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Divisions of 1 Medical Sciences and 2 Cancer Studies, School of Medicine, The University of Birmingham, and 3 West Midlands Regional Genetics Laboratory, Birmingham Women’s Hospital, Birmingham, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first mammalian Wnt was identified by its ability to promote mouse mammary tumorigenesis (14) . The canonical Wnt response pathway operates by stabilizing ß-catenin, enabling it to accumulate in the nucleus where it directs transcription of a range of genes in association with Lef/TCF factors (15) . In colorectal cancer, inactivating mutations of APC or stabilizing mutations of ß-catenin lead to constitutive activation of this pathway, and this has led to the assumption that Wnt signaling can make no additional contribution to tumor progression (16) .
However, two ß-catenin-independent Wnt response pathways have been described more recently. The planar cell polarity pathway, first identified in Drosophila (17) , diverges from the ß-catenin response upstream of APC and leads to activation of RhoA and JNK, whereas the Wnt/Ca pathway operates through PKC and CamKII (18) . It is possible that at least one of these contributes to tumor progression because Wnt3 has been shown to direct cyclooxygenase 2 transcription through a ß-catenin-independent route (19) .
ß-catenin regulates the expression of a number of Wnt pathway factors, including Axin 2 and hNkd1 (20) , the latter acting to direct the response toward the planar cell polarity pathway (21) . Thus, dysregulation of ß-catenin activity is likely to lead to a redirection of subsequent Wnt responses toward the noncanonical pathways, and this may play a role in progression of the tumor.
Wnt inhibitory factor 1 (22) , Dickkopfs (23) , and the secreted frizzled-related proteins (sFRPs; Refs. 13 , 24 ) are distinct classes of extracellular Wnt antagonists and may be expected to counter persistent or excessive stimulation by Wnts. Dickkopfs genes do not interact directly with Wnts but block the action of the lrp 5/6 coreceptor in canonical signaling (25) , whereas Wnt inhibitory factor 1 may be specific for Wnt8 (22) .
The sFRPs comprise an N-terminal domain homologous to the cysteine-rich domain (CRD) of the frizzled family of Wnt receptors and a COOH-terminal domain with some homology to netrin (13) . The CRD domain of the sFRPs competes with the frizzled receptors for Wnt binding, modulating the signal (26) .
The sFRP1 gene was proposed to lie at 8p11.2, within the region found to be deleted in our earlier tumor studies. Loss of expression has been shown recently to correlate with lymph node metastases and increased mortality in breast tumors (27) .
We present data on the localization, gene structure, a frequent polymorphism in the NH2 terminus of sFRP1, and tissue-specific alternative splicing of the COOH terminus. Mutational analysis of the coding region demonstrated homozygous inactivating mutations in 30% of a selected series of colorectal tumors, indicating that sFRP1 might act as a tumor suppressor gene in colorectal carcinogenesis. We proceeded to investigate sFRP1 in a larger cohort of colorectal tumors, looking at the influence of epigenetic phenomena on expression levels, and genomic alterations.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR conditions for all of the reactions were as follows: 95°C for 5 min, then 24 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 5 min. PCR products (1 µl) were combined with 1.5 µl loading buffer, size standard 0.5 µl 6-carboxytetramethylrhodamine, and 1 µl formamide (all supplied by Applied Biosystems, Foster City, CA). Products were denatured at 95°C for 4 min and resolved on an ABI 377 6% denaturing polyacrylamide gel (19:1; Applied Biosystems). Results were analyzed using ABI Genescan software (Applied Biosystems).
Immunohistochemistry.
The streptavidin-biotin indirect immunoperoxidase method was used as we have published previously (28)
. Briefly, 5-µm sections were dewaxed, rehydrated, and blocked by 10% H2O2 in methanol for 10 min. Microwave antigen retrieval was undertaken for 1 h. Sections were incubated overnight with primary antibody. Primary antibodies hMLH-1 and hMSH-2 were used at a dilution of 1:20 and 1:100, respectively (PharMingen, San Diego, CA). After washing with PBS, sections were incubated with biotinylated goat antimouse (Dako, Carpinteria, CA) according to the manufacturer’s instructions for 30 min. Serial PBS washing and incubation with streptavidin-peroxidase conjugate (Dako) was undertaken before incubation with diaminobenzidine tetrahydrochloride (Sigma, Poole, United Kingdom). Sections were counterstained with hemalum, dehydrated, and then analyzed by light microscopy.
