recovered and protein concentrations were determined using a Bradford protein assay (Bio-Rad, Hercules, CA). Proteins (50 µg) were separated by 7% SDS-PAGE at 200 V for 1 hour and transferred to a nitrocellulose membrane. For CDH11 expression, the membrane was blocked [1% bovine serum albumin in 1x TBS/0.05% Tween 20 (TBST) or 5% Blotto (Bio-Rad)] for 1 hour and incubated with either mouse OB-cadherin monoclonal antibody (clone E-5, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal anti-CDH11 (CDH113H, 0.001 µg/µL, kindly provided by Dr. St. John, ICOS Corp., Bothell, WA) for 1 to 2 hours followed by a goat anti-mouse IgG-alkaline phosphatase conjugated secondary antibody (1:8,000, Santa Cruz Biotechnology). Proteins were visualized using an alkaline phosphatase buffer containing nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate [100 mmol/L Tris (pH 9.5), 100 mmol/L NaCl, 5 mmol/L MgCl2, 375 µg/mL nitroblue tetrazolium, 250 µg/mL 5-bromo-4-chloro-3-indolyl phosphate, Roche Applied Science, Laval, Quebec, Canada]. For CDH13 expression, the membrane was washed in TBST, blocked, and incubated with rabbit T-cadherin polyclonal antibody (H-126, 1:100, Santa Cruz Biotechnology) for 1 hour followed by either a goat anti-mouse IgG-alkaline phosphatase conjugated secondary antibody (1:8,000, Bio-Rad) or a goat anti-rabbit IgG-horseradish peroxidase conjugated secondary antibody (1:8,000, Santa Cruz Biotechnology) for 1 hour. Proteins were visualized using either alkaline phosphatase buffer containing nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate or enhanced chemiluminescence reagents (Amersham, Little Chalfont, United Kingdom). As a loading control, ß-tubulin mouse monoclonal antibody was used (1:30,000, Sigma Chemical Co., St. Louis, MO).
Immunohistochemistry
Mouse (eyes or heads) and human eyes were fixed overnight in 4% formaldehyde from paraformaldehyde, paraffin embedded, and sectioned at 5 µm. Of five antibodies that were tested for immunohistochemistry, two were successful. Mouse and human sections were incubated with either rabbit anti-CDH11 (3 µg/mL, Zymed, South San Francisco, CA) or mouse monoclonal anti-CDH11 (CDH113H, 0.001 µg/µL) in 1% bovine serum albumin/TBST overnight at 4°C. The secondary antibody was biotinylated goat anti-rabbit or biotinylated horse anti-mouse IgG (H+L, 1:200, Vector Laboratories, Burlingame, CA). The color substrate was developed using a metal-enhanced diaminobenzidine substrate kit (Immunopure, Pierce Biotechnology, Rockford, IL). Double-stained mouse sections were further incubated with mouse monoclonal anti-SV40 TAg (1:200, Santa Cruz Biotechnology) followed by a biotinylated horse anti-mouse IgG (H+L) secondary antibody (1:200, Vector Laboratories). Streptavidin-Alexa488 was used as detection reagent. Slides were washed in TBST, mounted, and visualized with either light or fluorescence microscopy.
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received January 12, 2004; revised July 14, 2004; accepted July 19, 2004.
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