Loss of Heterozygosity Analysis.
The colorectal tumors were examined for loss of heterozygosity using four microsatellite markers (D8S505 forward, 5'-AGCCTGCTATTTGTAGATAATGTTT; reverse, 5'-AGTGCTAAGTCCCAGACCA; D8S1722 forward, 5'-CCTTGCTGGGAATTGTG; reverse, 5'-AGCTGCCTGGCTAAGAG; D8S532 forward, 5'-GCTCAAAGCCTCCAATGAC; reverse, 5'-GACTTCGTGATCCACCTGC; D8S519 forward, 5'-CTGTCACCCCAGCGTC; and reverse, 5'-AGTGGCCTTTCTGCTCC) found on chromosome 8p11.2 close to the sFRP1 gene. Oligonucleotide primers were obtained from Alta Biosciences (University of Birmingham, Birmingham, United Kingdom).
All of the PCR reactions were prepared to a final volume of 25 µl, containing 2.5 µl of 10x BIOTAQ NH4-based reaction buffer (Bioline, Randolph, MA), 1 mM MgCl2, 0.2 mM deoxynucleoside triphosphate stock (Amersham Pharmacia Biotech, Piscataway, NJ), 5 pmol 33P 
ATP-labeled forward primer, 5 pmol of unlabeled reverse primer, 0.5 units of BIOTAQ DNA polymerase (Bioline), and 100 ng of genomic DNA. Amplification involved an initial step of 2 min at 94°C, followed by 35 cycles of 94°C for 10 s, 55°C or 59°C for 20 s, and 72°C for 30 s, with a final extension of 5 min at 72°C. The DNA was denatured and separated on a 6% acrylamide gel followed by autoradiography.
Analysis of Promoter Methylation Status.
The method used for DNA modification was essentially that of Grunau et al. (29)
. In short, 10 µg tRNA (Sigma) were added to 1 µg of genomic DNA and made up to 100 µl. Freshly prepared NaOH (Sigma) was added to a final concentration of 0.3 M, and the sample was incubated at 42°C for 20 min. A total of 1.2 ml 5.2–5.69 M sodium bisulfite (Sigma) and 10 mM hydroquinone (Sigma; pH 5) was added, and the solution was overlaid with mineral oil and incubated at 55°C for 4 h. DNA was desalted and redissolved into 100 µl of Tris-Cl (pH 8; Sigma). NaOH was added to a final concentration of 0.3 M, and the solution then was incubated at 37°C for 20 min. After incubation, the solution was neutralized; 10 µg of tRNA were added; and nucleic acids were precipitated with ethanol at -20°C overnight. Precipitated DNA was washed with 70% ethanol, dried, and resuspended in 50 µl of 1 mM Tris-HCl (pH 8).
Modified DNA was amplified with oligonucleotides specific for unmethylated DNA (forward, 5'-GAGTTAGTGTTGTGTGTTTGTTGTTTTGT; reverse, 5'-CCCAACATTACCCAACTCCACAACCA) with cycle conditions of 95°C for 5 min, 35 cycles of 95°C for 1 min, 59°C for 1 min, 72°C for 1 min, and 72°C for 7 min. Oligonucleotides specific for methylated DNA used were forward 5'-GTGTCGCGCGTTCGTCGTTTCGC and reverse 5'-AACGTTACCCGACTCCGCGACCG. Cycle conditions were the same as with unmethylated oligonucleotides with an annealing temperature of 63°C. Amplified products were analyzed on a 2% agarose gel.
Methylation status also was analyzed using the combined bisulfite restriction analysis (COBRA) method (30) . The sFRP1 CpG island region was predicted using CpG plot (www.ebi.ac.uk). A strong CpG island (island size > 100 bp; GC percent > 50.0; and Obs/Exp > 0.6) was detected within the region -180 bp to +530 bp relative to the transcription start site of the sFRP1 gene. To analyze this region of the gene for methylation, PCR primers were designed that were specific for the bisulfite modified sequence. The sFRP1 CpG island was analyzed using nested PCR with the following primers: sFRP1 COBRA forward, 5'-GGTTAGTAGTTGGGTGTTTTTGTTTA and sFRP1 COBRA reverse, 5'-CCTTACCTTAAAACTTAAAAACTTC. One two hundred fiftieth of this then was used in a subsequent nested PCR reaction using the primers sFRP1 COBRA forward-nested 5'-TTGGGTGTTTTTGTTTAATAAGAATT and sFRP1 COBRA reverse-nested 5'-AAAACTTATCACACTTAAACATCTC. The PCR conditions used in both reactions were 94°C for 3 min; 30 cycles of 94°C for 20 s, 54°C for 30 s, 72°C for 40 s, and 72°C for 7 min; and 35 cycles were used in the nested PCR reaction. PCR products were incubated with restriction enzymes TaqI and BstUI for 2 h at 65°C and 60°C, respectively, to assay for methylation and were visualized on a 2% agarose gel.
Semiquantitative Real-Time PCR.
Total RNA was extracted using TRI reagent (Helena Biosciences, Sunderland, United Kingdom). First-strand cDNA was synthesized from 2 µg of DNase-treated total RNA using Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech) and random hexamers (Promega, Madison, WI).
Oligonucleotide primers and TaqMan probes were designed using Primer Express, version 1.5 (Applied Biosystems). The primers for sFRP1 (NM 003012) gene amplification were 5'-CCAATCCCACCGAAGCCT and 5'-ATGATGGCCTCAGATTTCAACTC. The sequence for the TaqMan fluorogenic probe for sFRP1 was 5'-CAAGCCCCAAGGCACAACGGTG. Data for the sFRP1 gene were normalized to the epithelial cell-specific gene keratin 8 (KRT8; NM 002273). For KRT8, the primers and probe were 5'-GATCGCCACCTACAGGAAGCT, 5'-ACTCATGTTCTGCATCCCAGACT, and 5'-CCGGCTCTCCTCGCCCTCCA, respectively. The TaqMan Universal PCR Master Mix and the TaqMan probes were purchased from Applied Biosystems. Primers were obtained from Alta Biosciences (University of Birmingham). Multiplex PCR amplifications were performed using an ABI PRISM 7700 sequence detector in a final volume of 25 µl (Applied Biosystems). Each reaction contained 12.5 µl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 90 nM KRT8 and sFRP1 primers, 150 nM sFRP1 TaqMan probe, 175 nM KRT8 TaqMan probe, 1 µl of cDNA sample, and water. The thermal cycling conditions comprised an initial step at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.
Informed consent was obtained from each patient for molecular analysis of the resected specimen. To analyze differences between the tumor and normal specimens, 
2 test was used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PAC clones were used to confirm the genomic map of sFRP1. A combination of PCR amplification and cycle sequencing revealed the presence of two introns in the coding sequence at nucleotides 540/541 and 618/619, as shown in Fig. 1
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Identification of Protein-Truncating Mutations in Tumor DNA.
Flanking primers were designed to amplify each exon from genomic DNA for analysis by direct cycle sequencing. Genomic DNA was analyzed from 10 advanced colorectal tumors shown previously to carry heterozygous interstitial deletions at 8p11.2.
Mutations leading to premature termination of the translation product were identified in 3 of the 10 samples. These were two single-base deletions (26delG and 67delG) and a single-base change (G450A), generating an in-frame stop codon (Fig. 2B)
. Each of these mutations was found within the first exon, shown previously to be sufficient for Wnt antagonist activity (32
, 26) . Of the 10 tumors analyzed, none contained truncating mutations in the second or third exons of sFRP1.
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An additional 51 tumors were analyzed by direct sequence analysis, of which 49 provided clearly interpretable results. Only the first exon was sequenced for stop codon mutations, but none were found. The remaining exons were not sequenced because no mutations had been identified in those regions in the preliminary analysis. This finding indicated that point mutation is not a frequent method of inactivation of the sFRP1 gene in colorectal cancer. The same bank of 51 locally advanced (T3/4) tumors then was analyzed for evidence of other methods of DNA disruption.
Exon 1 of sFRP1 Contains a Common Polymorphism.
In each of the seven tumors from the preliminary investigation without an identified truncating mutation, the retained sFRP1 allele contained an in-frame three-base insertion after nucleotide 37 (Fig. 1)
. This is predicted to lead to an extra alanine in the protein, after codon 13, and is represented in the expressed sequence tag (EST) database.
The presence of this variant was investigated by analysis of blood-derived DNA from 51 colorectal cancer patients (Table 1)
and a separate cohort of 102 patients without a history of colorectal neoplasia. Thirty-two of 102 (31%) colorectal cancer-free individuals carried the variant compared with 18 of 51 (35%) patients with colorectal cancer. No significant association between the development of colorectal cancer and the presence of the 3-bp insertion was identified in this cohort of patients.
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, of which 49 gave interpretable results. Investigation of the methylation status of the promotor region of sFRP1 by methylation-specific PCR and COBRA (shown in Fig. 3
) revealed hypermethylation in 40 (82%) cancers. Although these two approaches agreed broadly, COBRA was more sensitive and informative. We proceeded to analyze a selection of the matched normal colonic mucosal samples (n = 36; Table 1
). Gels were scored by eye for the presence or absence of a methylated band, and images were quantified using the GeneTools analysis package (Syngene, Cambridge, United Kingdom) to provide the percentages given in Table 1
. There were no cases in which sFRP1 was unmethylated in the tumor but methylated in the matched normal mucosa. Only 11 methylated matched normal mucosa samples (P < 0.001) were found. The mean (median) level of DNA modification differed between the groups, at 35% (33%) for the cancers and 10% (9%) for normal mucosae.
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The 3' End of the sFRP1 Coding Region Is Alternatively Spliced.
Attempts to amplify sFRP1 from colonic mucosa cDNA with 3' primers immediately downstream of the stop codon failed, whereas more distal primers yielded amplicons smaller than the predicted size (Fig. 4)
. Sequencing revealed that nucleotides 913-1005 were absent from this transcript, removing seven amino acids and the stop codon predicted in the original sequence and extending the reading frame by an additional 40 amino acids, terminating at nucleotide 1125.
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revealed that the extended protein is the predominant species. The unspliced form is the major species in only the lung and liver, whereas the prostate expresses another variant, which maintains the stop codon used in liver but lacks sequences from nucleotides 942-1092, downstream of the stop codon. Heart cDNA contains the colon and the prostate forms at low, but approximately equivalent, levels. The extended sequence contains a hydrophobic region, which may act as a transmembrane anchor, modifying the localization of the protein. This may influence the function of sFRP1 in different tissues because an untethered protein may be more effective in antagonizing Wnt signaling to tumor cells in trans than would a membrane-bound form.
sFRP1 Transcription Is Down-Regulated in Colorectal Tumors.
We compared the sFRP1 transcript expression level in the same cohort of tumors. Thirty-seven colorectal tumor samples with matched normal mucosa gave analyzable RNA (Table 1
; Fig. 5
). The sFRP1 TaqMan probe was designed to span the exon1/intron1 boundary of the gene. sFRP1 expression in each tumor and normal colon sample was standardized to cytokeratin 8 (KRT8) gene expression. KRT8 was used as an epithelial cell-specific marker because stromal and inflammatory cell components may vary in tumor and matched normal epithelium. The expression of KRT8 was consistent between normal and tumor samples. Expression of sFRP1 in tumors was normalized to the mean of sFRP1 expression in the matched normal mucosa.
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, sFRP1 mRNA was down-regulated by >10-fold in 28 of the 37 (76%) tumors compared with normal mucosa. In 6 of 37 (16%) tumors, there was a <10-fold change in expression level, and sFRP1 expression was up-regulated in 3 of 37 (7%) tumors. The clinical and pathologic data from these 37 tumors were investigated, but no correlation was found between sFRP1 expression level and patient age, sex, tumor site, serosal spread, or presence of lymph node metastases.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies (10, 11, 12) showed localized deletion of the sFRP1 locus in advanced colorectal, prostate, and squamous head and neck tumors, indicating that it plays a role in the progression of many solid tumors. Other studies have demonstrated reduced transcription of sFRP1 in breast cancer (27 , 33 , 34) and gastric cancer (35) .
sFRP1 levels were reduced in breast carcinomas and were maintained in benign breast tumors (34) . One study of 70 breast tumors found reduced levels of sFRP1 mRNA to be associated with lymph node metastases and increased mortality (27) . In our series of locally advanced colorectal tumors, we found no association between sFRP1 expression levels and the development of lymph node metastases or any other clinical or pathologic characteristics, although this had been suggested by our previous loss of heterozygosity studies (12) . This issue may be clarified by analysis of less advanced colorectal cancers and premalignant adenomas for levels of sFRP1 expression. One possibility is that sFRP1 influences tumor progression at an earlier stage of tumor development in colorectal cancer than in breast cancer.
We identified a high frequency (82%) of methylation, comparable with the 95% found by Suzuki et al. (37) , but there was not an absolute inverse correlation between methylation and transcription. There could be a number of reasons for this, including heterogeneity in the cancer samples, but the sFRP1 promotor has yet to be characterized fully; therefore, the contributions of each of its three CpG islands to transcriptional control are not known. Thus, our analysis provides an estimate of the frequency of methylation at the sFRP1 locus rather than a direct measurement of modification of a functional site. However, the observation that demethylation leads to expression of sFRP1 in RKO cells demonstrates that there can be a relationship between these processes, and our results show that methylation and transcriptional repression are common events in colorectal cancer (37) .
The primary translation product of sFRP1 contains an atypical signal sequence, in which the hydrophobic domain is preceded by a stretch of 15 hydrophilic amino acids. We have identified a common polymorphism, which results in an extra amino acid after codon 13. This insertion was over-represented in the retained alleles of our primary cohort of colorectal tumors with interstitial loss, but secondary analysis of a larger series of tumors failed to show a statistically significant correlation between its presence and the risk of developing colorectal cancer. Additional studies are underway to determine whether this polymorphism has any direct effect on protein activity.
The COOH-terminal domain of sFRP1 is related to netrin 1 (31) , a regulator of apoptosis via its interaction with DCC (38) , and this netrin-related motif also is found in a range of other proteins where it is thought to mediate protein-protein interactions (39) . The identification of sFRP1 as SARP2 (24) demonstrated its potential to promote apoptosis. If this is a response to Wnt occupancy of the N-terminal CRD, it may be modified by differences at the COOH terminus. We have identified alternative splicing, which leads to an extended COOH terminus in a range of tissues, including colon and prostate, and this may influence the role of sFRP1 in tumorigenesis.
According to the model of colorectal cancer progression that has emerged in recent years, APC and ß-catenin mutations lead to constitutive stimulation of ß-catenin transcription as an early or initiating event, and mutations in the canonical Wnt response pathway upstream of ß-catenin would have little effect (16) . However, Wnt signaling can act through at least two ß-catenin-independent, noncanonical pathways (40 , 41) . This suggests a model whereby chronic ß-catenin signaling leads to a shift in the Wnt response toward these alternative pathways and loss of the antagonist function of sFRP1 hypersensitizes the tumor to Wnt. This loss of stringency in growth factor responses could be an important step in tumor progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Notes: K. Gensberg is currently at the School of Biosciences, The University of Birmingham, Birmingham, United Kingdom; S. Jan is currently at the Birmingham Clinical Trials Unit, Birmingham, United Kingdom; R. G. Hardy is currently at the Department of Clinical and Surgical Sciences (Surgery), Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; S. Chughtai is currently at the Division of Medical Genetics, The University of Birmingham, Birmingham, United Kingdom.
Requests for reprints: Glenn Matthews, Department of Surgery, Queen Elizabeth Holspital, Birmingham, B15 2TH, United Kingdom.
Received 5/13/03. Revised 11/24/03. Accepted 11/26/03.
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